Methods for purifying heteromultimeric antibodies

Description

1 / 61 “METHODS FOR PURIFYING HETEROMULTIMERIC ANTIBODIES”

[0001] The present invention relates to methods for purifying polypeptides containing heteromultimeric Fc domains from mismatched variants thereof. Background of the Invention

[0002] Multispecific antibodies, such as bispecific antibodies, are very promising candidates for cancer treatment, and multiple different formats for bispecific antibodies have been developed. Many of them are heteromultimeric protein complexes generated by the co-expression of different polypeptide chains. With the development of new antibody formats, a new class of impurities, referred to as product-related impurities, has begun to appear in significant quantities. These product-related impurities are mismatched variants of heteromultimeric antibodies. In some situations, the correctly paired antibody constitutes only a minor fraction of all variants. Since correctly paired and mismatched antibodies often have very similar biochemical properties, they are difficult to separate using classical antibody purification methods.For example, in the case of a heteromultimeric antibody comprising an Fc domain, many mismatched variants will also comprise an Fc domain and thus cannot be easily removed by classical chromatography processes comprising a capture step involving binding to the Fc domain based on protein A, protein G, or their derivatives.

[0003] Furthermore, regarding any antibody production, it is an important objective to remove process-related impurities generated during production, such as host cell proteins or nucleic acids. Petition 870250087783, dated 09 / 29 / 2025, page 9 / 87 2 / 61

[0004] There is, therefore, a need for purification methods that can separate a multispecific antibody from both process-related impurities as well as product-related impurities. A solution to this problem is provided by the present matter of independent claims. Summary of the Invention

[0005] In a first aspect, the present invention relates to a method for purifying a polypeptide containing a heteromultimeric Fc domain, wherein the method comprises the following steps in the order indicated: a) providing a sample comprising the polypeptide containing a heteromultimeric Fc domain and one or more mismatched variants thereof; b) Place a protein chromatography matrix A in contact with the sample and bind the polypeptide containing the heteromultimeric Fc domain to the protein chromatography matrix A; c) placing the protein chromatography matrix A in contact with a washing solution, wherein the washing solution comprises octanoate; d) place the Protein A chromatography matrix in contact with an elution solution; and (e) collect an eluate comprising the polypeptide containing a heteromultimeric Fc domain.

[0006] In a second aspect, the present invention relates to the use of a washing solution comprising octanoate in a method of purifying a polypeptide containing a heteromultimeric Fc domain according to the first aspect.

[0007] In a third aspect, the present invention relates to a kit comprising a protein A chromatography matrix; and a washing solution comprising octanoate.

[0008] In a fourth aspect, the present invention relates to an eluate Petition 870250087783, dated 09 / 29 / 2025, page 10 / 87 3 / 61 obtained by purification method of a polypeptide containing a heteromultimeric Fc domain according to the first aspect, wherein the eluate comprises at least 90% of a purified polypeptide containing a heteromultimeric Fc domain; and less than 0.5% of mismatched variants. Detailed Description

[0009] Before the present invention is described in detail below, it should be understood that this invention is not limited to the particular methodology, protocols, and reagents described in this document, as these may vary. It should also be understood that the terminology used in this document is intended to describe only particular embodiments and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used in this document have the same meanings commonly understood by a person of ordinary skill in the art. Preferably, the terms used in this document are defined as described in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, Leuenberger, HGW, Nagel, B. and Kolbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

[0010] Several documents are cited throughout the text of this descriptive report. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, etc.), whether above or below, is hereby incorporated by reference in its entirety. Nothing in this document should be construed as an admission that the invention is not entitled to precede such invention by virtue of the prior invention.

[0011] To carry out the present invention, unless otherwise indicated, conventional methods of chemistry, biochemistry and recombinant DNA techniques are employed, which are explained in the literature in the field (see, Petition 870250087783, dated 09 / 29 / 2025, p. 11 / 87 4 / 61 for example, Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0012] Throughout this descriptive report and the claims that follow, unless the context requires otherwise, the word “includes” and variations such as “include” and “comprising” will be understood as implying the inclusion of a whole number or step or group of whole numbers or steps, but not the exclusion of any other whole number or step or group of whole numbers or steps. As used in this descriptive report and the appended claims, the singular forms “a”, “an”, “the” and “the” include plural references, except where the content clearly dictates otherwise.

[0013] The term antibody, as used in this document, is intended to include both native and genetically modified antibodies. The term genetically modified antibody includes functional antibody fragments, single-chain antibodies, single-domain antibodies, monospecific or multispecific (bispecific, trispecific, tetraspecific, pentaspecific, hexaspecific, etc.), monovalent or multivalent antibodies (bivalent, trivalent, tetravalent, etc.), as well as antibodies having more than one function (multifunctional antibodies), for example, antibodies comprising one or more variable domains and additional domains, such as an Fc domain capable of binding to the Fc receptor FcyRIII (also known as CD16).

[0014] Native antibodies are Y-shaped molecules comprising four polypeptide chains: two heavy chains and two light chains, in which the heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are five main classes (or isotypes) of heavy chains that determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA, and IgE. When referring to IgG in general, IgG1, IgG2, IgG3, and IgG4 are included. Petition 870250087783, dated 09 / 29 / 2025, p. 12 / 87 5 / 61 less than defined otherwise. Each light chain consists of two domains, the N-terminal domain being known as the variable or VL domain (or region) and the C-terminal domain being known as the constant (or Cl) domain (kappa constant (Ck) or lambda constant (CA)). Each heavy chain includes four or five domains depending on the antibody isotype: a variable domain (VH) followed by the first constant domain (CH1), the hinge region, and then constant domains CH2 and CH3, and in some isotypes CH4. In an assembled antibody, the Vl and Vh domains associate to form an antigen-binding domain. In addition, the Cl and CH1 domains associate to maintain a heavy chain associated with a light chain. The two heavy-light chain heterodimers associate through the interaction of the CH2 and CH3 domains and the interaction between the hinge regions of the two heavy chains.Constant domains confer important biological properties, such as antibody chain association, secretion, transplacental mobility, complement binding, and binding to Fc receptors (FcR).

[0015] Bispecific antibodies occur in a plurality of formats (Brinkmann and Kontermann, MAPS 2017, Vol. 9, No. 2, 182-212). Formats comprising only variable domains have the advantage of a very low molecular weight leading to satisfactory tumor penetrance, which is important for oncological applications. A disadvantage, however, is a low plasma half-life due to the lack of a constant domain, which mediates binding to FcRn.

[0016] In the following description, in some cases, amino acid numbers are used in relation to antibodies or antibody domains, which do not indicate a SEQ ID NO. These numbers refer to amino acid positions in antibodies according to the UniProtKB database (www.uniprot.org / uniprot) in the version as released on August 26, 2016. Unless otherwise specified, the number(s) correspond(s) to a position in human IgG, in particular in human IgG1. Petition 870250087783, dated 09 / 29 / 2025, page 13 / 87 6 / 61 The UniProtKB sequences (in the version as released on August 26, 2016) of antibody domains referred to herein, including those of human IgG1, are incorporated by reference as particular embodiments of the domains described herein, as well as their variants as defined below.

[0017] In the context of this descriptive report, the term “immunoglobulin (Ig) domain” is used to refer to a protein domain consisting of a 2-layer sandwich of 7-9 antiparallel β strands arranged in two β sheets with a Greek key topology. The Ig domain is probably the most frequently used “building block” in naturally occurring proteins. Proteins containing Ig domains are subsumed in the immunoglobulin superfamily. Not only antibodies, but also cell adhesion molecules, T cell receptors, Fcy receptors, and many more belong to this protein family.The immunoglobulin fold was fully described in a review article by Bork et al. (The immunoglobulin fold. Structural classification, sequence patterns and common core. September 1994; J. Mol. Biol. 242 (4): 309-20).

[0018] Specific binding means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. In certain embodiments, an antibody is said to bind specifically to an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. The ability of an antigen-binding molecule to bind to a specific antigen can be measured using an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to an individual skilled in the art, for example, Surface Plasmon Resonance (SPR) technique. As such, the specified ligand binds to its target molecule and does not bind in a substantial amount to other molecules present.Generally, an antibody that “specifically binds” to a target molecule has an equilibrium dissociation constant of less than about 10-5 (e.g., 106, 10-7, 10-8, 10-9, 10-10, 10-11, or 10-12) M for that target molecule. Petition 870250087783, dated 09 / 29 / 2025, p. 14 / 87 7 / 61

[0019] The term “antigen” is used to refer to a substance, preferably an immunogenic polypeptide comprising at least one epitope, preferably an epitope that induces a B cell or T cell response or a B cell and T cell response.

[0020] An “epitope,” also known as an antigenic determinant, is that part of a substance, for example, an immunogenic polypeptide, that is recognized by the immune system. Preferably, this recognition is mediated by the binding of antibodies, B cells, or T cells to the epitope in question. In this context, the term “binding” preferably refers to a specific binding. Epitopes generally consist of chemically active surface clusters of molecules such as amino acids or sugar side chains and generally have specific three-dimensional structural features as well as specific charge characteristics. The term “epitope” encompasses conformational and non-conformational epitopes. Conformational and non-conformational epitopes are distinguished by the fact that binding to the former, but not the latter, is lost in the presence of denaturing solvents.

[0021] An immunogenic polypeptide according to the present invention is a polypeptide that is specifically linked by the antigen-binding domain of an antibody or T-cell receptor. In the case of a T-cell receptor, the immunogenic polypeptide is linked in a complex with an MHC protein. In some embodiments, the immunogenic polypeptide is a tumor antigen, that is, a polypeptide specifically expressed by a tumor cell, preferably expressed on the surface of a tumor cell or presented on the surface of a tumor cell. In some embodiments, the immunogenic polypeptide is an antigen derived from a cell receptor. In some embodiments, the immunogenic polypeptide is an antigen derived from a cytokine. In some embodiments, the immunogenic polypeptide is an antigen derived from a pathogen selected from the Petition 870250087783, dated 09 / 29 / 2025, page 15 / 87 8 / 61 group consisting of viruses, bacteria, and protozoa. In some embodiments, the immunogenic polypeptide is an immune cell antigen, preferably an immune effector cell antigen, that is, a polypeptide expressed by an immune cell, preferably expressed on the surface of an immune cell.

[0022] A variable domain or immunoglobulin variable domain, as used herein, refers to each of a pair of domains that form an antigen-binding domain. An immunoglobulin variable domain can be a variable domain of an antibody or T-cell receptor (TCR). Antibody and TCR variable domains have the same general structure. Each variable domain comprises four framework regions (FRs) whose sequences are largely conserved, connected by three hypervariable regions (or complementarity-determining regions, CDRs). The framework regions adopt a β-sheet conformation, and the CDRs can form loops that connect the β-sheet structure and are maintained in their three-dimensional structure by the framework regions. In preferred embodiments, variable domain, as used herein, refers to antibody variable domain.

[0023] The term single variable domain immunoglobulin (ISVD), used interchangeably with single variable domain, refers to a single monomeric immunoglobulin variable domain that is capable of selectively binding to a specific antigen on its own, i.e., without a second immunoglobulin variable domain. For a further description relating to ISVD, reference is made to WO2021 / 110816. In view of the above definition, the antigen-binding domain of a conventional four-chain antibody (such as an IgG, IgM, IgA, IgD or IgE, known in the art) or of a Fab fragment, an F(ab')2 fragment, an Fv fragment such as a disulfide-linked Fv or an scFv fragment, or a diabody (known in the art) derived from a conventional four-chain antibody, would not normally be considered an ISVD, since in these cases the binding. Petition 870250087783, dated 09 / 29 / 2025, page 16 / 87 9 / 61 binding to the respective epitope of an antigen would not normally occur through a single immunoglobulin domain, but through a pair of immunoglobulin domains in association, such as variable light and heavy chain domains, i.e., a Vh-Vl pair of immunoglobulin domains, which together bind to an epitope of the respective antigen. On the other hand, ISVDs have the ability to bind specifically to an antigen epitope without pairing with an additional variable immunoglobulin domain. The binding site of an ISVD is formed by a single Vh, single VhH, or single Vl domain. A VhH domain refers to a variable domain comprised in a heavy chain antibody found in camelids.Thus, the ISVD can be a variable domain sequence of the light chain (e.g., a Vl sequence) or a suitable fragment thereof; or a variable domain sequence of the heavy chain (e.g., a Vh sequence or VhH sequence) or a suitable fragment thereof; provided that it is capable of forming a single antigen-binding unit / domain; that is, a functional antigen-binding unit / domain consisting essentially of the ISVD, such that the single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit. A single ISVD or variable domain can, for example, be a heavy-chain ISVD, such as a Vh, VhH, including a camelized Vh or humanized VhH. In one embodiment, it is a VhH, including a camelized Vh or humanized VhH. Heavy-chain ISVDs can be derived from a conventional four-chain antibody or from a heavy-chain antibody.For example, ISVD may be a single-domain antibody (or an amino acid sequence that is suitable for use as a single-domain antibody), a “dAb” (or an amino acid sequence that is suitable for use as a dAb), or a Nanobody® molecule (as defined in the prior art, notably in document no. WO2021 / 110816 and including, but not limited to, a VhH); other single variable domains or any fragment thereof. Petition 870250087783, dated 09 / 29 / 2025, page 17 / 87 10 / 61 In particular, ISVD can be a Nanobody® molecule (such as a VhH, including a humanized or camelized VhH) or a suitable fragment thereof.

[0024] The term Fc domain, as used in the context of the present invention, encompasses Fc and native Fc variants and includes monomeric, dimeric, and multimeric Fc domains, with or without a hinge region. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc domain is generally defined to extend from an amino acid residue at the C226 position, or from P230, to the carboxyl terminus thereof. An Fc domain comprises at least one, preferably two, polypeptide chains, each comprising a CH2 domain or a functional portion thereof and a CH3 domain or a functional portion thereof (collectively referred to herein as the CH2-CH3 region). The two polypeptide chains may be linked by covalent (i.e., disulfide bonds) and / or non-covalent association.The number of intermolecular disulfide bonds between monomeric subunits of native Fc domains ranges from 1 to 4 depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2). An example of a native Fc domain is a dimer resulting from the papain digestion of an IgG, in which the two polypeptide chains are associated by disulfide bonds.

[0025] The term CH2-CH3 region refers to a fraction comprising at least one functional portion of a CH2 domain and at least one functional portion of a CH3 domain. An example of a functional portion of a CH3 domain is a CH3 domain in which C-terminal amino acid residues, for example, the 5, 4, 3, 2, or 1 most C-terminal amino acid residues, have been removed or replaced with a linker. This modification does not impair the binding of the CH3 domain or a dimeric Fc domain comprising this CH3 domain to Protein A or Protein G. Petition 870250087783, dated 09 / 29 / 2025, page 18 / 87 11 / 61

[0026] Two CH2-CH3 regions together form a dimeric “Fc domain”.

[0027] “Protein A” is a 42 kDa protein that was originally isolated from Staphylococcus aureus and is capable of specifically binding to the Fc domain of many immunoglobulin molecules, including that of human IgG1, IgG2, and IgG4, through its five homologous immunoglobulin-binding domains, each comprising a set of three helices. In the context of the invention, Protein A refers to a ligand used in capture chromatography resins that may be native or manipulated (i.e., mutated) to improve their chromatographic performance (e.g., increased binding efficiency, increased selectivity, increased caustic stability).Thus, the term Protein A, as used in the context of the present invention, also refers to derivatives of Protein A that retain at least 10% of the ability of native Protein A to bind to a dimeric immunoglobulin Fc domain, such as proteins comprising multiple copies of a single immunoglobulin-binding domain of Protein A or proteins comprising immunoglobulin-binding domains of Protein A and Protein G or proteins comprising one or more genetically modified Protein A domains. Protein G is a 58-65 kDa protein that was originally isolated from streptococcal bacteria and is capable of specifically binding to the Fc domain of many immunoglobulin molecules, including human IgG1, IgG2, and IgG4. The term "Protein G", as used in the context of the present invention, also refers to derivatives of Protein G that retain at least 10% of the ability of native Protein G to bind to a dimeric immunoglobulin Fc domain.

[0028] A CH2-CH3 region, and in particular a dimeric Fc domain formed by two CH2-CH3 regions, as defined in the context of the invention, exhibits specific binding to protein A or protein G, in particular protein A, preferably with an affinity that is at least 0.01, at least 0.05, at least 0.1, at least 0.2, or at least 0.5 of Petition 870250087783, dated 09 / 29 / 2025, page 19 / 87 12 / 61 affinity of the CH2-CH3 region or Fc domain of IgG1, IgG2 or IgG4 for native Protein A or G, in particular Protein A, and preferably with no more than 100x or 10x the affinity of the CH2-CH3 region or Fc domain of IgG1, IgG2 or IgG4 for native Protein A or G, in particular Protein A. A CH2-CH3 region and, in particular, an Fc domain, as defined in the context of the invention, exhibits specific binding to commercially available Protein A or Protein G chromatography resins, including, but not limited to, MabSelect sure protein A resin (Cytiva).

[0029] Preferably, the CH2 and CH3 domains of the CH2-CH3 region are derived from IgG1, IgG2, or IgG4. If the CH2 and / or CH3 domains contain deletions, substitutions, and / or insertions or other modifications to the effect that the CH2-CH3 region is unable to bind specifically to protein A or protein G, in particular protein A, they are not considered functional portions of a CH2 or CH3 domain and do not form a CH2-CH3 region as defined in the invention. It has been reported that native human IgD, IgE, and IgGa exhibit no or only weak binding to protein A. Thus, a native CH2-CH3 region or Fc domain of IgD, IgE, or IgG3 would not be a functional CH2-CH3 region or Fc domain in the context of the first, second, third, or fourth aspect of the invention. Native human IgA, IgD, IgE, and IgM exhibit no or only weak binding to protein G.Thus, a native CH2-CH3 region or Fc domain of IgA, IgD, IgE, and IgM would not be a functional CH2-CH3 region or Fc domain in the context of alternative aspects of the invention related to purification using a protein G chromatography matrix.

[0030] Typically, but not necessarily, the CH2-CH3 region can also bind to an Fc receptor (e.g., an FcyR; or an FcRn), and / or can participate in complement activation.

[0031] In the context of different antibody formats, polypeptide chains comprising a CH2-CH3 region and at least one domain Petition 870250087783, dated 09 / 29 / 2025, page 20 / 87 13 / 61 variable are often referred to as “heavy chains”, whereas polypeptide chains comprising at least one variable domain but no CH2-CH3 region are often referred to as “light chains”.

[0032] The term “Fc domain,” as used herein, also includes an “Fc variant domain.” An Fc variant domain as used herein refers to a domain that is modified from a native Fc domain. In particular, an Fc variant domain is an Fc domain comprising a modification that results in an advantageous property that is novel or improved compared to the native Fc domain. The term Fc variant domain includes Fc domains comprising knob-into-hole mutations (see below). The term Fc variant domain may also refer to a domain that is humanized from a non-human native Fc domain. Furthermore, an Fc variant domain may also refer to an Fc domain in which regions have been removed that provide structural features or biological activity that are not required for the antigen-binding proteins of the invention.Thus, the term Fc variant domain may refer to an Fc domain in which one or more amino acid residues have been modified, affecting or being involved in: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity after expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than a salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the Fc variant domain is genetically modified, for example, by introducing mutations as defined in this document below.

[0033] When the Fc domain is dimeric, the two polypeptide chains are preferentially derived from the same isotype subclass or antibody isotype. Petition 870250087783, dated 09 / 29 / 2025, page 21 / 87 14 / 61

[0034] In some embodiments, the Fc domains comprise “knob-into-hole” mutations. The “knob-into-hole” technology refers to mutations at the CH2-CH3 interface of a dimeric Fc domain to create a “knob” in one CH3 domain and a “hole” in the other CH3 domain, promoting heteromultimer formation. By way of non-limiting example, particular “knob-into-hole” mutations are T366S, T366Y, L368A, Y407V, and Y407T. These knob-into-hole mutations can be further stabilized by the introduction of additional cysteine ​​amino acid substitutions Y349C and S354C. In the context of this descriptive report, a knob-into-hole Fc domain refers to the Fc domain comprising knob-into-hole mutations. In the context of this descriptive report, a "CH2-CH3 knob-into-hole region" is a CH2-CH3 region in which the CH3 domain comprises "knob-into-hole" mutations.

[0035] In some embodiments, the Fc domains comprise “RF mutations”. In the context of this descriptive report, the term “RF mutations” refers to the H435R and Y436F mutations (RF mutations) in one of a pair of CH3 domains.

[0036] In some embodiments, Fc domains comprise additional amino acid substitutions, such as charged pair substitutions, to enhance heterodimerization of the polypeptide chains that form the Fc domain.

[0037] In some embodiments, the Fc domain comprises one or more modifications that inhibit binding to the Fc gamma receptor (FcyR). Such modifications may be in one or both, preferably both, polypeptide chains that form the Fc domain. The modifications may include L234A and L235A as a non-limiting example.

[0038] In some embodiments, the Fc domain comprises an “N297Q”, “N297G”, or “N297A” mutation to remove the N-glycosylation site within the Fc portion. Such a mutation abolishes the interaction with the Fc-gamma receptor. The mutation may be in one or both, preferably both chains. Petition 870250087783, dated 09 / 29 / 2025, page 22 / 87 15 / 61 polypeptides that form the Fc domain.

[0039] The hinge, hinge region, or hinge domain typically refers to the flexible portion of a heavy chain located between the CH1 domain and the CH2 domain. It is approximately 25 amino acids long and is divided into an upper hinge, an intermediate hinge or central hinge, and a lower hinge.

[0040] In some embodiments, the Fc domain comprises or additionally comprises at least two additional cysteine ​​residues, for example, one in each polypeptide chain forming a dimeric Fc domain, to increase heterodimerization, or both in the same polypeptide chain, to form an intradomain disulfide bond.

[0041] In some embodiments, the Fc domain-containing polypeptide is fused to an ISVD as described above or to a cytokine that forms an immunocytokine. In particular, said ISVD or cytokine is fused to at least one of the CH2-CH3 regions of a polypeptide chain of the Fc domain-containing polypeptide. In some embodiments, the Fc domain-containing polypeptide comprises two polypeptide chains, each comprising a CH2-CH3 region, wherein an ISVD or cytokine is fused to each of the CH2-CH3 regions.

[0042] “Melted” or “fusion” in the context of the invention means that two polypeptides are covalently linked, in particular through a peptide bond. Preferably, two fused polypeptides are expressed as a single polypeptide chain.

[0043] The term cytokine in the context of the invention refers to small proteins (approximately 5-25 kDa) that are involved in autocrine, paracrine, and endocrine signaling as immunomodulatory agents. Cytokines cannot cross the lipid bilayer of cells to enter the cytoplasm and act through cell surface receptors. The term cytokine, as used herein, includes chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. Petition 870250087783, dated 09 / 29 / 2025, page 23 / 87 16 / 61

[0044] The term immunocytokine in the context of the invention refers to the molecule comprising a fusion protein comprising a cytokine and at least one variable immunoglobulin domain.

[0045] Unless otherwise specified, the binding proteins of the present invention are oriented with the amino-terminal direction (N-terminal end or N-terminal) on the left side and the carboxyl-terminal direction (C-terminal end or C-terminal) on the right side, in accordance with standard usage and convention.

[0046] The determination of the percentage of identity between two sequences is performed using the mathematical algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90, 5873-5877, 1993. This algorithm is incorporated into the BLASTN and BLASTP programs of Altschul et al. (1990) J. Mol. Biol. 215, 403-410. To obtain alignments with GAPPED for comparative purposes, Gapped BLAST is used as described in Altschul et al. (1997) Nucleic Acids Res. 25, 3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters of the respective programs are used. Alternatively, a variant may also be defined as having up to 20, 15, 10, 5, 4, 3, 2 or 1 amino acid substitutions, in particular conservative amino acid substitutions. Conservative substitutions are well known in the art (see, for example, Creighton (1984) Proteins. W.H. Freeman and Company).Families of amino acid residues with similar side chains are known in the art, and include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan).

[0047] The term chromatography matrix, as used in this document, refers to a solid-phase material that is capable of binding Petition 870250087783, dated 09 / 29 / 2025, p. 24 / 87 17 / 61 selective to one or more components of an applied charging fluid, as is well known in the art.

[0048] The expression "contacting a chromatography matrix with a solution" refers to applying the solution to a chromatography matrix and denotes a step in the claimed purification method in which a solution is contacted with a solid phase. This denotes that said solution is added to a chromatographic device in which the solid phase is located. In preferred embodiments, the solid phase is a stationary solid phase. Alternatively, in another embodiment, the solid-phase chromatography matrix may be directly added to the solution and recovered after centrifugation binding as a non-limited example. A solution comprising one or more substances, in particular the polypeptide containing heteromultimeric Fc domain and product- and process-related impurities, passes through the solid phase allowing interaction between the solid phase and the substances.Depending on conditions such as pH, conductivity, salt concentration, temperature, and / or flow rate, some substances in the solution are bound to the solid phase and are thus removed from the solution. Other substances remain in solution. The substances that remain in solution can be found in the passing stream.

[0049] The flow rate denotes the solution obtained after passing through the chromatographic device regardless of its origin. A washing step may optionally be applied to level the column. Subsequently, the application of an elution buffer may be used to cause the elution of one or more substances. The substance may be recovered from the solution by methods familiar to a person skilled in the art, such as, for example, precipitation, salting, ultrafiltration, diafiltration, lyophilization, affinity chromatography, or solvent volume reduction to obtain the substance of interest in purified or even substantially homogeneous form. Petition 870250087783, dated 09 / 29 / 2025, page 25 / 87 18 / 61

[0050] The term binding and elution mode denotes a way of performing a purification method by chromatography. In the present document, a solution comprising the protein to be purified and impurities is applied to a stationary phase, particularly a solid phase, where the protein to be purified interacts with the stationary phase and is retained therein. Some impurities are removed with the passing stream. The protein to be purified is then recovered from the stationary phase in a second step by applying an elution solution (typically a buffered solution), typically in a gradual or linear gradient (or a combination thereof) so that the protein to be purified elutes separately from impurities potentially bound to the stationary phase.

[0051] The term flow-through mode denotes an alternative way of performing a chromatographic purification method. In the present document, a solution comprising the protein to be purified and the impurities is applied to a stationary phase, particularly a solid phase, wherein the impurities, but not the protein to be purified, interact with the stationary phase and are retained therein. The protein of interest is eluted by flow-through.

[0052] As used in this document, buffer refers to a buffered solution that resists changes in pH through the action of its acid-base conjugate components. Loading buffer is used to load the protein mixture to be purified into the chromatography array, and washing buffer is used to wash the chromatography array in order to remove unbound material. Elution buffer is used to elute the protein to be purified from the column.

[0053] The term multimer (multi-, many and -mer, parts), as used in this document, denotes a molecule consisting of subunits (monomers) linked by covalent or non-covalent bonds, and Petition 870250087783, dated 09 / 29 / 2025, page 26 / 87 19 / 61 includes, for example, dimers (consisting of two monomers) and tetramers (consisting of four monomers).

[0054] The term “homodimer”, as used herein, denotes a dimer consisting of two identical subunits, while the term “heterodimer”, as used herein, denotes a dimer consisting of two different subunits.

[0055] The term “heteromultimeric polypeptide”, as used in this document, denotes a protein complex comprising more than one, preferably at least three, polypeptide chains. The polypeptide chains comprised in the heteromultimeric polypeptide are not identical. In other words, each of the polypeptide chains comprised in the heteromultimeric polypeptide is different from at least one other polypeptide chain, preferably from all other polypeptide chains comprised in the heteromultimeric polypeptide.

[0056] The elements of the present invention will be described below. These elements are listed with specific embodiments; however, it should be understood that they can be combined in any way and in any number to create additional embodiments. The various examples and preferred embodiments described should not be interpreted as limiting the present invention only to the embodiments explicitly described. This description should be understood as supporting and encompassing embodiments that combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all the elements described in this application should be considered disclosed by the description of the present application, unless the context indicates otherwise.

[0057] In a first aspect, the present invention relates to a method for purifying a polypeptide containing a heteromultimeric Fc domain, wherein the method comprises the following steps in the order indicated: a) providing a sample comprising the polypeptide containing Petition 870250087783, dated 09 / 29 / 2025, page 27 / 87 20 / 61 heteromultimeric Fc domain and one or more mismatched variants thereof; b) Place a protein chromatography matrix A in contact with the sample and bind the polypeptide containing the heteromultimeric Fc domain to the protein chromatography matrix A; c) placing the protein chromatography matrix A in contact with a washing solution, wherein the washing solution comprises octanoate; d) place the Protein A chromatography matrix in contact with an elution solution; and (e) collect an eluate comprising the polypeptide containing a heteromultimeric Fc domain.

[0058] In an alternative aspect, a method is provided for purifying a polypeptide containing a heteromultimeric Fc domain according to the first aspect, wherein steps b) to d) comprise the use of a protein A or protein G chromatography matrix, in particular a protein G chromatography matrix. All embodiments specified for the first aspect are also provided for this alternative aspect, wherein every reference to protein A is intended to refer to protein A or protein G.

[0059] The expression of a polypeptide containing a heteromultimeric Fc domain requires co-expression of the different polypeptide chains comprised within the heteromultimeric polypeptide. Due to this co-expression, not only the desired heteromultimeric polypeptide but also mismatched variants thereof are secreted into the cell culture supernatant. These mismatched variants are also referred to in this document as “product-related impurities.” The presence of product-related impurities can affect the activity and safety of the product. If the polypeptide containing a heteromultimeric Fc domain is a bis antibody Petition 870250087783, dated 09 / 29 / 2025, page 28 / 87 21 / 61 specific, for example, a mismatched variant exhibiting binding activity to only one target antigen would block the binding of the fully functional bispecific antibody, thus antagonizing the desired activity of the bispecific molecule. At least, mismatched variants would likely reduce the efficacy of the final product if they were not separated. Additionally, many mismatched variants have exposed regions that normally promote peptide-peptide interaction, and thus may exhibit a tendency towards immunogenicity and aggregation. Several approaches have been developed to force the correct pairing of polypeptide chains, such as knob-into-hole technology (Ridgway JB et al., Protein Eng 1996; 9: 617-621) or CrossMab technology (Schaefer, W. et al, PNAS, 108 (2011) 111871191). However, none of these approaches can completely prevent the formation of mismatched variants.Furthermore, in existing formats that do not use knob-into-hole, CrossMab, or other technologies to prevent mismatches, the separation of mismatched variants of the protein of interest is even more important. Thus, there is a general need to separate mismatched variants from the correctly paired heteromultimeric polypeptide.

[0060] In addition to product-related impurities, the sample may comprise impurities that occur due to recombinant production of the heteromultimeric Fc domain-containing polypeptide in a host cell, such as nucleic acids, components of the cell culture medium, endotoxins, viruses, host cell lipids or host cell proteins, for example, phospholipases, clusterin, serine proteases, elongation factors, and / or any combination thereof. Such impurities are referred to herein as process-related impurities. The claimed method has the additional effect of separating these process-related impurities from the correctly paired heteromultimeric polypeptide. Sample Petition 870250087783, dated 09 / 29 / 2025, page 29 / 87 22 / 61

[0061] The sample is derived from cultured cells recombinantly expressing the heteromultimeric Fc domain-containing polypeptide. In some embodiments, the sample is an unprocessed cell culture supernatant from cultured cells recombinantly expressing the heteromultimeric Fc domain-containing polypeptide. Such a sample is also referred to as “bulk harvest” in this document. By way of non-limiting example, the sample may be conditioned cell culture supernatant, clarified conditioned cell culture supernatant, or clarified homogenized / lysed cell cultures. As used in this document, the terms “clarified” and “clarification” refer to the removal of particulate matter from a solution, including, but not limited to, filtration, preferably using a 0.2 polyethersulfone (PES) filter, sterilization, and / or centrifugation.Thus, a sample referred to as clarified bulk harvest in this document is a liquid material comprising the polypeptide containing a heteromultimeric Fc domain, product-related impurities, and process-related impurities, which has been extracted from a cell culture, for example, a fermentation bioreactor, after being subjected to centrifugation to remove large solid particles and / or subsequent filtration to remove finer solid particles and impurities from the material. The cultured cells may be any cells that are suitable for expressing a recombinant nucleic acid sequence including, for example, prokaryotic cells (such as E. coli cells, A. niger cells, etc.).Eukaryotic cells (such as yeast cells, plant cells, insect cells (e.g., SI cells) and / or mammalian cells (e.g., CHO cells, HEK cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, or hybridoma cells) (mouse, rat, hamster, rabbit, human, non-human primate, etc.). In preferred embodiments, the cultured cells are CHO or HEK293 cells, preferably CHO cells. Petition 870250087783, dated 09 / 29 / 2025, page 30 / 87 23 / 61 Polypeptide containing Fc domain

[0062] In some embodiments, the polypeptide containing a heteromultimeric Fc domain is a secreted polypeptide. In some embodiments of the first aspect of the invention, the polypeptide containing a heteromultimeric Fc domain is an antibody as defined above. In some embodiments, the antibody is a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the antibody is a monoclonal antibody. In preferred embodiments, the antibody is a multispecific antibody, preferably a bispecific, trispecific, tetraspecific, pentaspecific, or hexaspecific antibody, more preferably a bispecific or trispecific antibody.

[0063] The polypeptide containing a heteromultimeric Fc domain may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 immunoglobulin variable domains forming 1, 2, 3, 4, 5, or 6 antigen-binding domains. The antigen-binding domains may comprise or consist of an ISVD as defined above. In a preferred embodiment, the antigen-binding domains comprise or consist of two variable domains, in particular an immunoglobulin light chain variable domain and an immunoglobulin heavy chain variable domain, each comprising three complementary determinant regions (CDR-1 to CDR-3). Preferably, the polypeptide containing a heteromultimeric Fc domain comprises 4 or 6 immunoglobulin variable domains forming 2 or 3 antigen-binding domains.

[0064] The polypeptide containing a heteromultimeric Fc domain may comprise heavy and / or light chains of de-immunized, murine, chimeric, humanized or human antibodies, as well as combinations of variable and / or constant domains derived from de-immunized, murine, chimeric, humanized or human antibodies and fragments thereof. The polypeptide containing a heteromultimeric Fc domain may also comprise variable domains derived from a TCR. Petition 870250087783, dated 09 / 29 / 2025, page 31 / 87 24 / 61

[0065] Preferably, the Fc domain comprised in the polypeptide containing the heteromultimeric Fc domain is a dimeric Fc domain formed by two polypeptide chains, each comprising a CH2-CH3 region. The dimeric Fc domain may be homodimeric or heterodimeric. The bispecific antigen-binding molecule of the invention comprises no more than one Fc domain. In one embodiment, the Fc domain is an IgG Fc domain, preferably an IgG1 Fc domain or an IgG4 Fc domain.

[0066] In preferred embodiments, the polypeptide containing the domain The Fc domain preferably comprises or consists of a first and a second polypeptide chain, each comprising a CH2-CH3 region, and a third polypeptide chain that does not comprise a CH2CH3 region. The polypeptide containing the heteromultimeric Fc domain comprises no more than one copy of a polypeptide chain.

[0067] In another embodiment, the Fc domain-containing polypeptide comprises an ISVD fused to a CH2-CH3 region of a polypeptide chain of the Fc domain-containing polypeptide. In another embodiment, the Fc domain-containing polypeptide comprises more than one ISVD fused to a CH2-CH3 region of a polypeptide chain of the Fc domain-containing polypeptide. In another embodiment, the Fc domain-containing polypeptide comprises two CH2-CH3 regions, wherein an ISVD is fused to each of the CH2-CH3 regions. In another embodiment, the Fc domain-containing polypeptide comprises a cytokine fused to a CH2-CH3 region of a polypeptide chain of the Fc domain-containing polypeptide. In another embodiment, the Fc domain-containing polypeptide comprises more than one cytokine fused to a CH2-CH3 region of a polypeptide chain of the Fc domain-containing polypeptide.In another embodiment, the polypeptide containing the Fc domain comprises two CH2-CH3 regions, in which a cytokine is fused to each of the CH2-CH3 regions. Petition 870250087783, dated 09 / 29 / 2025, page 32 / 87 25 / 61

[0068] In some embodiments, the polypeptide components containing heteromultimeric Fc domains (e.g., CH2-CH3 region, constant domains, variable domains) can be linked directly or through various linkers, particularly peptide linkers comprising one or more amino acids, typically about 2-20 amino acids, which are described in this document or are known in the art. If two domains are coupled by a peptide linker having a length of 0 aa, this means that the two domains are linked directly through a peptide bond between the two domains. For this, the term fused can also be used instead of linked. A peptide linker is, in particular, a flexible peptide linker, that is, it provides flexibility between the domains that are linked together.Such flexibility is generally increased if the amino acids are small and do not have bulky side chains that impede rotation or bending of the amino acid chain. Thus, preferably, the peptide linker of the present invention has an increased content of small amino acids, in particular glycines, alanines, serines, threonines, leucines, and isoleucines. Preferably, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the amino acids in the peptide linker are such small amino acids. In a particular embodiment, the amino acids of the linker are selected from glycines and serines, that is, said linker is a polyglycine, a polyserine, or a polyglycine / serine linker, wherein poly means a proportion of at least 50%, 60%, 70%, 80%, 90%, or even 100% of glycine and / or serine residues in the linker.Exemplary polyglycine / serine ligands are, for example, STGS (SEQ ID NO: 7), or [GwSxGy]z, where w is an integer between 0 and 20, in some embodiments between 2 and 5, x is an integer between 0 and 10, in some embodiments between 0 and 3, y is an integer between 0 and 20, in some embodiments between 0 and 5, and z is an integer between 0 and 10, in some embodiments between 0 and 4. In the context of this descriptive report, the term polyglycine / serine ligand may also refer to a ligand consisting of... Petition 870250087783, dated 09 / 29 / 2025, p. 33 / 87 26 / 61 only one amino acid selected from G or S.

[0069] The inventors have surprisingly discovered that the method according to the invention can effectively separate the heteromultimeric Fc domain-containing polypeptide from mismatched variants. Thus, the invention provides a method for purifying the heteromultimeric Fc domain-containing polypeptide from mismatched variants thereof. The method results in the heteromultimeric Fc domain-containing polypeptide being purified away from mismatched variants to a higher degree than a corresponding method without the step of washing the matrix with the washing solution according to the invention. The prior art describes that particular washing buffers for Protein A chromatography can be effective in removing process-related impurities, in particular host cell proteins. Such host cell proteins frequently interact with Fc domain-containing polypeptides bound to the Protein A chromatography matrix.To remove these process-related impurities, the interactions between host cell proteins and Fc domain-containing polypeptides have to be broken. This situation is very different in the case of product-related impurities comprising an Fc domain itself. It was thus an unexpected finding that, using the method of the invention, which comprises the use of a washing buffer comprising octanoate, mismatched variants containing an Fc domain can be rotationally separated from a correctly matched heteromultimeric Fc domain-containing polypeptide.

[0070] In some embodiments, the sample is a clarified bulk harvest sample obtained from tissue culture and the purity of the polypeptide containing heteromultimeric Fc domain is - at least 40% or 45% of the total protein concentration after step e); - at least 50%, 60%, 70%, 80%, 90% or 95% of the total concentration Petition 870250087783, dated 09 / 29 / 2025, page 34 / 87 27 / 61 of protein after step h); or - at least 90%, 92%, 94%, 95%, 96% or 97% of the total protein concentration after step j),

[0071] wherein the concentration is preferably determined by capillary electrophoresis or capillary gel electrophoresis. Poorly paired variants

[0072] The co-expression of the different polypeptide chains that form the heteromultimeric polypeptide containing the Fc domain leads to the assembly of mismatched variants of the heteromultimeric polypeptide containing the Fc domain.

[0073] In some modalities, the poorly paired variants are selected from the group consisting of: a) monomers of the first, second or third polypeptide chains; b) homodimers or homomultimers of a polypeptide chain comprised in the polypeptide containing a heteromultimeric Fc domain; c) heteromultimers comprising more than one copy of the first, second, or third polypeptide chain; d) heteromultimers that do not comprise the first, second, or third polypeptide chain; and e) aggregates of mismatched variants according to a) ad) and / or the polypeptide containing a heteromultimeric Fc domain.

[0074] The embodiment described in e) in particular refers to heteromultimers comprising paired variable domains that are specific for different antigens and together do not form an antigen-binding domain that specifically binds to an antigen, for example, a variable domain specific for a tumor antigen is paired with a variable domain specific for an immune effector cell. Such mismatched variants may exhibit correct pairing of dimerization domains, for example, pairing of two Fc domains to form a dimeric Fc domain and / or pairing of a CH1 domain with Petition 870250087783, dated 09 / 29 / 2025, page 35 / 87 28 / 61 a Ck or CÃ domain.

[0075] In the context of this descriptive report, an aggregate is a cluster of multiple copies of the polypeptide containing a heteromultimeric Fc domain and / or its mismatched variants. Aggregates may, for example, comprise 2, 3, 4, 5, 10, 25, 50 or 100 copies of the polypeptide containing a heteromultimeric Fc domain and / or its mismatched variants.

[0076] In some embodiments, the method results in the separation of the polypeptide containing the heteromultimeric Fc domain from at least one mismatched variant selected from a) ae). In some embodiments, the method results in the separation of the polypeptide containing the heteromultimeric Fc domain from at least two, more preferably at least three mismatched variants selected from a) ae), even more preferably from all of said mismatched variants. Format

[0077] In some embodiments, the polypeptide containing a heteromultimeric Fc domain consists of a first, a second, and a third polypeptide chain, each comprising a variable immunoglobulin domain. Preferably, the second polypeptide chain comprises two variable immunoglobulin domains.

[0078] In some embodiments, the polypeptide containing heteromultimeric Fc domain comprises three polypeptide chains (1), (2) and (3): - V1-C1-CH2-CH3(1), - V2-C2-CH2-CH3-V3-C3 (2);e - V4-C4(3);

[0079] wherein V1 to V4 are variable immunoglobulin domains, wherein the antigen-binding domains are formed by V1 and V2 and by V3 and V4; and

[0080] Where C1 to C4 are heterodimerization domains, preferably selected from CH1, CK and CL domains, preferably where one of C1 and C2 is a CH1 domain and the other is CK or CL, and one of Petition 870250087783, dated 09 / 29 / 2025, page 36 / 87 29 / 61 C3 and C4 is one CH1 domain and the other is Ck or Cl, even more preferably where C2 and C3 are CH1 and C1 and C4 are Ck or Cl, or where C2 and C3 are CH1 and Ck or Cl, or C1 and C4 are Ck or Cl and CH1.

[0081] For the format described above comprising three polypeptide chains (1), (2) and (3), the following mismatched variants may occur: - “Very light” mismatched variants: single polypeptide chains (1), (2) or (3) (i.e., mismatched variants as defined in a) above), or dimers of (3), (i.e., mismatched variants as defined in b) above). - “Light” mispaired variants: multimers consisting of 2 copies of (1) (i.e., mispaired variants as defined in b) above) and optionally one or two copies of (3) (i.e., mispaired variants as defined in c) or d) above). These “light” mispaired variants have a molecular weight within the same range, but lower than the correctly paired heteromultimeric Fc domain-containing polypeptide. - “Heavy” mismatched variants: Multimers comprising, for example, two copies of (2) plus additional chains, or one copy each of (1), (2) and (3) plus additional chains, for example, (3)-(2)(2)-(3), (1)-(2)-(3)-(3) or (1)-(2)-(3)-(3)-(3), (i.e., mismatched variants as defined in c) or d) above). These “heavy” mismatched variants have a molecular weight within the same range, but higher than the correctly matched Fc domain-containing heteromultimeric polypeptide. - Aggregates comprising multiples of all three polypeptide chains (i.e., mismatched variants as defined in e) above).

[0082] These mismatched variants can be efficiently separated from the heteromultimeric Fc domain-containing polypeptide of interest using the method of the invention.

[0083] In preferred embodiments, the polypeptide containing the domain The heteromultimeric Fc comprises three polypeptide chains (I), (II) and (III): - V1A-C1A-L3-(CH2-CH3)a (I), Petition 870250087783, dated 09 / 29 / 2025, page 37 / 87 30 / 61 - V1B-C1B-L4-(CH2-CH3)b-L1-V2A-C2A-L2 (II); and - V2B-C2B (Ill); in which: VIA and V1B form a Vi-linking pair; V2A and V2B form a V2 bonding pair; C1A and C1B form a Ci (CH1 / CL) pair, and C2A and C2B form a C2 (CH1 / CL) pair, where CH1 is a constant domain of immunoglobulin 1 heavy chain and Cl is a constant domain of immunoglobulin light chain;

[0084] (CH2-CH3)ae (CH2-CH3)b are identical or different, and comprise a constant immunoglobulin heavy chain domain 2 (CH2) and a constant immunoglobulin heavy chain domain 3 (CH3);

[0085] L1, L2, L3, L4 are optional independent amino acid ligands, which may be identical or different.

[0086] For the format described above comprising three polypeptide chains (I), (II) and (III), the same mismatched variants may occur as described for the format comprising three polypeptide chains (1), (2) and (3).

[0087] In preferred embodiments, a V1 linking pair specifically binds to CD123 and a V2 linking pair specifically binds to NKp46.

[0088] In some embodiments, the polypeptide containing a heteromultimeric Fc domain comprises a first antigen-binding domain specific for NKp46 and a second antigen-binding domain specific for CD123. In some embodiments, the polypeptide containing a heteromultimeric Fc domain comprises: - a polypeptide chain (I) comprising an amino acid sequence from SEQ ID NO: 1, a polypeptide chain (II) comprising an amino acid sequence from SEQ ID NO: 2 and a polypeptide chain (III) comprising an amino acid sequence from SEQ ID NO: 3, or a variant of polypeptide chains (I), (II) and (III) having at least 80%, by Petition 870250087783, dated 09 / 29 / 2025, page 38 / 87 31 / 61 less than 85%, at least 90%, at least 95% sequence identity with SEQ IDs 1, 2, and 3, respectively; or - a polypeptide chain (I) comprising an amino acid sequence from SEQ ID NO: 4, a polypeptide chain (II) comprising an amino acid sequence from SEQ ID NO: 5 and a polypeptide chain (III) comprising an amino acid sequence from SEQ ID NO: 6, or variants of polypeptide chains (I), (II) and (III) having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NOs: 4, 5 and 6, respectively.

[0089] In some embodiments, the polypeptide containing the heteromultimeric Fc domain comprises at least two polypeptide chains linked by at least one disulfide bridge. In some embodiments, the polypeptide chains (I), (II) and (III) are characterized by: the polypeptide chain (I) is covalently linked to the polypeptide chain (II), in particular covalently linked to the polypeptide (II) by one or more disulfide bonds. According to some of these particular embodiments, the polypeptide chains (I), (II) and (III) are characterized by: the polypeptide chain (II) is covalently linked to the polypeptide chain (III) by one or more disulfide bonds. In some embodiments, the heteromultimeric Fc domain-containing polypeptide is characterized by the Fc domain that binds to a human Fc-γ receptor polypeptide comprising a CH2 heavy chain constant domain with an N-linked glycosylation at residue N297 according to EU numbering.In some embodiments, the polypeptide containing a heteromultimeric Fc domain is characterized by the N297 residue of the Fc region or its EU numbering variant comprising an N-linked glycosylation. In some embodiments, the polypeptide containing a heteromultimeric Fc domain is characterized by all or part of the Fc domain binding to a human Fc-γ receptor polypeptide. In some embodiments, the polypeptide containing a heteromultimeric Fc domain is characterized by... Petition 870250087783, dated 09 / 29 / 2025, page 39 / 87 32 / 61 all or part of the Fc domain binds to a human CD16A (FcyRIII) polypeptide.

[0090] Exemplary configurations are shown in Figure 3. Protein chromatography

[0091] In the context of this descriptive report, the term “protein A chromatography” refers to a chromatographic method useful for the purification of a protein of interest from a protein mixture, wherein the protein mixture preferably comprises proteins that are product-related impurities and / or proteins that are process-related impurities. Protein A chromatography relies on the specific and reversible binding of proteins comprising a CH2 domain (or its functional portion), a CH3 domain (or its functional portion), preferably a CH2-CH3 region, and more preferably a dimeric Fc domain, to protein A. In the method according to the invention, the protein A chromatography matrix is ​​brought into contact with the sample under conditions suitable for the polypeptide containing the heteromultimeric Fc domain in the sample to bind to protein A.For the alternative aspect, please refer to the paragraph regarding protein G chromatography below. Suitable methods and conditions for contacting and binding an Fc domain-containing polypeptide to a protein A matrix or resin are readily understood by a person skilled in the art (e.g., methods as described in the manufacturer's protocol for a commercially available protein A matrix or resin). Any suitable protein A matrix or resin known in the art may be used in the methods of the present invention, including, for example: Mab Select, Mab Select Xtra, Mab Select Sure, Mab Select Sure LX Protein A, Mab Select PCC, Mab Select PrismA, rProtein A Sepharose CL-4B, and nProtein A Sepharose 4 FF (Cytiva); EshmunoA, ProSep A, ProSep-vA High Capacity, ProSep-vA Ultra, and ProSep-vA UltraPlus (Millipore); Porns A and Mabcapture A (Porns); IPA-300, IPA-400, and IP A-500 (RepliGen Corp.); Affigel A protein and Affiprep A protein (Bio-Rad); MABsorbent AIPP e. Petition 870250087783, dated 09 / 29 / 2025, p. 40 / 87 33 / 61 MABsorbent A2P (Affinity Chromatography Ltd.); Protein A Ceramic Hyper DF (Pall Corp.); Ultralink Immobilized protein A and Agarose Protein A (PIERCE); Protein A Cellthru 300 and Protein A Ultraflow (Bioseparation); Amsphere A3 (JSR); Fibro PrismA (Cytiva); Praesto Jetted A50, Praesto AP+ and Praesto APc (Purolite); Sartobind Rapid A membrane (Sartorius) and / or Toyopearl AF-rProtein A HC-650F (Tosoh Biosciences). In some embodiments, the protein A chromatography matrix is ​​used in a column chromatography format. In some embodiments, one or more parameters of the protein A chromatography matrix (such as pH, ionic strength, temperature, addition of other substances) are adjusted before the protein A matrix or resin contacts a sample. In some embodiments, the protein A matrix or resin is washed, rinsed, balanced, removed, and / or sanitized before and / or after contact of the protein A matrix or resin with the sample.In some embodiments, the protein chromatography matrix is ​​equilibrated and / or washed before contact of protein chromatography matrix A with the sample.

[0092] In some embodiments, the Protein A matrix or resin is sanitised, removed and / or regenerated between uses.

[0093] The term octanoate in the context of the present invention refers to a chemical compound of the formula CH3-(CH2)6-COOH. Octanoate is also known as octanoic acid, octyl acid, octoic acid, caprylic acid, (1-)1-heptanocarboxylate ion, n-octanoate, (n-)octylate, n-octoate, (n-)caprylate or caprylate.

[0094] In preferred embodiments, the octanoate in the washing solution of step c) is an octanoate salt. In some embodiments, the concentration of octanoate, preferably the octanoate salt, is about 10 mM to about 500 mM, preferably about 25 mM to about 250 mM, more preferably about 50 mM to about 150 mM. For example, the concentration of octanoate, preferably the octanoate salt, can be about 50 mM, 60 mM, 70 mM, 75 mM, 80 mM, 90 mM, 100 mM, 110 Petition 870250087783, dated 09 / 29 / 2025, page 41 / 87 34 / 61 mM, 120 mM, 130 mM, 140 mM, or 150 mM. In preferred embodiments, the concentration is 75 mM. Any suitable source or form of an octanoate salt (e.g., an alkaline salt) known in the art may be used in the washing solutions of the present invention, including, for example, sodium octanoate, potassium octanoate, lithium octanoate, calcium octanoate, magnesium octanoate, beryllium octanoate, barium octanoate, strontium octanoate, rubidium octanoate, cesium octanoate, and / or any combinations thereof. In some embodiments, the octanoate salt is an alkaline octanoate salt. In some embodiments, the octanoate salt is sodium octanoate or potassium octanoate. In some embodiments, the octanoate salt is sodium octanoate.

[0095] In some embodiments, the washing solution of step c) additionally comprises one or more (for example, one or more, two or more, three or more, four or more, or all) of the following additives: benzenesulfonate, caprylic acid, hexylene glycol, propylene glycol, benzyl alcohol, a non-buffering salt and / or creatine. In some embodiments, the washing solution of step c) additionally comprises benzyl alcohol, hexylene glycol and / or propylene glycol. In some embodiments, the washing solution of step c) additionally comprises hexylene glycol or propylene glycol at a concentration of about 5% to about 25%, preferably about 10% to about 20%, more preferably about 15% volume / volume. In some embodiments, the washing solution of step c) additionally comprises benzyl alcohol at a concentration of about 0.5% to about 4%, preferably about 1% to about 3%, more preferably about 2% volume / volume.It is preferable that the washing solution in step c) does not contain benzoate, for example, any benzoate salt.

[0096] In some embodiments, the washing solution additionally comprises a buffering agent. Any suitable buffering agent known in the art may be used in the washing solutions of Petition 870250087783, dated 09 / 29 / 2025, page 42 / 87 35 / 61 present invention, including, for example, phosphate, tris(tris(hydroxymethyl)methylamine), bis-tris, bis-trispropane, arginine, histidine, triethanolamine, diethanolamine, formate, acetate, MES carbonate (2-(N-morpholino)ethanesulfonic acid), citrate, HEPES (4-2-methyl-1-piperazineethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), TAPS (3-{[tris(hydroxymethyl)methyl]amino}propanesulfonic acid), Bicine (N,N-bis(2-hydroxyethyl)glycine), Tricine (N-tris(hydroxymethyl)methylglycine), TES (2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid), cacodylae (dimethylarsinic acid), SSC (sodium citrate saline) and / or any combination thereof. In some embodiments, the buffering agent is selected from phosphate, tris, arginine, acetate, and citrate. In some embodiments, the buffering agent is at a concentration of about 10 mM to about 500 mM.In some embodiments, the washing solution of step c) comprises tris, preferably at a concentration of about 25 to about 100 mM, more preferably about 50 mM.

[0097] In some embodiments, the washing solution of step c) has a pH of about 8.5 to about 10.0, such as about 7.0, about 7.5, about 8.0, about 7.0, about 9.0, about 9.5, or about 10.0, preferably about 7.5 or about 9.0.

[0098] In some embodiments, the washing solution of step c) additionally comprises NaCl, preferably at a concentration of about 0.5 to about 2.0 M, such as about 0.5 M, about 0.75 M, about 1.0 M, about 1.25 M, about 1.5 M, about 1.75 M or about 2.0 M, preferably about 1.0 M.

[0099] In some embodiments, the washing solution of step c) is a solution selected from BON buffer (50 mM Tris, 75 mM sodium octanoate, 1000 mM NaCl, 2% v / v benzyl alcohol, pH 9.0), HON buffer (50 mM Tris, 75 mM sodium octanoate, 1000 mM NaCl, 15% v / v hexylene glycol, pH 9.0) and PON buffer (50 mM Tris, 75 mM sodium octanoate, NaCl Petition 870250087783, dated 09 / 29 / 2025, page 43 / 87 36 / 61 1000 mM, propylene glycol 15% by v, pH 9.0).

[0100] In some modalities, the protein chromatography matrix A is contacted with an equilibrium buffer (or a “priming wash solution”) between steps b) and c). In some embodiments, the protein chromatography matrix A is contacted with an equilibrium buffer (or a subsequent wash solution) between steps c) and d). In some embodiments, the equilibrium buffer (or subsequent priming / wash solution) comprises a buffer selected from a phosphate buffer, a tris buffer, an acetate buffer, a carbonate buffer, a citrate buffer, and any combinations thereof, preferably 50 mM Tris, 20 mM NaCl at pH 7.5.

[0101] The method comprises a contact step of the protein chromatography matrix A with an elution solution after one or more washing steps. In some embodiments, the elution solution of step d) comprises 25 mM acetic acid. In some embodiments, the elution solution of step d) has a pH between 4.0 and 4.5, preferably 4.2.

[0102] Using the method of the present invention, the polypeptide containing a heteromultimeric Fc domain and its mismatched variants can be eluted separately from the protein chromatography matrix A, thus separating the polypeptide containing a heteromultimeric Fc domain from its mismatched variants. In particular, the polypeptide containing a heteromultimeric Fc domain is eluted by the elution solution, while the mismatched variants are not eluted with the washing buffer or are not eluted during the washing and elution steps, but remain bound to the chromatography matrix.

[0103] In some embodiments, the method further comprises a filtration step of the eluate by depth filtration. In some embodiments, the method further comprises a viral inactivation step at a pH of about 3.0 to about 4.0, preferably about 3.2 to about 3.8, more preferably about 3.5, between the Petition 870250087783, dated 09 / 29 / 2025, page 44 / 87 37 / 61 stage e) ef).

[0104] After Protein A chromatography, remaining product and / or process-related impurities can be removed based on differences in size, charge (e.g., isoelectric point or IEP), solubility, and / or degree of hydrophobicity. Thus, the sample can be further purified using multimodal chromatography and / or ion-exchange chromatography, preferably multimodal chromatography and ion-exchange chromatography. The method also removes product-related impurities present in the sample. In some embodiments, the method additionally comprises the steps of: f) contact a multimodal chromatography array with the eluate from step e) and link the polypeptide containing the heteromultimeric Fc domain to the multimodal chromatography array; g) place the multimodal chromatography matrix in contact with an elution solution; and h) collect an eluate comprising the polypeptide containing a heteromultimeric Fc domain.

[0105] In some modalities, the method additionally includes the following steps: i) place an ion-exchange chromatography matrix in contact with the eluate from step e) or h); and j) collect an eluate or streamflow comprising the polypeptide containing a heteromultimeric Fc domain. Multimodal chromatography

[0106] In the context of this descriptive report, the term “multimodal chromatography” refers to a chromatographic method useful for the purification of a protein of interest from a protein mixture, wherein the protein mixture preferably comprises proteins that are product-related impurities and / or proteins that are process-related impurities. Multimodal chromatography (also referred to as chromatography) Petition 870250087783, dated 09 / 29 / 2025, page 45 / 87 38 / 61 (mixed mode) is based on a combination of at least two modes of interactions between the protein of interest and the chromatography matrix, including two or more ion exchange, hydroxyapatite, electrostatic forces, calcium coordination complexes, affinity, and hydrophobic interactions. Multimodal chromatography may also include size exclusion chromatography as one of at least two modes. In the method according to the invention, the multimodal chromatography matrix is ​​brought into contact with the sample under conditions suitable for the polypeptide containing the heteromultimeric Fc domain in the sample to bind to the multimodal chromatography matrix.Appropriate methods and conditions for using a multimodal chromatography matrix or resin to purify a polypeptide containing an Fc domain are readily understood by an individual of ordinary skill in the art (e.g., methods as described in the manufacturer's protocol for a commercially available multimodal chromatography matrix or resin). The use of any suitable multimodal chromatography matrix or resin known in the art is provided for herein. In some embodiments, the multimodal chromatography matrix is ​​used in a column chromatography format. In some embodiments, one or more parameters of the multimodal chromatography matrix (such as pH, ionic strength, temperature, the addition of other substances) are adjusted before the multimodal chromatography matrix or resin contacts a sample.In some embodiments, the multimodal chromatography matrix or resin is washed, rinsed, equilibrated, removed and / or sanitized before and / or after contact of the multimodal chromatography matrix or resin with the sample.

[0107] In some embodiments, a multimodal chromatography purification (fah steps) is performed after purification by Protein A chromatography (or, alternatively, after purification by Protein G chromatography).

[0108] In some embodiments, the eluate from step e) is adjusted to a pH of about 4.5 to about 5.5 and a salt concentration of about Petition 870250087783, dated 09 / 29 / 2025, page 46 / 87 39 / 61 at about 200 mM NaCI before step f).

[0109] In some embodiments, the multimodal chromatography matrix comprises one or more ligands comprising at least 2, preferably at least 3, more preferably at least 4 different linking chemical moieties selected from the group consisting of a hydrophobic chemical moiety, an ionic chemical moiety, a hydrogen bond donor chemical moiety and a sulfur-containing chemical moiety.

[0110] In some embodiments, the multimodal chromatography matrix comprises Mep HyperCel, Capto MMC ImpRes, Capto Phenyl ImpRes, Capto Adhere ImpRes (all available from GE Healthcare), HEA HyperCel, PPA HyperCel, CHT ceramic hydroxyapatite, Toyopearl NH2-750F (available from Tosoh) or Nuvia cPrime, preferably Toyopearl NH2-750F or Capto MMC ImpRes.

[0111] In some embodiments, the multimodal chromatography matrix comprises a ligand of formula (1)

[0112] In some embodiments, the multimodal chromatography matrix is ​​brought into contact with a washing solution between steps f) and g), preferably wherein said washing solution has a pH of about 6.0 to about 8.0.

[0113] In some embodiments, the elution solution of step g) has a pH of about 7.0 to about 8.0 and a salt concentration of about 0 to about 200 mM NaCl. Ion exchange chromatography

[0114] In the context of this descriptive report, the term “chromatography” Petition 870250087783, dated 09 / 29 / 2025, p. 47 / 87 40 / 61 ion exchange chromatography refers to a chromatographic method useful for purifying a protein of interest from a mixture of proteins, wherein the protein mixture preferably comprises proteins that are product-related impurities and / or proteins that are process-related impurities. Ion exchange chromatography relies on electrostatic interactions between the protein of interest and the ion exchange chromatography matrix comprising charged ions. Ion exchange chromatography includes anion exchange chromatography and cation exchange chromatography.In the method according to the invention, the ion-exchange chromatography matrix is ​​brought into contact with the sample under suitable conditions for the heteromultimeric Fc-domain-containing polypeptide in the sample to flow through the ion-exchange chromatography matrix (in other words, the heteromultimeric Fc-domain-containing polypeptide passes through the ion-exchange chromatography matrix with the through-flow). Suitable methods and conditions for using an ion-exchange chromatography matrix or resin to purify an Fc-domain-containing polypeptide are readily understood by an individual of ordinary skill in the art (e.g., methods as described in the manufacturer's protocol for a commercially available ion-exchange chromatography matrix or resin). The use of any suitable ion-exchange chromatography matrix or resin known in the art is provided for herein.In some embodiments, the ion-exchange chromatography matrix is ​​used in a column chromatography format. In some embodiments, one or more parameters of the ion-exchange chromatography matrix (such as pH, ionic strength, temperature, the addition of other substances) are adjusted before the ion-exchange chromatography matrix or resin comes into contact with a sample. In some embodiments, the ion-exchange chromatography matrix or resin is washed, rinsed, equilibrated, removed, and / or sanitized before and / or after the ion-exchange chromatography matrix or resin comes into contact with the sample.

[0115] In some embodiments, a purification by chromatography of Petition 870250087783, dated 09 / 29 / 2025, page 48 / 87 41 / 61 ion exchange (steps iaj) is performed after purification by Protein A chromatography (or, alternatively, after purification by Protein G chromatography).

[0116] In some embodiments, the eluate from step h) is adjusted to a pH of about 6.0 to about 8.0 and a salt concentration of about 0 to about 400 mM NaCl before step i).

[0117] In some embodiments, the ion-exchange chromatography matrix is ​​an anion-exchange chromatography matrix.

[0118] In some embodiments, step j) comprises placing the ion-exchange chromatography array in contact with an elution solution and collecting an eluate comprising the polypeptide containing a heteromultimeric Fc domain; or collecting a passing stream comprising the polypeptide containing a heteromultimeric Fc domain.

[0119] The polypeptide containing a heteromultimeric Fc domain can be purified using the method provided in this document alone or in conjunction with any other suitable separation techniques, such as, by way of example, not limited to and not exhaustive, membrane filtration techniques and protein precipitation techniques. The method may further comprise the step of determining the purity and proportions of the polypeptide containing a heteromultimeric Fc domain.This step can be performed using any of a variety of techniques recognized in the art, such as, by way of non-limiting and non-exhaustive example, non-reduced capillary electrophoresis (NR-CE) using high-throughput microfluidic electrophoretic separation on a chip or traditional capillary electrophoresis, high-performance size-exclusion-liquid chromatography (SECHPLC), high-performance hydrophobic-liquid interaction chromatography (HIC-HPLC), high-performance ion-exchange-liquid chromatography (IEXHPLC), or high-performance reverse-phase-liquid chromatography (RPHPLC).

[0120] Alternatively, a method is provided for purifying Petition 870250087783, dated 09 / 29 / 2025, page 49 / 87 42 / 61 a polypeptide containing a heteromultimeric Fc domain according to the first aspect, wherein step b) comprises placing a protein A or protein G chromatography matrix with the sample. All embodiments specified for the first aspect are also provided for in this alternative aspect, wherein every reference to protein A is intended to refer to protein A or protein G, in particular protein G. G-protein chromatography.

[0121] In the alternative aspect, the protein A or protein G chromatography matrix is ​​contacted with the sample under conditions suitable for the heteromultimeric Fc-domain-containing polypeptide in the sample to bind to protein A or protein G, respectively. Suitable methods and conditions for contacting and binding an Fc-domain-containing polypeptide to a protein A or protein G matrix or resin are readily understood by one skilled in the art (e.g., methods such as those described in the manufacturer's protocol for a commercially available protein A or protein G matrix or resin). Any suitable protein A or protein G matrix or resin known in the art may be used.

[0122] All modalities specified in the paragraph “Protein A chromatography” are also provided for Protein G chromatography, where applicable.

[0123] All terms used in relation to the second, third and fourth aspects of the invention below have the meanings as defined in relation to the first aspect of the invention, unless specifically defined otherwise. In addition, all embodiments specified for the first aspect that are applicable to the second, third and fourth aspects are also provided for those aspects.

[0124] In a second aspect, the present invention relates to the use of a washing solution comprising octanoate in a method for purifying a polypeptide containing a heteromultimeric Fc domain according to the first aspect of the invention. In some embodiments, the Petition 870250087783, dated 09 / 29 / 2025, p. 50 / 87 43 / 61 The octanoate concentration in the washing solution is about 10 mM to about 500 mM, preferably about 25 mM to about 250 mM, more preferably about 50 mM to about 150 mM. For example, the octanoate concentration may be about 50 mM, 60 mM, 70 mM, 75 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, or 150 mM. In preferred embodiments, the concentration is 75 mM.

[0125] In a third aspect, the present invention relates to a kit comprising a protein A chromatography matrix; and a washing solution comprising octanoate. In some embodiments, the octanoate concentration in the washing solution is from about 10 mM to about 500 mM, preferably from about 25 mM to about 250 mM, more preferably from about 50 mM to about 150 mM. For example, the octanoate concentration may be about 50 mM, 60 mM, 70 mM, 75 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM or 150 mM. In preferred embodiments, the concentration is 75 mM. In some embodiments, the kit further comprises a multimodal chromatography matrix and / or an ion-exchange chromatography matrix.

[0126] In a fourth aspect, the present invention relates to an eluate obtained by the method according to the first aspect of the invention, wherein the eluate comprises at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% of a purified heteromultimeric Fc domain-containing polypeptide; and less than 0.5%, 0.4%, 0.3%, 0.2%, 0.1% of mismatched variants.

[0127] In addition, alternative aspects to the second, third and fourth aspects of the invention are provided: These alternative aspects correspond to the second, third or fourth, but refer to purification using a protein A or protein G chromatography matrix, in particular protein G, instead of a protein A chromatography matrix. All embodiments specified for the second, third or fourth aspect are also provided for these alternative aspects, wherein each reference Petition 870250087783, dated 09 / 29 / 2025, p. 51 / 87 44 / 61 to protein A is intended to refer to protein A or protein G, in particular protein G.

[0128] In another aspect, the present invention relates to a purified polypeptide containing a heteromultimeric Fc domain obtained by a method according to the invention. Brief Description of the Drawings Figure 1: The purity of the polypeptide containing the heteromultimeric Fc domain obtained with different Protein A washing solutions determined by microfluidic electrophoretic separation. Figure 2: The effect of different protein A washing solutions on the removal of product-related impurities determined by microfluidic electrophoretic separation. Virtual gel image generated from the LabChip GXII System showing the ladder and analysis of protein A samples with the different washing solutions. Figure 3: Illustrative configurations of a polypeptide containing a heteromultimeric Fc domain. Example Section Example 1: Washing solutions to improve the removal of product-related impurities. Sample preparation

[0129] Recombinant Chinese hamster ovary (CHO) cells genetically modified to express a polypeptide containing a heteromultimeric Fc domain having a shape as represented in Figure 3 (in the illustrative section referred to as “protein of interest”) were cultured in a cell culture bioreactor. The recombinant product was secreted into the culture medium which was then clarified by depth filtration for downstream processing. The clarified harvest material was filtered with a 0.2 polyethersulfone (PES) filter before loading onto the protein A column. Protein chromatography Petition 870250087783, dated 09 / 29 / 2025, page 52 / 87 45 / 61

[0130] Protein resin A was prepared as follows: The resin MabSelect Sure Protein A (GE Healthcare Life Science) was obtained pre-packaged from the supplier in robocolumn format (200 μl). Before loading the harvested material, the columns were washed with equilibration buffer (50 mM Tris, 20 mM NaCl pH 7.5) to remove storage solution. Subsequently, the column was purged with 0.5 M acetic acid to ensure the removal of any bound entities, then cleaned using 0.5 M sodium hydroxide. The columns were then equilibrated until the pH was >7. The prepared samples were then loaded onto the Protein A columns. The columns were loaded onto a 20 g / l resin target with a heterospecific Fc domain-containing polypeptide that has a shape as illustrated in Figure 3. The loaded columns were washed with equilibration buffer (also referred to as preparation wash buffer in this document).Next, the columns were washed with the test washing solution (Table 2) followed by the equilibrium buffer. Finally, the protein of interest was eluted from the column using a solution containing 25 mM acetic acid at a pH of 3.5 to 3.9. Table 1. Example of a Protein A chromatography process. Buffer Stage Volume (CV) Equilibrium Tris 50 mM NaCl 20 mM at pH 7.5 5 CV Loading Loading Initiation Wash Tris 50 mM NaCl 20 mM at pH 7.5 5 CV Washing Test Solutions 5 CV Subsequent Washing Tris 50 mM NaCl 20 mM at pH 7.5 5 CV Elution Variable 10 CV Table 2. Washing Solution Compositions Name Composition Control Tris 50 mM NaCl 1000 mM at pH 7.5 Petition 870250087783, dated 09 / 29 / 2025, page 53 / 87 46 / 61 BON Tris 50 mM NaOctanoate 75 mM NaCl 1000 mM 2% benzyl alcohol at pH 9.0 Carbonate 200 mM Carbonate at pH 9.0 Arginine 750 mM Arginine at pH 9.0 PON Tris 50 mM NaOctanoate 75 mM NaCl 1000 mM Propylene Glycol at pH 9.0 HON Tris 50 mM NaOctanoate 75 mM NaCl 1000 mM Hexylene Glycol at pH 9.0 BEBE Tris 50 mM NaBenzoate 500 mM 2% Benzyl Alcohol at pH 9.0 Determination of purity

[0131] The analytical procedure for purity determination uses the Labchip® GXII high-throughput microfluidic capillary electrophoresis (CE) platform under non-reducing conditions with the HT Protein Express200 Reagent kit. The CE technique separates denatured proteins based on size, and fluorescence detection allows for the determination of the relative amount of purity. Reagents, sample buffer, and the chip are prepared according to the vendor protocol for the HT Protein Express200 Reagent kit. Prior to analysis, samples that have undergone at least one purification stage (e.g., Protein A purification) are diluted to approximately ~1 g / l in water. Samples are then prepared by mixing 4 pl of diluted sample with 16.5 pl of sample buffer in a 96-well plate and heat-denatured at 70°C for 10 min. After incubation, 35 pl of water are added to each sample.The 96-well plate is then loaded into the instrument for analysis along with the previously prepared chip. The determination of the relative amount of purity (main form, "protein of interest") is obtained by dividing the individual peak area by the sum of all the integrated peak areas in the measured samples. The values ​​in Figure 1 and Table 3, as well as the image in Figure 2, were... Petition 870250087783, dated 09 / 29 / 2025, page 54 / 87 47 / 61 respectively obtained by microfluidic electrophoretic separation using the LabChip GXII System and virtual gel image generated from the LabChip GXII System.

[0132] The results were confirmed by size exclusion chromatography (SEC) or SEC-HPLC. Results

[0133] Impurities related to the product, such as variants of the protein of interest whose chains are incorrectly paired with each other, have properties very similar to the protein of interest. They share affinity for the immobilized Protein A ligand and are thus normally co-eluted with the protein of interest. To identify potential washing additives capable of reducing the amount of mismatched molecules co-eluted with a monoclonal antibody (mAb) of interest, 200 µl protein A chromatography roboclumns were loaded with a sample containing a secreted protein of interest harvested from CHO cells that were cleared by depth filtration before downstream processing. The loaded columns were first washed with a Tris equilibrium buffer.Next, the columns were washed with one of several test washing solutions containing an additive, carbonate, octanoate, sodium benzoate, arginine, and alcohol, individually or in combination, as shown in Table 2. Finally, the antibody was eluted from the column and the pH adjusted to pH 5.0 using 2 M Tris base before analysis.

[0134] Interestingly, washing solutions containing octanoate (HON, PON, BON) enhanced the purity of the protein of interest in the eluted fraction, compared to the control, as well as compared to washing solutions containing only carbonate or arginine (Figures 1 and 2, Table 3). When both octanoate and benzyl alcohol are present in the washing solution, purity is maximized. These specific washes thus significantly improve purification performance. Petition 870250087783, dated 09 / 29 / 2025, page 55 / 87 48 / 61 removing impurities related to the product. Table 3. Effect of Protein A washing solutions on the removal of product-related impurities determined by microfluidic electrophoretic separation. Washing Solution | Product-Related Impurities | Target | < 100 kDa | 120 kDa | 120-150 kDa | > 155 kDa | BON | 3.0% | 28.6% | 1.7% | 6.0% | 60.8% | Control | 11.7% | 48.5% | 2.1% | 12.5% ​​| 25.2% | Carbonate | 13.7% | 50.2% | 2.4% | 8.5% | 25.3% | Arginine | 4.4% | 60.9% | 1.3% | 8.4% | 25.0% | HON | 1.0% | 34.5% | 1.2% | 39.7% | 23.6% | PON | 2.5% | 33.3% | 1.1% | 39.1% 24.0%

[0135] In this experiment, impurities related to the product < 100 kDa are single polypeptide chains (1), (2) or (3), or dimers of (3). Impurities related to the product of about 120 kDa and 120-150 kDa are multimers comprising two copies of (1) and optionally one or two copies of (3). Impurities related to the product > 155 kDa are multimers comprising, for example, two copies of (2) plus additional chains, or one copy each of (1), (2) and (3) plus additional chains, for example (3)-(2)-(2)-(3), (1)-(2)-(3)-(3) or (1)-(2)-(3)-(3)-(3). This group of product-related impurities also includes aggregates of the polypeptide containing a heteromultimeric Fc domain and mismatched variants thereof. Example 2: Polishing Screening

[0136] The following example describes the use of various polishing resins to evaluate the potential for removing product-related impurities (mismatch) during purification. Sample preparation

[0137] Human monoclonal antibody collection materials were Petition 870250087783, dated 09 / 29 / 2025, pp. 56 / 87 49 / 61 prepared as described in Example 1. The harvest was generated in a suspension culture of recombinant CHO cells genetically modified to express the protein of interest. The recombinant product was secreted into the culture medium which was then clarified by depth filtration for downstream processing. The clarified harvest material was filtered with a 0.22 µm polyethersulfone (PES) filter before loading onto the chromatographic column. The material was then captured on a protein A resin and eluted as described in Example 1 to generate materials for evaluation of the polishing resins. Mixed Mode Chromatography

[0138] Next, several polishing resins were evaluated to further improve the purity of the protein of interest by removing product-related impurities (Table 4). The resins evaluated are used in flow-through mode or bind and elute. A range of conditions for the pH (between 6 and 8) and salt concentration (NaCl from 0 to 400 mM) of the loading material was evaluated for each resin used in flow-through mode. For the resins used in bind and elute, the pH of the loading (4.5 to 5.5), the pH of the washing buffer (6 to 8), and the pH of the elution buffer (7 to 8) were evaluated, as well as the salt concentration for the loading and elution buffer (NaCl from 0 to 200 mM). Before loading the Protein A eluate material adjusted to the corresponding pH and salt concentration, the columns were equilibrated with the corresponding equilibrium buffer for at least 5 CV (and until the pH and conductivity reached the defined point).After loading, the unbound material was pushed using equilibrium buffer for at least 10 CV. The flow-through resins were then cleaned using WFI for hydrophobic interaction resins, or 50 mM Tris 1000 mM NaCl at pH 7.5 for the other resins, followed by 0.5 M sodium hydroxide. For binding and eluting resins, the column was then washed using the corresponding washing buffer, and the molecule eluted using the elution buffer as described above for at least 5 CV. The column was then... Petition 870250087783, dated 09 / 29 / 2025, pp. 57 / 87 50 / 61 clean using 50 mM Tris and 1000 mM NaCl at pH 7.5 followed by 0.5 M sodium hydroxide. Table 4. High-Performance Screening of Flow-Through Polishing Resins Abbr. Name Format Mode Technology Capto Adhere (GE) CA Resin Flow-through Multimodal Capto Adhere ImpRes (GE) CAi Resin Flow-through Multimodal Toyopearl NH2-750F (Tosoh) Tosoh Resin Flow-through Anion Exchange Capto Phenyl High Sub (GE) CPhHS Resin Flow-through Hydrophobic Capto MMC ImpRes (GE) CMMCi Resin Bind and Elute Multimodal Capto Phenyl ImpRes (GE) CPhi Resin Flow-through Hydrophobic Results.

[0139] Impurities related to the product have very similar biochemical properties to the protein of interest. To identify potential polishing resins that can separate the protein of interest from mismatched fragments, several polishing resins with specific physicochemical characteristics were evaluated using 200 μL of robocolumns. The resins were loaded with a Protein A eluate produced as described in Example 1. Several operating conditions (loading pH, loading conductivity, washing pH, elution pH, elution conductivity) were tested to evaluate the operating range of each resin and to find ideal operating conditions. The samples obtained were Petition 870250087783, dated 09 / 29 / 2025, pp. 58 / 87 51 / 61 were then analyzed and compared across all different conditions and with different resins. It was found that, using suitable polishing resins under optimized operating conditions, it is possible to further remove mismatched variants and increase the purity of the protein of interest.

[0140] When using Capto Adhere and Capto Phenyl High Sub resins, no conditions were found that could increase purity to levels higher than those observed in the previous chromatography (capture) step, with approximately 30% purity in the passing flow fractions. The derivatives of the previous resins (Capto Adhere ImpRes and Capto Phenyl ImpRes) perform similarly to the previously mentioned resins, with approximately 30-40% purity achieved. However, when using Capto MMC ImpRes and Toyopearl NH2-750F, significantly better results were obtained with 47% purity for Toyopearl NH2-750F and 97% purity for Capto MMC ImpRes. These two resins showed a significant reduction in mismatch content, significantly improving the target purity. These two resins were then selected to be used in combination for a complete process aimed at providing efficient decontamination of product-related impurities. Table 5. Results Resin Purity (SEC) Purity (EC) Medium Purity (EC) Capto Adhere (GE) 31.7 % 29.5 % 35.1 % 34.6 % 33.2 % 45.9 % 58.0 % 34.9 % 31.3 % 34.5 % 31.3 % 30.5 % 27.3 % 49.6% 63.3% 40.4% Petition 870250087783, dated 09 / 29 / 2025, page 59 / 87 52 / 61 Capto Adhere ImpRes (GE) 37.6 % 35.7 % 41.3 % 44.2 % 30.7 % 29.3 % 34.4 % 29.4 % Capto MMC ImpRes (GE) 88.1 % 96.8 % 96.9 % 96.8 % 98.7 % 96.8 % 98.7 % 85.3 % 95.8 % 96.2 % 98.8 % 94.2 % 98.3 % 98.3 % 98.6 % 94.1 % 98.7 % 98.3 % 98.5 % 88.8 % 91.9 % 91.9 % 90.9 % Capto Phenyl High Sub (GE) 31.6 % 25.8 % 27.9 % 33.0 % 28.1 % 32.8 % 24.8 % 31.6 % 27.7 % 31.8 % 28.1 % 31.1 % 27.6 % 34.1 % 28.0 % 33.0 % 29.4 % 31.6 % 31.3 % 32.7 % 26.6 % 33.2 % 30.0 % Capto Phenyl ImpRes (GE) 32.4 % 24.5 % 27.9 % 29.3 % 25.0 % 28.3 % 24.5 % Petition 870250087783, dated 09 / 29 / 2025, pp. 60 / 87 53 / 61 30.8 % 27.2 % 31.3 % 29.6 % 29.2 % 28.6 % 32.0 % 30.6 % 30.9 % 28.9 % 31.3 % 28.4 % 30.4 % 26.5 % 30.9 % 30.7 % 31.8 % 29.9 % Toyopearl NH2-750F (Tosoh) 35.2 % 39.3 % 46.8 % 49.9 % 67.1 % 30.4 % 27.3 % 62.8 % 73.5 % 28.5 % 26.8 % Example 3: Pilot Scale Tests

[0141] The method was used for pilot-scale batch purification of a polypeptide containing a heteromultimeric Fc domain (protein of interest). The aim was to improve the purity of the protein of interest and remove product-related impurities (mismatches) as well as process-related impurities (host cell proteins (HCPs), DNA, etc.). The following example describes the purification of a protein of interest using the methods as described above. The protein of interest was first captured and purified by Protein A chromatography. An intermediate wash containing octanoate and benzyl alcohol was used to improve / intensify the removal of mismatch impurities. Then, mixed-mode chromatography was used to further purify the target molecule. Finally, an anion exchange resin was used to further purify the target molecule. Sample preparation Petition 870250087783, dated 09 / 29 / 2025, pp. 61 / 87 54 / 61

[0142] Harvesting materials of human monoclonal antibodies were prepared as described in Example 1. The harvest was generated in a suspension culture of recombinant CHO cells engineered to express the monoclonal antibody. The recombinant protein of interest was secreted into the culture medium which was then clarified by depth filtration for downstream processing. The clarified harvesting material was filtered with a 0.22 µm polyethersulfone (PES) filter before loading onto the chromatographic column. Protein chromatography

[0143] Protein resin A was prepared as described in example 1. Mabselect Sure Protein A (Cytiva) chromatography resin was packed using a 140 mm diameter column (Axichrom 140 / 300 column). The resin was packed to a bed height of 20 cm + / - 2 cm. Column efficiency was determined and measured at >6000 theoretical plates per meter and a skewness of 1.0. Before loading the harvest material, the column was purged with 0.5 M sodium hydroxide and then equilibrated with 50 mM Tris and 20 mM NaCl at pH 7.5. Prepared samples were loaded onto the column. The column was loaded to a target of 30 g / l, then washed as described above with equilibration buffer. The column was then washed with a washing solution containing 50 mM Tris, 75 mM octanoate, 1 M NaCl, and 2% benzyl alcohol at pH 9.0, followed again by equilibration buffer. Finally, the protein of interest was eluted from the column using a solution containing 20 mM acetic acid at pH 4.2. Table 1 provides an example chromatography process.The protein A step is followed by a viral inactivation step comprised of an acidic adjustment to an inactivation pH, halting inactivation for a defined amount of time, then an alkaline adjustment before proceeding to the subsequent step. Table 6. Purification by protein chromatography A Buffer Phase Volume Time Petition 870250087783, dated 09 / 29 / 2025, pp. 62 / 87 55 / 61 Equilibrium Treatment: Tris 50 mM, NaCl 20 mM at pH 7.5, 0.5 CV, 4 min. Charge: Clarified volume - 4 min. Initiation Wash: Tris 50 mM, NaCl 20 mM at pH 7.5, 2 CV, 4 min. Washing: Tris 50 mM, Octanoate 75 mM, NaCl 1 M, Benzyl Alcohol 2% at pH 9.0, 3 CV, 4 min. Subsequent Washing: Tris 50 mM, NaCl 20 mM at pH 7.5, 10 CV, 4 min. Elution: Acetic Acid 25 mM at pH 4.2, 7 CV*, 4 min. Stripping: Acetic Acid 0.5 M, 2 CV, 4 min. Cleaning: NaOH 0.5 M, 2 CV, 4 min. Equilibrium Treatment: Tris 50 mM, NaCl 20 mM at pH 7.5, 2.5 CV, 4 min. *UV Collection Criterion: 50 Au / m (top) - 100 Au / m (bottom) - meaning 250 mAu (top) and 500 mAu for an optical path length = 5 mm Multimodal chromatography

[0144] The Capto MMC ImpRes step is a ligation and elution step that binds the protein of interest and some of the impurities while other impurities are directed to the flow-through load and equilibrium stream. The Capto MMC ImpRes chromatography resin (Cytiva) was packed using a 140 mm diameter column (Axichrom 140 / 300 column). The resin was packed to a bed height of 20 cm + / - 2 cm. The column efficiency was determined and measured at >14000 theoretical plates per meter, and an asymmetry of 1.1. Before loading the protein A eluate, the column was purged with 0.5 M sodium hydroxide and then equilibrated with 50 mM acetic acid at pH 5.0. The sample from protein A was loaded onto the column. The column was loaded onto a 30 g / L target, then washed using 50 mM Tris at pH 8.0. Finally, the antibody was eluted from the column using a solution containing 50 mM Tris and 100 mM NaCl at pH 8.0. Table 7. Purification by multimodal chromatography Petition 870250087783, dated 09 / 29 / 2025, pp. 63 / 87 56 / 61 Buffer Phase Volume Residence Time Equilibrium Acetic acid 50 mM at pH 5.0 1 CV 5 min Loading Grouping after IV - 5 min Equilibrium Acetic acid 50 mM at pH 5.0 3 CV 5 min Washing Tris 50 mM at pH 8.0 3 CV 5 min Elution* Tris 50 mM NaCl 100 mM at pH 8.0 5 CV 5 min Removal** Tris 50 mM NaCl 1 M at pH 7.5 2 CV 5 min Cleaning** NaOH 0.5 M 2 CV 5 min Regeneration** Tris 50 mM NaCl 1 M at pH 7.5 2 CV 5 min Equilibrium Acetic acid 50 mM at pH 5.0 2.5 CV 5 min *UV Collection Criteria: 50 Au / m (top) - 150 Au / m (bottom) - meaning 0.25 Au (top) and 0.75 Au (bottom) for an optical path length = 5 mm ** Upward column flux Anion Exchange Chromatography.

[0145] The Toyopearl NH2-750F step is a flow-through step that binds impurities (additionally removed during cleaning) while the protein of interest is directed to the flow-through charge and equilibrium. The Toyopearl NH2-750F chromatography resin (Tosoh Bioscience) was packed using a 50 mm diameter column (Axichrom 50 / 300 column). The resin was packed to a bed height of 20 cm + / - 2 cm. The column efficiency was determined and measured at >6800 theoretical plates per meter and a Petition 870250087783, dated 09 / 29 / 2025, pp. 64 / 87 57 / 61 skewness of 1.1. Before loading the Capto MMC eluate, the column was purged with 0.5 M sodium hydroxide and then equilibrated with 50 mM Tris and 100 mM NaCl at pH 8.0. Sample from Capto MMC was loaded onto the column. The column was loaded to a target of 150 g / L, then the load was pushed using equilibrium buffer. Table 8. Purification by anion exchange chromatography Buffer Phase Volume Residence Time Equilibrium Tris 50 mM NaCl 100 mM at pH 8.0 1 CV 5 min Charge * Clustering after CMMCi - 5 min Impulse-Charge* Tris 50 mM NaCl 100 mM at pH 8.0 6 CV 5 min Removal** Tris 50 mM NaCl 1 M at pH 7.5 3 CV 5 min Cleaning** NaOH 0.5 M 2 CV 5 min Regeneration** Tris 50 mM NaCl 1 M at pH 7.5 2 CV 5 min Equilibrium Tris 50 mM NaCl 100 mM at pH 8.0 2.5 CV 5 min *UV Collection Criterion: 50 Au / m (top) - 50 Au / m (bottom) - meaning 0.10 Au (top) and 0.10 Au (bottom) for an optical path length = 2 mm ** Upward column flow Results

[0146] A pilot-scale proof of concept was successfully performed, dispensing approximately 50 grams of purified protein of interest. The initial purity in the clarified bulk harvest is unknown, but estimated to be approximately 30% at best. Purity was increased to 50.4% (SEC: 49.9%) after capture, then increased to 94.3% (SEC: 97.9%) after mixed mode and finally 97.9% (SEC: 100.0%) after anion exchange (Table 9). Petition 870250087783, dated 09 / 29 / 2025, pp. 65 / 87 58 / 61 Table 9. Results Target concentration (g / l) Purity by % of capillary electrophoresis area (non-reduced conditions; % target) Cleared volume After protein A chromatography + viral inactivation (clustering) 4.63 50.4 After multimodal chromatography 2.52 94.3 After anion exchange chromatography 2.37 97.9 Sequences SEQ ID NO: Cadeia polipeptídica 1 - F25 (I) divmtqspdslavslgeratincessqsllssgnqknyltwyqqkpgqppkpliywastresgvpdrfsgsgsgtdftltisslqaedvavyycqndysypytfgqgtklei krtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyacevthqglsspvtksfnrgecd kthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalp apiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealh nhytqkslslspgk SEQ ID NO: Cadeia polipeptídica 2 - F25 (II) evqlvqsgaevkkpgeslkisckgsgysftdyymkwarqmpgkglewmPetição 870250087783, de 09 / 29 / 2025, pág. 66 / 87 59 / 61 GDIIPSSGATFYNQKFKGQVTISADKSISTTYLQWSSLKASDTAMYYCARSHLLRASWFAYWG QGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGSTGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSDYVINWVRQAPGQGLEWMGEIYPGSGTNYYNEKFKAKATITADKSTSTAYMELSSLRS EDTAVYYCARRGRYGLYAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKRVEPKSCDKTH SEQ ID NO: Cadeia polypeptídica 3 - F25 (III) DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFFTISSLQPEDIATYFCQQGNTRPWTFGGGTKVEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQGSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECC SEQ ID NO: Cadeia polypeptídica 4 - F5 (I) DIVMTQSPDSLAVSLGERATINCESSQSLLSSGNQKNYLTWYQQKPGQPPKPLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPYTFGQGTKLEI Petição 870250087783, de 29 / 09 / 2025, pág. 67 / 87 60 / 61 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK SEQ ID NO: Cadeia polipeptídica 5 - F5 (II) EVQLVQSGAEVKKPGESLKISCKGSGYSFTDYYMKWARQMPGKGLEWMGDIIPSSGATFYNQKFKGQVTISADKSISTTYLQWSSLKASDTAMYYCARSHLLRASWFAYWG QGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGSTGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSDYVINWVRQAPGQGLEWMGEIYPGSGTNYYNEKFKAKATITADKSTSTAYMELSSLRS EDTAVYYCARRGRYGLYAMDYWGQGTTVTVSSRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKKVYACEVTHQGLSSPVTKSFNRGEC Petition 870250087783, de 29 / 09 / 2025, pág. 68 / 87 61 / 61 SEQ ID NO: Cadeia polypeptídica 6 - F5 (III) DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQQGNTRPWTFGGGTKVEIKASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHS SEQ ID NO: 7 - linker STGS Petition 870250087783, dated 09 / 29 / 2025, pp. 69 / 87

Claims

1 / 5 CLAIMS 1. A method for purifying a polypeptide containing a heteromultimeric Fc domain, characterized in that it comprises the following steps in the order indicated: a) providing a sample comprising the polypeptide containing a heteromultimeric Fc domain and one or more mismatched variants thereof; b) contacting a protein chromatography matrix A with the sample and binding the polypeptide containing a heteromultimeric Fc domain to the protein chromatography matrix A; c) contacting the protein chromatography matrix A with a washing solution, wherein the washing solution comprises octanoate; d) contacting the protein chromatography matrix A with an elution solution; e) collecting an eluate comprising the polypeptide containing a heteromultimeric Fc domain.

2. Method according to claim 1, characterized in that it further comprises the steps of: f) placing a multimodal chromatography matrix in contact with the eluate of step e) and linking the polypeptide containing a heteromultimeric Fc domain to the multimodal chromatography matrix; g) placing the multimodal chromatography matrix in contact with an elution solution; and h) collecting an eluate comprising the polypeptide containing a heteromultimeric Fc domain.

3. Method, according to claim 1 or 2, characterized in that it further comprises the steps of: i) placing an ion-exchange chromatography matrix in contact with the eluate of step e) or h); and Petition 870250087783, dated 09 / 29 / 2025, page 70 / 87 2 / 5 j) collecting an eluate or streamflow comprising the polypeptide containing a heteromultimeric Fc domain.

4. Method according to claim 1, characterized in that it is intended to purify the polypeptide containing a heteromultimeric Fc domain from its mismatched variants.

5. Method according to claim 1, characterized in that the Fc domain-containing polypeptide comprises a first and a second polypeptide chain, each comprising a CH2-CH3 region, and a third polypeptide chain that does not comprise a CH2-CH3 region.

6. Method according to claim 1, characterized in that it also removes process-related impurities.

7. A method according to any one of claims 1 to 6, characterized in that the polypeptide containing a heteromultimeric Fc domain is a multispecific antibody, preferably a bispecific, trispecific, tetraspecific, pentaspecific or hexaspecific antibody, more preferably a bispecific or trispecific antibody, most preferably in that the polypeptide containing a heteromultimeric Fc domain comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 variable immunoglobulin domains that form 2, 3, 4, 5 or 6 antigen-binding domains, in particular 4 or 6 variable immunoglobulin domains that form 2 or 3 antigen-binding domains.

8. A method according to any one of claims 1 to 7, characterized in that the polypeptide containing the heteromultimeric Fc domain comprises no more than one copy of a polypeptide chain.

9. Method, according to any one of claims 1 to 8, characterized in that the mismatched variants are selected from the group consisting of Petition 870250087783, dated 09 / 29 / 2025, page 71 / 87 3 / 5 a) monomers of the first, second or third polypeptide chains; b) homodimers or homomultimers of a polypeptide chain comprised in the polypeptide containing a heteromultimeric Fc domain; c) heteromultimers comprising more than one copy of the first, second or third polypeptide chain; d) heteromultimers not comprising the first, second or third polypeptide chain; e) aggregates of the mismatched variants according to a) ad) and / or the polypeptide containing a heteromultimeric Fc domain.

10. Method according to claim 9, characterized in that it results in separation of the polypeptide containing a heteromultimeric Fc domain from at least one, preferably at least two, more preferably at least three, even more preferably all of said mismatched variants.

11. Method, according to any one of claims 1 to 10, characterized in that the heteromultimeric Fc domain-containing polypeptide consists of a first, a second and a third polypeptide chain, each comprising a variable immunoglobulin domain, preferably wherein the second polypeptide chain comprises two variable immunoglobulin domains.

12. Method according to claim 11, characterized in that: - the first polypeptide chain is represented by formula (1): V1-C1-CH2-CH3 (1), - the second polypeptide chain is represented by formula (2): V2-C2-CH2-CH3-V3-C3 (2); and - the third polypeptide chain is represented by formula Petition 870250087783, dated 09 / 29 / 2025, page 72 / 87 4 / 5 (3): V4-C4 (3); wherein V1 to V4 are variable immunoglobulin domains, wherein the antigen-binding domains are formed by V1 and V2 and by V3 and V4; and wherein C1 to C4 are heterodimerization domains, preferably selected from CH1, Ck and Cl domains, preferably wherein one of C1 and C2 is a CH1 domain and the other is Ck or Cl, and one of C3 and C4 is a CH1 domain and the other is Ck or Cl, even more preferably wherein C2 and C3 are CH1 and C1 and C4 are Ck or Cl, or wherein C2 and C3 are CH1 and Ck or Cl and C1 and C4 are Ck or Cl and CH1.

13. Method, according to any one of claims 1 to 12, characterized in that the purity of the polypeptide containing heteromultimeric Fc domain is - at least 40%, 45%, 50% or 55% of the total protein concentration after step e); - at least 50%, 60%, 70%, 80%, 90% or 95% of the total protein concentration after step h); or - at least 90%, 92%, 94%, 95%, 96% or 97% of the total protein concentration after step j), wherein the concentration is preferably determined by capillary electrophoresis.

14. Method, according to any one of claims 1 to 13, characterized in that the octanoate concentration in the washing solution of step c) is about 50 mM to about 150 mM, preferably 75 mM, and wherein preferably the octanoate is an octanoate salt.

15. Method, according to any one of claims 1 to 14, characterized in that the washing solution of step c) - further comprises benzyl alcohol, hexylene glycol and / or propylene glycol; - further comprises benzyl alcohol at a concentration of about 0.5% to about 4%, preferably about 1% to about 3%, more preferably about 2% volume / volume; - further comprises hexylene glycol or propylene glycol at a concentration of about 5% to about 25%, preferably about 10% to about 20%, more preferably about 15% volume / volume; and / or - does not comprise benzoate. Petition 870250087783, dated 09 / 29 / 2025, pp. 74 / 87

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