Kappa light chain binding convection matrix

Abstract

The present invention relates to the isolation and separation of kappa light chain-containing proteins. More specifically, the present invention relates to a separation matrix comprising a kappa light chain-binding ligand covalently coupled to a porous support, wherein the kappa light chain-binding ligand comprises, consists essentially of, or consists of a multimer of an alkali-stabilized Protein L domain of Finegoldia magna (formerly Peptostreptococcus magnus), and wherein the porous support is a convection-based chromatography matrix.

Description

Technical Field

[0001] The present invention relates to the field of separation of biomolecules. More specifically, the present invention relates to a separation matrix for affinity chromatography and to the separation of biomolecules based on the presence of kappa light chains, such as immunoglobulins and immunoglobulin fractions. The present invention also relates to methods of using said separation matrix.

Background Art

[0002] Immunoglobulins and immunoglobulin fragments represent some of the most prevalent biopharmaceuticals in manufacturing or development worldwide. Due to the high commercial demand for this particular therapeutic market and thus their value, pharmaceutical companies have focused on maximizing the productivity of their respective manufacturing processes while controlling associated costs.

[0003] Affinity chromatography, typically in a matrix containing staphylococcal protein A or variants thereof, is commonly used as one of the important steps in the purification of intact immunoglobulin molecules. The highly selective binding of protein A to the Fc chain of immunoglobulins results in a very high clearance of impurities and contaminants in a common process.

[0004] Immunoglobulin fragments, or antibody fragments, such as Fab, single-chain variable fragments (scFv), bispecific T cell engagers (BiTE), domain antibodies, etc., that lack the Fc chain but have a subclass 1, 3, or 4 kappa light chain, and matrices containing protein L derived from Finegoldia magna (formerly Peptostreptococcus Magnus) (B Akerstrom, L Bjorck: J. Biol. Chem. 264, 19740 - 19746, 1989; W Kastem et al: J. Biol. Chem. 267, 12820 - 12825, 1992; B HK Nilson et al: J. Biol. Chem. 267, 2234 - 2239, 1992 and U.S. Patent No. 6,822,075) show great promise as a purification platform that provides the required high selectivity.

[0005] The protein L matrix is commercially available, for example, from Cytiva(™) as Capto(™) L and can be used for the separation of kappa light chain-containing proteins such as intact antibodies, Fab fragments, scFv fragments, domain antibodies, etc. Approximately 75% of the antibodies produced by healthy humans have a kappa light chain, and approximately 90% of therapeutic monoclonal antibodies and antibody fragments contain a kappa light chain (Carter, P., Lazar, G. Next generation antibody drugs: pursuit of the 'high-hanging fruit'. Nat Rev Drug Discov 17, 197 - 223 (2018). https: / / doi.org / 10.1038 / nrd.2017.227).

[0006] Any application of bioprocess chromatography requires comprehensive attention to the reliable removal of impurities and / or contaminants. Such impurities and / or contaminants can be, for example, non-eluting molecules adsorbed to the stationary phase or matrix in a chromatography procedure, such as unwanted biomolecules or microorganisms, such as proteins, carbohydrates, lipids, bacteria, and viruses. The removal of such impurities and / or contaminants from the matrix is usually carried out after the desired product has been eluted first, in order to regenerate the matrix before subsequent use. Such removal usually involves a procedure known as clean-in-place (CIP), and agents capable of inactivating or eluting impurities from the stationary phase are used. One such class of agents that is often used with chromatography media is an alkaline solution that passes over the matrix. Currently, the most widely used cleaning and sanitizing agent is NaOH, and it is desirable to use NaOH at a concentration ranging from 0.05 M up to, for example, 1 M at most, depending on the degree and nature of the contamination and impurities. However, Protein L is a protein that is considerably more alkali-sensitive compared to, for example, Protein A, and has a tolerance of only up to approximately 15 mM NaOH over a number of cycles. This means that additional, less desirable cleaning solutions, such as urea or guanidinium salts, may have to be used to ensure sufficient cleaning.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-Patent Documents

[0008] [Non-Patent Document 1] B Akerstrom, L Bjorck: J. Biol. Chem. 264, 19740 - 19746, 1989 [Non-Patent Document 2] W Kastem et al.: J. Biol. Chem. 267, 12820 - 12825, 1992 [Non-Patent Document 3] B HK Nilson et al.: J. Biol. Chem. 267, 2234 - 2239, 1992 [Non-Patent Document 4] Carter, P., Lazar, G. Next generation antibody drugs: pursuit of the 'high-hanging fruit'. Nat Rev Drug Discov 17, 197 - 223 (2018). https: / / doi.org / 10.1038 / nrd.2017.227 [Non-Patent Document 5] Good et al. (1966) Biochemistry 5:467 [Non-Patent Document 6] Good and Izawa (1972) Methods Enzymol. 24:62 [Non-Patent Document 7] "Recombinant production of a VL single domain antibody in Escherichia coli and analysis of its interaction with Peptostreptococcal protein L" (Protein Expression and Purification, Vol. 51, No. 2, February 2007, pp. 253 - 259) [Summary of the Invention] [Problems to be Solved by the Invention]

[0009] Accordingly, there remains a need in the art to obtain a separation matrix containing a Protein L-derived ligand having improved stability against alkaline cleaning procedures.

Means for Solving the Problems

[0010] The present inventors have provided a separation matrix containing a kappa light chain binding ligand covalently coupled to a porous support, wherein the kappa light chain binding ligand comprises, consists essentially of, or consists of a multimer of an alkali-stabilized Finegoldia magna (formerly Peptostreptococcus Magnus) Protein L domain, and the porous support is a convection-based chromatography matrix, thereby achieving the solution to the above problems.

[0011] The convection-based chromatography matrix may be a fibrous substrate. The fibrous substrate may be based on electrospun polymeric fibers or cellulose fibers, optionally non-woven fibers.

[0012] The polymer may be selected from the group consisting of cellulose, cellulose acetate, polysulfone, polyamide, polyacrylic acid, polymethacrylic acid, polyacrylonitrile, polystyrene, polyethylene oxide, and mixtures thereof. According to one embodiment, the fibrous substrate is a fibrous non-woven polymer matrix.

[0013] The fibers contained in the fibrous substrate may have a cross-sectional diameter of 10 to 1000 nm, such as 200 to 800 nm, 200 to 400 nm, or 300 to 400 nm.

[0014] The ligand may be bound to a divinylsulfone functional group coupled to a glycidol group grafted onto the fibrous substrate.

[0015] The kappa light chain binding ligand may comprise at least two alkali-stabilized protein L domains. The alkali-stabilized protein L domain may be selected from the group consisting of functional variants of the B1 domain, B2 domain, B3 domain, B4 domain, B5 domain, C2 domain, C3 domain, C4 domain and D1 domain of Protein L from Finegoldia magna (formerly Peptostreptococcus magnus), and the corresponding positions in the alignment at positions 10 and 45 in the B2 domain (SEQ ID NO: 1) are histidine, and the corresponding position in the alignment at position 60 in the B2 domain (SEQ ID NO: 1) is tyrosine or glutamine. In one embodiment, the alkali-stabilized protein L domain is selected from the group consisting of the B2 domain, B3 domain, B4 domain, B5 domain, C2 domain, C3 domain, C4 domain and D1 domain.

[0016] The alkali-stabilized protein L domain is determined by the BLOSUM matrix of 75, using a gap open penalty of 12 and a gap extension penalty of 3, and has at least 90%, 95% or 98% sequence identity, or 77.5% sequence similarity with any one of the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19, and the positions corresponding in the alignment to positions 10 and 45 in SEQ ID NO: 1, and the position corresponding in the alignment to position 60 in SEQ ID NO: 1 do not change. The alkali-stabilized protein L domain is determined by the BLOSUM matrix of 75, using a gap open penalty of 12 and a gap extension penalty of 3, and may have at least 90%, 95% or 98% sequence identity, or 77.5% sequence similarity with any one of the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID NO: 37.

[0017] The ligand density for the separation matrix may be at least 20 mg / ml of the porous support, or at least 25 mg / ml of the porous support, or at least 30 mg / ml of the porous support, or at least 35 mg / ml of the porous support, or at least 40 mg / ml of the porous support, or at least 45 mg / ml of the porous support, or at least 50 mg / ml of the porous support.

[0018] The separation matrix may have a dynamic binding capacity (DBC) of 25 g / mL at 10% breakthrough for a kappa light chain-comprising antibody, such as trastuzumab, when flowed at a flow rate of 10 mL / min in a 0.4 mL HiTrap™ device.

[0019] The separation matrix may have a dynamic binding capacity (DBC) of 45 g / mL at 10% breakthrough for a kappa light chain-comprising antibody, such as trastuzumab, when flowed at a flow rate of 10 mL / min in a 0.4 mL HiTrap™ device.

[0020] Further provided is a method for isolating a kappa light chain-comprising protein, comprising: a) contacting a liquid sample containing a kappa light chain-comprising protein with a separation matrix; b) washing the separation matrix with one washing liquid or a combination of several washing liquids; c) eluting the kappa light chain-comprising protein from the separation matrix using an eluent; and d) cleaning the separation matrix using a cleaning liquid, wherein the separation matrix has a dynamic binding capacity (DBC) of 25 g / mL at 10% breakthrough for a kappa light chain-comprising antibody, such as trastuzumab, when flowed at a flow rate of 10 mL / min in a 0.4 mL HiTrap™ device.

[0021] Further provided is a method for separation for isolating a kappa light chain-binding protein derived from a lambda light chain-comprising protein, comprising: a) contacting a liquid sample containing a kappa light chain-comprising protein with a separation matrix; b) washing the separation matrix with one washing liquid or a combination of several washing liquids; c) eluting the kappa light chain-comprising protein from the separation matrix using an eluent; and d) cleaning the separation matrix using a cleaning liquid, wherein the separation matrix has a dynamic binding capacity (DBC) of 25 g / mL at 10% breakthrough for a kappa light chain-comprising antibody, such as trastuzumab, when flowed at a flow rate of 10 mL / min in a 0.4 mL HiTrap™ device.

[0022] Furthermore, a method for the separation of bispecific antibodies from monospecific antibodies, comprising: a) contacting a liquid sample containing a bispecific antibody with a separation matrix; b) washing the separation matrix with one washing liquid or a combination of several washing liquids; c) eluting the bispecific antibody from the separation matrix using an eluent; and d) cleaning the separation matrix using a cleaning liquid, wherein the separation matrix has a dynamic binding capacity (DBC) of 25 g / mL with 10% breakthrough when flowed at a flow rate of 10 mL / min in a 0.4 mL HiTrap™ device for an antibody containing a kappa light chain, such as trastuzumab, is provided.

[0023] In any of the separation methods disclosed above, the separation is carried out by applying a volume gradient or a pH gradient in step c).

[0024] In any of the methods according to the above, the separation matrix may be as described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0025]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

DETAILED DESCRIPTION OF THE INVENTION

[0026] Definitions The terms "antibody" and "immunoglobulin" may be used interchangeably herein and refer to an antigen-binding protein having a basic structure of four polypeptide chains consisting of two heavy (H) chains and two light (L) chains, the chains being stabilized by interchain or intrachain disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region. The light chain constant region consists of one domain, CL. There are two types of light chains in humans, kappa chains and lambda chains. This term is to be understood to include any antibody, for example, but not limited to, monoclonal antibodies and bispecific antibodies, as well as antibody fragments, fusion proteins containing an antibody or antibody fragment, and conjugates containing an antibody or antibody fragment.

[0027] The terms "kappa light chain binding polypeptide" and "kappa light chain binding protein" are used herein to mean a polypeptide or protein, respectively, that is capable of binding to antibody subclasses 1, 3, or 4 kappa light chains (as in B H K Nilson et al.: J. Biol. Chem. 267, 2234-2239, 1992, also referred to as V κI , V κIII and V κIV ), and include, for example, protein L, and any variant, fragment, or fusion protein thereof that maintains said binding properties.

[0028] The term "kappa light chain-containing protein" is used as a synonym for "immunoglobulin kappa light chain-containing protein" and herein refers to antibody-derived subclasses 1, 3, or 4 kappa light chains (as in B H K Nilson et al.: J. Biol. Chem. 267, 10 2234-2239, 1992, also referred to as V κI , V κIII and VκIV refers to a protein (also referred to as such), and includes any intact antibody, antibody fragment, fusion protein, conjugate or recombinant protein containing a subclass 1, 3 or 4 kappa light chain.

[0029] The term "mAb" represents a monoclonal antibody.

[0030] The term "Fab" represents an antigen-binding fragment derived from an immunoglobulin, including a kappa or lambda light chain.

[0031] The term "bispecific antibody" represents an antibody capable of binding two different types of antigens or two different epitopes on the same antigen. Similarly, a trispecific antibody represents an antibody capable of binding three different types of antigens or three different epitopes on the same antigen.

[0032] "DBC" means dynamic binding capacity and is the binding capacity in a packed affinity chromatography column under operating conditions, i.e., during sample application. The DBC of a chromatography resin is the amount of target protein that binds to the resin under a given flow condition before significant breakthrough of unbound protein occurs. DBC is determined by loading a sample containing a known concentration of target protein and monitoring the flow-through. The protein binds to the resin up to a certain limit point before the unbound protein flows through the column.

[0033] DBC can be determined, for example, from the breakthrough curve at 10% protein loss. This is referred to as the Qb10% value, or simply Qb10%. The sample is applied to the chromatography resin column for a specific residence time, and the dynamic binding capacity for each resin is calculated at 10% protein breakthrough, i.e., the amount of target sample loaded onto the column until the concentration of the target sample in the column effluent reaches 10% of the target sample concentration in the liquid sample. When the dynamic binding capacity for each resin is calculated at 80% of the breakthrough capacity, this is referred to as the Qb80% value.

[0034] As used herein, the term "liquid sample" refers to a liquid containing at least one target substance that is required to be purified from other substances that are also present. The liquid sample can be, for example, an aqueous solution, an organic solvent system, or an aqueous / organic solvent mixture or solution. A stock solution is often a complex mixture or solution containing many biomolecules (e.g., proteins, antibodies, hormones, and viruses), small molecules (e.g., salts, sugars, lipids, etc.) and even particulate matter. A typical stock solution of biological origin can start as an aqueous solution or suspension, but it can also contain organic solvents used in earlier separation steps such as solvent precipitation, extraction, etc. Examples of liquid samples that can contain beneficial biological substances suitable for purification according to various embodiments of the present invention include, but are not limited to, culture supernatants from bioreactors, homogenized cell suspensions, plasma, plasma fractions, and milk. Alternatively, the liquid sample can be referred to as a "clarified cell culture feed" or "CCF".

[0035] A "buffer" is a substance that, by its presence in a solution, increases the amount of acid or base that must be added to cause a unit change in pH. A buffer solution resists changes in pH by the action of its acid-base conjugate components. A buffer solution for use with biological reagents can generally maintain a certain concentration of hydrogen ions so that the pH of the solution is within the physiological range. The term "physiological pH" refers to the pH of mammalian blood (i.e., 7.38 or about 7.4). Thus, the physiological pH range is from about 7.2 to 7.6. Conventional buffer components include, but are not limited to, organic and inorganic salts, acids and bases. Exemplary buffers for use in the purification of biomolecules (e.g., protein molecules) include zwitterionic or "Good" buffers, see, for example, Good et al. (1966) Biochemistry 5:467 and Good and Izawa (1972) Methods Enzymol. 24:62.

[0036] "Equilibration buffer" is a buffer used to prepare a binding reagent, a solid phase, or both, for loading a stock solution containing a target protein. The equilibration buffer is preferably isotonic and generally has a pH in the range of about 6 to about 8. "Loading buffer" is a buffer used to load a stock solution or a liquid sample containing a protein containing a binding region and impurities onto a solid phase to which a binder is immobilized. Often, the equilibration buffer and the loading buffer are the same.

[0037] As used herein, all of "washing solution" or "washing buffer" refer to a liquid used herein to remove impurities from a chromatographic resin to which a target substance is bound. For example, two or more washing solutions can be used sequentially, using a continuous washing solution having various properties such as pH, conductivity, solvent concentration, etc., designed to dissociate and remove various types of impurities non-specifically associated with the chromatographic resin.

[0038] "Eluent" or "elution buffer" are used interchangeably herein and refer to a liquid used herein to dissociate a target substance from a chromatographic resin and thereby elute a protein containing a binding region from an immobilized binder after washing with one or more washing solutions. The eluent acts to dissociate the target substance without irreversibly denaturing it. Typical eluents are well known in chromatography techniques and can have different pHs (typically lower pH), higher salt concentrations, free affinity ligands or analogs, or other substances that promote dissociation of the target substance from the chromatographic resin. "Elution conditions" refer to the process conditions imposed on a target substance-binding chromatographic resin to dissociate the target substance from the chromatographic resin, e.g., contacting the target substance-binding chromatographic resin with an eluent or elution buffer to effect such dissociation.

[0039] Preferably, the elution buffer has a low pH, thereby interfering with the interaction between the kappa light chain binding separation matrix and the protein of interest. Preferably, the low pH elution buffer has a pH in the range from about 2 to about 5, most preferably in the range from about 3 to about 4. Examples of buffers that control the pH within this range include glycine, phosphate, acetate, and citrate buffers, and combinations thereof. Preferred such buffers are citrate and acetate buffers, most preferably sodium citrate or sodium acetate buffers.

[0040] The cleaning solution can be an acidic or alkaline solution for removing resin residues after elution of the target substance. For example, precipitated proteins, hydrophobic proteins, nucleic acids, endotoxins and viruses can be removed by the cleaning solution. Most commonly, an alkaline solution is used for this purpose.

[0041] Clean-in-Place (CIP) is an important process for the efficient use of chromatography columns. In order to maximize the number of cycles in which the column can be reused, a cleaning procedure is required that efficiently removes impurities without being harmful to the chromatography resin.

[0042] As used herein, the terms "comprises", "comprising", "containing", "having", etc. may mean "includes", "including", etc.; "consisting essentially of" or "consists essentially" are open-ended terms that allow for the presence of entities beyond those described, provided that the basic or novel features of what is described are not changed by the presence of entities beyond those described, except for prior art embodiments.

[0043] Detailed Description The inventors aimed to invent a separation matrix containing a Protein L-derived ligand that has sufficient efficiency in binding capacity and fluidity characteristics in chromatography settings, while maintaining improved stability against alkaline cleaning procedures, preferably superior to currently commercially available separation matrices.

[0044] According to one aspect, this object was achieved by providing a separation matrix comprising a kappa light chain-binding ligand covalently coupled to a porous support, wherein the kappa light chain-binding ligand comprises, consists essentially of, or consists of a multimer of an alkali-stabilized F. magna (formerly Peptostreptococcus magnus) Protein L domain, and the porous support is a convection-based chromatography matrix.

[0045] The kappa light chain-binding ligand contained in the separation matrix of the present invention comprises, consists essentially of, or consists of a multimer of an alkali-stabilized Protein L domain. The Protein L domain may be any functional Protein L-derived domain as long as it is alkali-stabilized. The Protein L domain is selected from functional variants of the B1 domain, B2 domain, B3 domain, B4 domain, B5 domain, C2 domain, C3 domain, C4 domain, and D1 domain, and the corresponding positions in the alignment at positions 10 and 45 in the B2 domain (SEQ ID NO: 1) are histidine, and the corresponding position in the alignment at position 60 in the B2 wt domain (SEQ ID NO: 1) is tyrosine or glutamine. Accordingly, the above-mentioned positions corresponding to positions 10, 45, and 60 in the B2 wt domain (SEQ ID NO: 1) do not vary within the functional Protein L domain. SEQ ID NO: 1 (wt B2) PKEEVTIKANLIYADGKTQTAEFKGTFEEAAAEAYRYADALKKDNGEYTVDVADKGYTLNIKFAGKEKTPEE

[0046] Examples of such functional protein L domains can be as follows: SEQ ID NO: 2 (B2:N10H, N45H, N60Y mutation) PKEEVTIKAHLIYADGKTQTAEFKGTFEEAAAEAYRYADALKKDHGEYTVDVADKGYTLYIKFAGKEKTPEE SEQ ID NO: 3 (B2:N10H, N45H, N60Q mutation) PKEEVTIKAHLIYADGKTQTAEFKGTFEEAAAEAYRYADALKKDHGEYTVDVADKGYTLQIKFAGKEKTPEE SEQ ID NO: 4 (B3:N10H, N45H, N60Y mutation) PKEEVTIKAH LIYADGKTQT AEFKGTFEEA TAEAYRYADL LAKEHGKYTV DVADKGYTLY IKFAGKEKTP EE SEQ ID NO: 5 (B3:N10H, N45H, N60Q mutation) PKEEVTIKAH LIYADGKTQT AEFKGTFEEA TAEAYRYADL LAKEHGKYTV DVADKGYTLQ IKFAGKEKTP EE SEQ ID NO: 6 (B1:N10H, N45H, N60Y mutation) SEEEVTIKAHLIFANGSTQTAEFKGTFEKATSEAYAYADTLKKDHGEYTVDVADKGYTLYIKFAGKEKTPEE SEQ ID NO: 7 (B1:N10H, N45H, N60Q mutation) SEEEVTIKAHLIFANGSTQTAEFKGTFEKATSEAYAYADTLKKDHGEYTVDVADKGYTLQIKFAGKEKTPEE SEQ ID NO: 8 (B4:N10H, N45H, N60Y mutation) PKEEVTIKAHLIYADGKTQTAEFKGTFAEATAEAYRYADLLAKEHGKYTADLEDGGYTIYIRFAGKKVDEKPEE SEQ ID NO: 9 (B4:N10H, N45H, N60Q mutation) PKEEVTIKAHLIYADGKTQTAEFKGTFAEATAEAYRYADLLAKEHGKYTADLEDGGYTIQIRFAGKKVDEKPEE SEQ ID NO: 10 (B5: N9H, N44H, N59Y mutation) KEQVTIKEH IYFEDGTVQTATFKGTFAEATAEAYRYADLLSKEHGKYTADLEDGGYTIQIRFAGKEEPEE SEQ ID NO: 11 (B5: N9H, N44H, N59Q mutation) KEQVTIKEHIYFEDGTVQTATFKGTFAEATAEAYRYADLLSKEHGKYTADLEDGGYTIQIRFAGKEEPEE SEQ ID NO: 12 (C2b N57Y: N10H, N45H, N60Y mutation) PKEEVTIKVHLIFADGKTQTAEFKGTFEEATAKAYAYADLLAKEHGEYTADLEDGGYTIYIKFAGKETPETPEE SEQ ID NO: 13 (C2b N57Y: N10H, N45H, N60Q mutation) PKEEVTIKVHLIFADGKTQTAEFKGTFEEATAKAYAYADLLAKEHGEYTADLEDGGYTIQIKFAGKETPETPEE SEQ ID NO: 14 (C3b N39D, N57Y: N10H, N45H, N60Y mutation) PKEEVTIKVHLIFADGKIQTAEFKGTFEEATAKAYAYADLLAKEHGEYTADLEDGGYTIYIKFAGKETPETPEE SEQ ID NO: 15 (C3b N39D, N57Y: N10H, N45H, N60Q mutation) PKEEVTIKVHLIFADGKIQTAEFKGTFEEATAKAYAYADLLAKEHGEYTADLEDGGYTIQIKFAGKETPETPEE SEQ ID NO: 16 (C4: N10H, N45H, N60Y mutation) PKEEVTIKVHLIFADGKTQTAEFKGTFEEATAEAYRYADLLAKVHGEYTADLEDGGYTIYIKFAGKEQPGENPG Accession number 17 (C4:N10H, N45H, N60Q mutation) PKEEVTIKVHLIFADGKTQTAEFKGTFEEATAEAYRYADLLAKVHGEYTADLEDGGYTIQIKFAGKEQPGENPG Accession number 18 (D1:N10H, N45H, N60Y mutation) PKEEVTIKAHLIFADGKTQTAEFKGTFEEATAEAYRYADLLAKVHGEYTADLEDGGYTIYIKFAGKEQPGEN Accession number 19 (D1:N10H, N45H, N60Q mutation) PKEEVTIKAHLIFADGKTQTAEFKGTFEEATAEAYRYADLLAKVHGEYTADLEDGGYTIQIKFAGKEQPGEN

[0047] Preferably, the protein L domain is selected from the group consisting of the B3 domain, C2 domain, C3 domain and D domain, the positions corresponding in the alignment to positions 10 and 45 in the B2 wt domain (Accession number 1) are histidine, and the position corresponding in the alignment to position 60 in the B2 wt domain (Accession number 1) is tyrosine or glutamine. The above-mentioned positions corresponding to positions 10, 45 and 60 in the B2 wt domain (Accession number 1) do not change within the functional protein L domain.

[0048] The remaining positions in such a functional protein L domain can vary as long as the three-dimensional structure does not change compared to the three-dimensional structure of the B2 wt domain (Accession number 1), and as long as it retains at least the kappa light chain binding capacity and is alkali-stabilized compared to the B2 wt domain (Accession number 1). The variations may be conservative amino acid substitutions for amino acids having similar or identical charge, hydrophobicity, etc., and those skilled in the art can determine what such variations of amino acids can be.

[0049] The protein L domain is determined using a BLOSUM matrix of 75, a gap opening penalty of 12, and a gap extension penalty of 3, and may have at least 90%, 95%, or 98% sequence identity, or 77.5% sequence similarity with any one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19 of the amino acid sequence.

[0050] The functional protein L domain may be a truncated sequence at the ends. For example, the positions corresponding to positions 1 to 4 in the B2 wt domain (SEQ ID NO: 1) may be deleted. For example, the positions corresponding to the positions following position 65 in the B2 wt domain (SEQ ID NO: 1) may be deleted.

[0051] SEQ ID NO: 20 (B1_trunc, N6H, N41H, N56Y mutation) VTIKAHLIFANGSTQTAEFKGTFEKATSEAYAYADTLKKDHGEYTVDVADKGYTLYIKFAG SEQ ID NO: 21 (B1_trunc, N6H, N41H, N56Q mutation) VTIKAHLIFANGSTQTAEFKGTFEKATSEAYAYADTLKKDHGEYTVDVADKGYTLQIKFAG SEQ ID NO: 22 (B2_trunc, N6H, N41H, N56Y mutation) VTIKAHLIYADGKTQTAEFKGTFEEAAAEAYRYADALKKDHGEYTVDVADKGYTLYIKFAG SEQ ID NO: 23 (B2_trunc, N6H, N41H, N56Q mutation) VTIKAHLIYADGKTQTAEFKGTFEEAAAEAYRYADALKKDHGEYTVDVADKGYTLQIKFAG SEQ ID NO: 24 (B3_trunc, N6H, N41H, N56Y mutation) VTIKAHLIYADGKTQTAEFKGTFEEATAEAYRYADLLAKEHGKYTVDVADKGYTLYIKFAG SEQ ID NO: 25 (B3_trunc, N6H, N41H, N56Q mutation) VTIKAHLIYADGKTQTAEFKGTFEEATAEAYRYADLLAKEHGKYTVDVADKGYTLQIKFAG SEQ ID NO: 26 (B4_trunc, N6H, N41H, N56Y mutation) VTIKAHLIYADGKTQTAEFKGTFAEATAEAYRYADLLAKEHGKYTADLEDGGYTIYIRFAG SEQ ID NO: 27 (B4_trunc, N6H, N41H, N56Q mutation) VTIKAHLIYADGKTQTAEFKGTFAEATAEAYRYADLLAKEHGKYTADLEDGGYTIQIRFAG SEQ ID NO: 28 (B5_trunc, N6H, N41H, N56Y mutation) VTIKEHIYFEDGTVQTATFKGTFAEATAEAYRYADLLSKEHGKYTADLEDGGYTIYIRFAG SEQ ID NO: 29 (B5_trunc, N6H, N41H, N56Q mutation) VTIKEHIYFEDGTVQTATFKGTFAEATAEAYRYADLLSKEHGKYTADLEDGGYTIQIRFAG SEQ ID NO: 30 (C2b N57Y_trunc, N6H, N41H, N56Y mutation) VTIKVHLIFADGKTQTAEFKGTFEEATAKAYAYADLLAKEHGEYTADLEDGGYTIYIKFAG SEQ ID NO: 31 (C2b N57Y_trunc, N6H, N41H, N56Q mutation) VTIKVHLIFADGKTQTAEFKGTFEEATAKAYAYADLLAKEHGEYTADLEDGGYTIQIKFAG SEQ ID NO: 32 (C3b N39D, N57Y trunc, N6H, N41H, N56Y mutation) VTIKVHLIFADGKIQTAEFKGTFEEATAKAYAYADLLAKEHGEYTADLEDGGYTIYIKFAG SEQ ID NO: 33 (C3b N39D, N57Y_trunc, N6H, N41H, N56Q mutation) VTIKVHLIFADGKIQTAEFKGTFEEATAKAYAYADLLAKEHGEYTADLEDGGYTIQIKFAG SEQ ID NO: 34 (C4_trunc, N6H, N41H, N56Y mutation) VTIKVHLIFADGKTQTAEFKGTFEEATAEAYRYADLLAKVHGEYTADLEDGGYTIYIKFAG SEQ ID NO: 35 (C4_trunc, N6H, N41H, N56Q mutation) VTIKVHLIFADGKTQTAEFKGTFEEATAEAYRYADLLAKVHGEYTADLEDGGYTIQIKFAG SEQ ID NO: 36 (D1_trunc, N6H, N41H, N56Y mutation) VTIKAHLIFADGKTQTAEFKGTFEEATAEAYRYADLLAKVHGEYTADLEDGGYTIYIKFAG SEQ ID NO: 37 (D1_trunc, N6H, N41H, N56Q mutation) VTIKAHLIFADGKTQTAEFKGTFEEATAEAYRYADLLAKVHGEYTADLEDGGYTIQIKFAG

[0052] The Protein L domain is determined using a 75 BLOSUM matrix, a 12 gap open penalty, and a 3 gap extension penalty, and has at least 90%, 95%, or 98% sequence identity, or 77.5% sequence similarity, with any one of SEQ ID NOs: 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37 of the amino acid sequences.

[0053] As can be seen above, the C2 domain and the C3 domain contain additional mutations. The C2 domain scaffold contains an additional N57Y mutation and is designated herein as C2b. The C3 domain scaffold contains additional N39D and N57Y mutations and is designated herein as C3b.

[0054] As described above, the kappa light chain binding ligand contained in the separation matrix comprises, consists essentially of, or consists of a multimer of an alkali-stabilized protein L domain. The multimer may contain 2, 3, 4, 5, 6, 7, 8, or 9 alkali-stabilized protein L domains. In other words, the multimer may be a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, or nonamer. Preferably, the ligand contains 4, 5, 6, or 7 alkali-stabilized protein L domains, such as 5 or 6 alkali-stabilized protein L domains.

[0055] The multimer may further contain a linker, spacer, or additional amino acids. The additional amino acids may, for example, be derived from the cloning process and expression of the ligand or may constitute residues from a cleaved signaling sequence. One of ordinary skill in the art will recognize and understand that such additional amino acids may vary without affecting the function of the kappa light chain binding function of the multimer.

[0056] The porous support comprises a convection-based chromatography matrix. The convection-based chromatography matrix may be a fibrous substrate. The fibrous substrate may be based on electrospun polymer fibers or cellulose fibers, optionally non-woven fibers, which form a stationary phase containing a plurality of pores that can penetrate the stationary phase during use. The fibrous substrate may thus be a non-woven polymer matrix. Such a substrate can be seen in the HiTrap™ unit from Cytiva™.

[0057] Convection-based chromatography supports enable a combination of high flow rates and high binding capacities and are therefore desirable for use, for example, in mAb purification.

[0058] The polymer fibers may be non-woven fibers. By using a randomly deposited fiber mat (non-woven) structure, flow disruption is promoted, thereby preventing channeling.

[0059] The polymer fibers can be electrospun. Electrospinning provides fibers with a constant diameter and can be easily adjusted to create different ratios of fibers (e.g., by changing the atmospheric properties during spinning). Electrospinning is also a technique that exhibits excellent distribution, which is particularly useful when creating layered membranes. The mass transfer characteristics of the fibers have been shown to be similar to those of monolithic structures that enable flow rate-independent separation.

[0060] The polymers used in the present invention are not limited to any particular polymer and can be adapted to specific uses. The polymers can be, for example, nylon, poly(acrylic acid), polyacrylonitrile, polystyrene, polysulfone, polyacrylonitrile, polycaprolactone, collagen, chitosan, agarose, and polyethylene oxide, and combinations thereof. The polymers may be derivatized to enhance the solubility and / or other properties of the polymer to improve its suitability for electrospinning. The derivatized polymers (e.g., polyethersulfone, cellulose acetate, or poly(acrylonitrile-co-acrylic acid)) may be treated after electrospinning to regenerate the original polymer or to be derivatized to yield further new functionality. The polymers used in the present invention are typically cellulose. Cellulose is often used because it is readily available, inexpensive, biodegradable, biocompatible, and has a hydrophilic surface that results in low non-specific binding.

[0061] The polymer fibers may be crosslinked by covalent bonds. Once the electrospun fiber network is created, the fibers can fuse together at the points where the fibers cross each other, by heat, chemical, or other means. This results in improved layer-making characteristics by hand.

[0062] The fibers may have a diameter of 10 nm to 1000 nm. The fibers may have a diameter of 200 nm to 800 nm, and further may have a diameter of 300 nm to 400 nm. Fibers of this size result in an improvement in the pore size and the consistency of the particle size distribution.

[0063] The fibers may have an average length greater than 10 cm. Fibers produced by electrospinning are typically much longer than the fibers found in conventional chromatography media. Longer fibers result in improved lamination characteristics. In some cases, including where electrospinning emits fibers from a single source, a single continuous fiber can be produced, and a membrane formed of only this fiber, or a few (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) long fibers, can be formed.

[0064] The pores of the stationary phase may have a diameter of 10 nm to 10 μm, often may have a diameter of 25 nm to 5 μm, and may have a diameter of 50 nm to 2 μm. The use of pore diameters within these size ranges can help minimize clogging of the chromatographic medium and reduce product loss due to polarization, concentration, and exclusion at the particle interface. However, the pores remain small enough to minimize the loss of target components passing through the membrane without contacting the medium. The selection of these pore diameters ensures good utilization of capacity and a sharper breakthrough curve. For membrane structures with similar pore diameters, it has previously been shown that fibrous structures have 1.5 to 2 times higher permeability to aqueous flow than typical membranes produced by phase inversion. This is due to the relatively high surface porosity resulting from the electrospinning process. It has previously been shown that a non-woven fiber membrane with an average fiber diameter of 300 nm contains an average pore diameter of approximately 500 nm and results in a further porosity of 49%. It is possible to achieve a sufficiently high level of surface porosity distribution, similar to that expected for typically formed membranes, where the relationship between decreasing pore diameter and decreasing surface porosity is dominant, with an electrospun fiber membrane.

[0065] The pores may have a narrow size distribution, where the standard deviation of the pore diameter is preferably 250 nm or less. The uniformity of the pore diameter has been shown to affect axial diffusion, radial diffusion, and adsorption kinetics, as well as an important performance coefficient in chromatography (especially affinity chromatography) such as the sharpness of the breakthrough curve (BTC).

[0066] Fibers such as those disclosed above are functionalized before the ligand is immobilized thereon. The preparation of a convection-based chromatographic matrix is disclosed, for example, in WO / 2019 / 137869 and WO / 2013 / 068741, which are hereby incorporated by reference in their entirety.

[0067] In the present disclosure, "membrane" is often used interchangeably with "porous support". Whenever "membrane" is used, it should be clear that it refers to a porous support as disclosed above. The ligand density achieved on the porous support is at least 20 mg ligand / ml porous support, or at least 25 mg ligand / ml porous support, such as 30 mg ligand / ml porous support, such as at least 35 mg ligand / ml porous support, or at least 40 mg ligand / ml porous support, or at least 45 mg ligand / ml porous support, or at least 50 mg / ml porous support.

[0068] The separation matrix of the present invention has a dynamic binding capacity (DBC) of 25 g / mL of a kappa light chain-containing antibody, such as trastuzumab, with 10% breakthrough when flowed at a flow rate of 10 mL / min in a 0.4 mL HiTrap™ device. According to a preferred embodiment, the separation matrix of the present invention has a dynamic binding capacity (DBC) of 45 g / mL of a kappa light chain-containing antibody, such as trastuzumab, with 10% breakthrough when flowed at a flow rate of 10 mL / min in a 0.4 mL HiTrap™ device.

[0069] The present invention also relates to a method for the isolation of kappa light chain binding proteins. The method is preferably carried out using a separation matrix as disclosed above. The isolation can be from a cell culture or other liquid containing the kappa light chain binding protein.

[0070] Thus, the present disclosure provides a) contacting a liquid sample containing a kappa light chain-containing protein with a separation matrix; b) washing the separation matrix with one washing liquid or a combination of several washing liquids; c) eluting the kappa light chain-containing protein from the separation matrix using an eluent; and d) cleaning the separation matrix using a cleaning liquid and provides a method for isolating a kappa light chain-containing protein.

[0071] The separation matrix has a dynamic binding capacity (DBC) for kappa light chain-containing proteins, such as trastuzumab, of 28 g / mL with 10% breakthrough when flowed at a flow rate of 10 mL / min in a 0.4 mL HiTrap™ device. According to one embodiment, a separation matrix as disclosed above is used in the method.

[0072] The production of bispecific antibodies or IgG molecules is difficult because the pairing of the light and heavy chains, and as a result the pairing of the variable domains (VL; VH) therein, can be random. Usually, only one specific asymmetric combination is desired, and many of the combinations achieved are non-functional or undesirable molecules, such as monospecific homodimers, so the pairing of two different light chains and two different heavy chains can result in a large number of mispairings. Therefore, there is an increasing need for improved tools and methods for separating bispecific antibodies from monospecific antibodies and for separating mismatched bispecific antibodies from correctly matched bispecific antibodies. This can be done by separation based on the different light chains contained in the bispecific antibody.

[0073] According to yet another aspect, a) contacting a liquid sample containing a kappa light chain-containing protein with a separation matrix; b) washing the separation matrix with one washing liquid or a combination of several washing liquids; c) eluting the kappa light chain-containing protein from the separation matrix using an eluent; and d) cleaning the separation matrix using a cleaning liquid is provided herein a method for separating bispecific antibodies, comprising.

[0074] The separation matrix has a dynamic binding capacity (DBC) of 28 g / mL at 10% breakthrough for kappa light chain-containing proteins, such as trastuzumab, when flowed at a flow rate of 10 mL / min in a 0.4 mL HiTrap™ device.

[0075] According to one embodiment, the separation matrix disclosed above is used in the method.

[0076] The elution step can be carried out using a volume gradient. This is shown in Example X. Alternatively, the elution step can be carried out using a pH gradient.

[0077] This method enables the separation of bispecific antibodies from monospecific or mismatched antibodies based on the presence of kappa light chains. Monoclonal antibodies have two identical kappa light chains. Bispecific antibodies can be designed to contain two different kappa light chains.

[0078] Any antibody that does not contain a kappa light chain, such as two lambda light chains, does not bind to the separation matrix and as a result is present in the effluent stream during step a) or washed away during step b). Any antibody that has at least one kappa light chain binds to the separation matrix. Upon elution, antibodies that have only one kappa light chain elute before antibodies that have two kappa light chains.

[0079] By using the disclosed separation matrix that binds to subclasses 1, 3, or 4 kappa light chains, it is also possible to separate an antibody or antibody fragment containing one or two kappa light chains of subclass 2 from an antibody or antibody fragment containing one or two kappa light chains of subclass 1, 3, or 4. By the same principle as above, an antibody containing two kappa light chains of subclass 2, or an antibody containing one kappa light chain of subclass 2 and a lambda light chain, does not bind to the separation matrix and as a result, is present in the effluent stream during step a) or is washed away during step b). An antibody having one kappa light chain of subclass 2 and one kappa light chain of any of subclasses 1, 3, or 4 elutes before an antibody having two kappa light chains of any of subclasses 1, 3, and 4.

Example

[0080] The general method for preparing the materials used in the following examples was 1: obtaining a substrate formed by one or more polymer fibers (cellulose acetate (CA)) 2: subjecting CA to glycidol polymerization and saponification 3: functionalizing the material of step 2 with divinyl sulfone (DVS) 4: reducing and desalting the kappa light chain binding ligand, immobilizing the reduced kappa light chain binding ligand on the material obtained in step 3 on a shaker washing the material with the kappa light chain binding ligand immobilized thereon immobilizing the kappa light chain binding ligand on the material obtained in step 3, including 5: inactivating the remaining vinyl groups using 1-thioglycerol 6: washing the material to remove the unbound kappa light chain binding ligand as much as possible is.

[0081] Unless otherwise specified herein, the steps were carried out according to the methods disclosed in Example 6 of WO / 2019 / 137869 and WO / 2018 / 011600.

[0082] Protein reduction Sodium sulfate (1.4 M) was added to the protein solution, and when all had dissolved, dithiothreitol (DTT, 0.1 M) was added. The reaction mixture was placed on a shaker (23 °C, 500 rpm) and left to reduce overnight.

[0083] Desalting Desalting was carried out by using a prepacked PD10 column containing Sephadex-G-25 medium. Before loading the protein (maximum 2.5 mL), the column was equilibrated with four column volumes of degassed desalting solution (0.15 NaCl, 1 mM EDTA). The eluted fractions were collected and combined.

[0084] The desalted solution was diluted 20-fold with desalting solution, the absorbance at 276 nm was measured, and corrected by the blank of the desalting buffer. By using the determined protein concentration, it was then possible to calculate the desired amount of desalted protein solution to be used during immobilization.

[0085] For immobilization, a solution with a concentration of 25 - 30 mg ligand / ml was used and added to the activated material according to the method referred to above. Depending on the temperature, pH and reaction time for ligand immobilization, 20 mg ligand to at least 50 mg ligand per 1 ml of the porous support could be immobilized. Longer reaction times such as in Example 6 of WO / 2019 / 137869 and WO / 2018 / 011600 generally result in higher ligand densities than shorter reaction times such as in Example 5 of the same disclosure.

[0086] (Example 1) Dynamic binding capacity The separation matrix according to the present invention was prepared as described above and the dynamic binding capacity was analyzed.

[0087] Sample preparation 0.5 mg / mL of IgG in 500 mL or 250 mL of 20 mM Tris-HCl, 150 mM NaCl, pH 7.5 (binding buffer) was prepared by diluting 1.515 mL or 0.758 mL of Gammanorm (165 mg / mL) in the binding buffer and adjusting the volume to 500 mL or 250 mL in a volumetric flask. Immediately before analysis, the Gammanorm solution was filtered using a Sterivex filter. After filtration, the concentration was checked by measuring the UV absorbance at 280 nm. Beer's law: A = εLc was used to calculate the IgG concentration, where A is the UV absorbance at 280 nm, ε is the extinction coefficient (ε is 1.38 ml / (mg×cm) for polyclonal IgG), L is the path length of the cell holder, and c is the concentration of the solution.

[0088] 3.4 mL of purified trastuzumab (30 g / L) was diluted in a total volume of 200 mL with 20 mM phosphate, 150 mM NaCl, pH 7.2. The concentration was determined by UV measurement at 280 nm using a 96-well UV plate, 200 μL / well. The blank was 20 mM phosphate, 150 mM NaCl, pH 7.2. The trastuzumab concentration was calculated using a extinction coefficient of 1.48.

[0089] dAB is a domain antibody containing a variable light chain and was generated according to the method described in the paper "Recombinant production of a V L single domain antibody in Escherichia coli and analysis of its interaction with Peptostreptococcal protein L" (Protein Expression and Purification, Vol. 51, No. 2, February 2007, pp. 253-259).

[0090] 3.45 mL of purified dAb (14.5 g / L) was diluted in a total volume of 100 mL with 20 mM phosphate 150 mM NaCl pH 7.2. The concentration was determined by UV measurement at 280 nm using a 96-well UV plate, 200 μL / well. The blank was 20 mM phosphate 150 mM NaCl pH 7.2. The dAb concentration was calculated using an extinction coefficient of 1.6.

[0091] As disclosed in Example 6 of WO / 2019 / 137869 and WO / 2013 / 068741, the ligand was coupled to a porous support.

[0092] [Table 1]

[0093] The membrane single disk of the porous support coupled with the ligand was attached to the device and connected to a chromatography system AKTAavant 25 IP31154, 10 mm cell. The membrane was equilibrated with equilibration / binding buffer before loading the protein sample with either Gammanorm or trastuzumab respectively. The volume at 10% breakthrough (Qb10%) was calculated and reported. The flow rate used during the equilibration and washing of the membrane was 20 mL / min. The flow rate was decreased to 10 mL / min during protein loading. The analysis was performed with a double membrane disk and each disk was analyzed twice. The first run was a blank without protein loading, followed by two front-end analysis runs with protein.

[0094] The breakthrough volume was calculated using Exctensions-DBC Calculations-Analyze, an evaluation tool in the Unicorn software used in AKTAavant 25 IP31154.

[0095] For the calculation of the breakthrough capacity (Qb10) at 10%, the following equation was used. This is, namely, the amount of trastuzumab loaded onto the column until the concentration of trastuzumab in the column effluent becomes 10% of the trastuzumab concentration in the liquid sample.

[0096]

Number

[0097] A 100% = 100% UV signal A sub = Absorbance contribution from unbound mAb A(V) = Absorbance at a given applied volume V C = Column volume V app = Volume applied until 10% breakthrough V sys = Dead volume of the system C0 = Liquid sample concentration

[0098] For all experiments, the sample solution contained 0.5 mg of protein.

[0099]

Table 2

[0100] The DBC decreased between runs 1 and 2, increased again for run 3, and then decreased again for run 4. This is due to the fact that no blank runs were performed between analyses 1 and 2 and between analyses 3 and 4, respectively.

[0101]

Table 3

[0102] In this experiment, a blank run was added between two runs, Run 1 and Run 2. This indicates that the blank run should be carried out between each run.

[0103] Without being bound by any theory, the higher DBC achieved in Test 2 may be due to the fact that CIP with 0.1M NaOH was carried out on the material before Test 2, while Test 1 was carried out without a prior CIP wash.

[0104] [Table 4]

[0105] [Table 5]

[0106] Next, the DBC of the prototype according to the present invention was compared with a commercial product having the same convection-based matrix. As shown in Figure 1, the matrix was included in the HiTrap™ device. The HiTrap™ device (1) has a volume of 0.4 mL. The device (1) includes two stacked Fibro prototype membranes (2) having a non-woven material layer (3) as a filler in the middle. The disk diameter is 26.4 mm. Prototype 1 included a separation matrix with 52 mg ligand / ml membrane. Prototype 2 included a separation matrix with 34 mg ligand / ml membrane.

[0107] [Table 6]

[0108] As can be seen above, the prototype shows at least as good DBC as the commercial product Fibro™ PrismA for both Qb5 and Qb10.

[0109] (Example 2) Analysis between Fab fragment and trastuzumab based on volume gradient In the prototype according to the present invention, it was further investigated whether analysis between the Fab fragment and trastuzumab could be achieved.

[0110] The Fab fragment (Fab) was generated from trastuzumab by papain cleavage. The trastuzumab solution was adjusted to pH 7.4 by the addition of 0.5 M sodium phosphate and then diluted 1+1 in digestion buffer (25 mM Na phosphate, 1 mM EDTA, 5 mM mercaptoethanol, pH 7.5). The final volume was approximately 100 mL. Papain crystals were added to the solution. The solution was incubated overnight at 37 °C. Thereafter, antipain (papain inhibitor) was added to the digested trastuzumab. The solution was left at room temperature for 30 minutes and then applied onto a Capto(™) L HiScale 26 column to remove Fc-containing molecules (Fc or partially digested trastuzumab collected in the flow-through). Fab was collected during elution.

[0111] Fab and trastuzumab were separated using a linear pH gradient of pH 5.0 to 2.3 with 50 mM citrate buffer. Resolution experiments were performed with a linear gradient of 20 mL to 100 mL. For example, the gradient increased from 20 mL to 40, 60, and 100 mL with a run time of 5.5 to 13.5 minutes. The results are shown in Figures 2 to 5.

[0112] The results show that when the pH gradient slope is 20 mL, Fab and trastuzumab co-elute. Figure 2.

[0113] Regarding the separation between Fab and trastuzumab in the prototype, when the gradient is flat and when it increases up to 40 mL, the shoulder of the peak increases. Figure 3.

[0114] When the pH gradient slope is 60 mL, better separation between Fab and trastuzumab is obtained. Figure 4.

[0115] A better separation with a resolution of 0.88 between Fab and trastuzumab is achieved with Fibro at a pH gradient of 100 mL. See Figure 5 where the Fab peak is located on the left of the chromatogram and the trastuzumab peak is on the right.

Explanation of Signs

[0116] 1 HiTrap (trademark) device 2 Fibro prototype membrane 3 Non-woven material layer

Claims

**Claim 1** A separation matrix comprising a kappa light chain binding ligand coupled by a covalent bond to a porous support, wherein the kappa light chain binding ligand comprises, consists essentially of, or consists of a multimer of an alkali-stabilized Protein L domain of Finegoldia magna (formerly Peptostreptococcus magnus), and wherein the porous support is a convection-based chromatography matrix. **Claim 2** The separation matrix according to claim 1, wherein the convection-based chromatography matrix is a fibrous substrate. **Claim 3** The separation matrix according to claim 2, wherein the fibrous substrate is based on electrospun polymer fibers or cellulose fibers, optionally non-woven fibers. **Claim 4** The separation matrix according to claim 3, wherein the polymer is selected from the group consisting of cellulose, cellulose acetate, polysulfone, polyamide, polyacrylic acid, polymethacrylic acid, polyacrylonitrile, polystyrene, polyethylene oxide, and mixtures thereof. **Claim 5** The separation matrix according to claim 3 or 4, wherein the fibrous substrate is a fibrous non-woven polymer matrix. **Claim 6** The separation matrix according to any one of claims 2 to 5, wherein the fibers comprised in the fibrous substrate have a cross-sectional diameter of 10 to 1000 nm, such as 200 to 800 nm, 200 to 400 nm, or 300 to 400 nm. **Claim 7** The separation matrix according to any one of claims 2 to 6, wherein the ligand is bound to a divinylsulfone functional group coupled to a glycidol group grafted onto the fibrous substrate. **Claim 8** The separation matrix according to any one of claims 1 to 7, wherein the kappa light chain binding ligand comprises at least two alkali-stabilized Protein L domains. **Claim 9** The alkali-stabilized protein L domain is selected from the group consisting of functional variants of the B1 domain, B2 domain, B3 domain, B4 domain, B5 domain, C2 domain, C3 domain, C4 domain and D1 domain of Protein L from Finegoldia magna (formerly Peptostreptococcus magnus), and the corresponding positions in the alignment at positions 10 and 45 in the B2 domain (SEQ ID NO: 1) are histidine, and the corresponding position in the alignment at position 60 in the B2 domain (SEQ ID NO: 1) is tyrosine or glutamine. The isolated matrix according to claim 8.

10. The isolated matrix according to claim 9, wherein the alkali-stabilized protein L domain is selected from the group consisting of the B2 domain, B3 domain, B4 domain, C2 domain, C3 domain, C4 domain and D1 domain.

11. The alkali-stabilized protein L domain is determined by the BLOSUM matrix of 75, using a gap opening penalty of 12 and a gap extension penalty of 3, and has at least 90%, 95% or 98% sequence identity, or 77.5% sequence similarity with any one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19 of the amino acid sequence, and the corresponding positions in the alignment at positions 10 and 45 in SEQ ID NO: 1, and the corresponding position in the alignment at position 60 in SEQ ID NO: 1 do not change. The isolated matrix according to any one of claims 8 to 10.

12. The alkali-stabilized protein L domain is determined by the 75 BLOSUM matrix using a 12 gap open penalty and a 3 gap extension penalty, and has at least 90%, 95% or 98% sequence identity, or 77.5% sequence similarity with any one of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID NO: 37 of the amino acid sequence, and the separated matrix according to any one of claims 8 to 10.

13. The ligand density is at least 20 mg / ml of the porous support, or at least 25 mg / ml of the porous support, or at least 30 mg / ml of the porous support, or at least 35 mg / ml of the porous support, or at least 40 mg / ml of the porous support, or at least 45 mg / ml of the porous support, or at least 50 mg / ml of the porous support, and the separated matrix according to any one of claims 1 to 12.

14. The separated matrix according to any one of claims 1 to 13, which has a dynamic binding capacity (DBC) of 25 g / ml of a kappa light chain-containing antibody, such as trastuzumab, with 10% breakthrough when flowed at a flow rate of 10 ml / min in a 0.4 mL HiTrap™ device.

15. The separated matrix according to any one of claims 1 to 14, which has a dynamic binding capacity (DBC) of 45 g / ml of a kappa light chain-containing antibody, such as trastuzumab, with 10% breakthrough when flowed at a flow rate of 10 ml / min in a 0.4 mL HiTrap™ device.

16. a) contacting a liquid sample containing a kappa light chain-containing protein with the separation matrix; b) washing the separation matrix with one washing solution or a combination of several washing solutions; c) eluting the kappa light chain-containing protein from the separation matrix using an eluent; and d) cleaning the separation matrix using a cleaning solution A method for isolating a kappa light chain-containing protein, comprising a separation matrix having a dynamic binding capacity (DBC) of 25 g / mL at 10% breakthrough when flowed at a flow rate of 10 mL / min in a 0.4 mL HiTrap™ device, for a kappa light chain-containing antibody, such as trastuzumab.

17. a) contacting a liquid sample containing a kappa light chain-containing protein with a separation matrix; b) washing the separation matrix with one washing solution or a combination of several washing solutions; c) eluting the kappa light chain-containing protein from the separation matrix using an eluent; and d) cleaning the separation matrix using a cleaning solution A method for separation for isolating a kappa light chain-binding protein derived from a lambda light chain-containing protein, comprising a separation matrix having a dynamic binding capacity (DBC) of 25 g / mL at 10% breakthrough when flowed at a flow rate of 10 mL / min in a 0.4 mL HiTrap™ device, for a kappa light chain-containing antibody, such as trastuzumab.

18. a) contacting a liquid sample containing a bispecific antibody with a separation matrix; b) washing the separation matrix with one washing solution or a combination of several washing solutions; c) eluting the bispecific antibody from the separation matrix using an eluent; and d) cleaning the separation matrix using a cleaning solution A method for separation for isolating a bispecific antibody from a monospecific antibody, comprising a separation matrix having a dynamic binding capacity (DBC) of 25 g / mL at 10% breakthrough when flowed at a flow rate of 10 mL / min in a 0.4 mL HiTrap™ device, for a kappa light chain-containing antibody, such as trastuzumab.

19. The method according to claim 17 or 18, wherein the separation is carried out by applying a volume gradient or a pH gradient in step c).

20. The method according to any one of claims 16 to 19, wherein the separation matrix is as described in any one of claims 1 to 15.

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