Kappa Light Chain Binding Convection Substrate

Abstract

The present invention relates to the isolation (isolation) and the isolation (separation) of a protein containing a kappa light chain. More specifically, it 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 the L domain of the alkali-stabilized Vinclorda (previously referred to as peptostreptococcus bovis) protein; and the porous support is a convection-based chromatographic matrix.

Description

Technical Field

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

[0002] Immunoglobulins and immunoglobulin fragments represent the most prevalent biopharmaceutical products produced or developed worldwide. The high commercial demand for, and therefore the value of, this specific therapeutic market has led to a focus on pharmaceutical companies to maximize the productivity of their respective production processes while controlling associated costs.

[0003] Affinity chromatography (typically on a matrix containing Staphylococcus Protein A or its variants) is commonly used as one of the key steps in the purification of intact immunoglobulin molecules. The highly selective binding of Protein A to the Fc chains of immunoglobulins provides a versatile procedure with very high clearance of impurities and contaminants.

[0004] For immunoglobulin fragments or antibody fragments lacking Fc chains but having 1, 3 or 4 subclass kappa light chains, such as Fab, single-chain variable fragment (scFv), bispecific T-cell engagers (BiTE), domain antibodies, etc., a matrix (B) comprising protein L derived from Finegoldia magna (formerly known as Peptoscoccus Magnus) L 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. Pat. No. 6,822,075) show great promise as a purification platform that provides the desired high selectivity.

[0005] Protein L matrices are commercially available, for example as TM Capto TML, and can be used to separate proteins containing κ light chains, such as complete antibodies, Fab fragments, scFv fragments, domain antibodies, etc. About 75% of antibodies produced by healthy people have κ light chains, and about 90% of therapeutic monoclonal antibodies and antibody fragments contain κ light chains (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 bioprocess chromatography application requires comprehensive attention to the clear 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 the chromatographic procedure, such as undesirable biomolecules or microorganisms, including, for example, proteins, carbohydrates, lipids, bacteria and viruses. Removal of such impurities and / or contaminants from the matrix is ​​usually carried out after the first elution of the desired product, so that the matrix can be regenerated before subsequent use. This removal usually includes a procedure called cleaning in place (CIP), in which a reagent that can inactivate impurities or elute impurities from the stationary phase is used. A class of such reagents that are usually used with chromatographic media is an alkaline solution, which is passed on the matrix. At present, the most widely used cleaning and disinfecting agent is NaOH, and according to the degree and nature of contamination and impurities, and it is ideal to use NaOH in a concentration ranging from 0.05M to, for example, 1M. However, compared with, for example, protein A, protein L is a protein that is quite sensitive to alkali, and only tolerates up to about 15mM NaOH in many cycles. This means that, to ensure adequate cleaning, additional, less ideal cleaning solutions, such as urea or guanidine salts, may have to be used.

[0007] Therefore, there remains a need in the art to obtain separation matrices containing Protein L-derived ligands having improved stability towards alkaline cleaning procedures. Summary of the invention

[0008] The present inventors have achieved a solution to the above-mentioned problems by providing a separation matrix comprising a κ light chain binding ligand covalently coupled to a porous support, wherein the κ light chain binding ligand comprises, consists essentially of, or consists of a polymer of the alkali-stable Peptostreptococcus major (formerly known as Peptostreptococcus major) protein L domain; and the porous support is a convection-based chromatography matrix.

[0009] The convection-based chromatography matrix may be a fibrous substrate. The fibrous substrate may be based on electrospun polymer fibers or cellulose fibers, optionally based on nonwoven fibers.

[0010] The polymer may be selected from 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 nonwoven polymer matrix.

[0011] The fibers contained in the fibrous substrate may have a cross-sectional diameter of 10-1000 nm, for example, 200-800 nm, 200-400 nm, or 300-400 nm.

[0012] The ligand may be bound to a divinyl sulfone functional group coupled to a glycidyl group grafted onto the fiber substrate.

[0013] The κ light chain binding ligand may include at least two alkali-stable protein L domains. The alkali-stable protein L domain may be selected from the 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 of large Fingold's bacteria (formerly known as large peptostreptococcus), wherein the positions corresponding to positions 10 and 45 in the B2 domain (SEQ ID NO 1) in the alignment are histidine, and the position corresponding to position 60 in the B2 domain (SEQ ID NO 1) in the alignment is tyrosine or glutamine. In one embodiment, the alkali-stable protein L domain is selected from B2 domain, B3 domain, B4 domain, C2 domain, C3 domain, C4 domain and D1 domain.

[0014] The alkali-stable protein L domain may have at least 90%, 95% or 98% sequence identity or 77.5% sequence similarity to any of the amino acid sequences 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, as determined by a BLOSUM matrix of 75, wherein the gap opening penalty is 12 and the gap extension penalty is 3, wherein the positions corresponding to positions 10 and 45 in the alignment and the position corresponding to position 60 in the alignment are invariant. The alkali-stable protein L domain may have at least 90%, 95% or 98% sequence identity or 77.5% sequence similarity to any of the amino acid sequences 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, as determined by a BLOSUM matrix of 75, wherein the gap opening penalty is 12 and the gap extension penalty is 3.

[0015] The ligand density of the separation matrix can be at least 20 mg / ml porous support, or at least 25 mg / ml porous support, or at least 30 mg / ml porous support, or at least 35 mg / ml porous support, or at least 40 mg / ml porous support, or at least 45 mg / ml porous support, or at least 50 mg / ml porous support.

[0016] The separation matrix was used in 0.4 mL HiTrap TM The device can have a dynamic binding capacity (DBC) of 25 g / mL at 10% breakthrough for a kappa light chain-containing antibody, such as trastuzumab, when run at a flow rate of 10 mL / min.

[0017] The separation matrix was used in 0.4 mL HiTrap TM The device can have a dynamic binding capacity (DBC) of 45 g / mL at 10% breakthrough for a kappa light chain-containing antibody, such as trastuzumab, when run at a flow rate of 10 mL / min.

[0018] In addition, a method for separating proteins containing κ light chains is provided, the method comprising the following steps: a) contacting a liquid sample containing proteins containing κ light chains with a separation matrix; b) washing the separation matrix with a washing solution or a combination of washing solutions; c) eluting the proteins containing κ light chains from the separation matrix with an elution solution; and d) cleaning the separation matrix with a cleaning solution; wherein when the separation matrix is ​​heated to 0.4 mL HiTrap TM The separation matrix has a dynamic binding capacity (DBC) of 25 g / mL for a kappa light chain-containing antibody, such as trastuzumab, at 10% breakthrough when run at a flow rate of 10 mL / min in the device.

[0019] In addition, a method for separating a κ light chain binding protein from a λ light chain-containing protein is provided, the method comprising the following steps: a) contacting a liquid sample containing a κ light chain-containing protein with a separation matrix; b) washing the separation matrix with a washing solution or a combination of several washing solutions; c) eluting the κ light chain-containing protein from the separation matrix with an elution solution; and d) cleaning the separation matrix with a cleaning solution; wherein when the separation matrix is ​​heated to 0.4 mL HiTrap TM The separation matrix has a dynamic binding capacity (DBC) of 25 g / mL for a kappa light chain-containing antibody, such as trastuzumab, at 10% breakthrough when run at a flow rate of 10 mL / min in the device.

[0020] In addition, a method for separating bispecific antibodies from monospecific antibodies is provided, the method comprising the following steps: a) contacting a liquid sample containing the bispecific antibodies with a separation matrix; b) washing the separation matrix with a washing solution or a combination of washing solutions; c) eluting the bispecific antibodies from the separation matrix with an elution solution; and d) cleaning the separation matrix with a cleaning solution; wherein when the bispecific antibodies are separated from the separation matrix in 0.4 mL HiTrap TM The separation matrix has a dynamic binding capacity (DBC) of 25 g / mL for a kappa light chain-containing antibody, such as trastuzumab, at 10% breakthrough when run at a flow rate of 10 mL / min in the device.

[0021] In any of the separation methods disclosed above, the separation is performed by applying a volume gradient or a pH gradient in step c).

[0022] In any of the methods according to above, the separation matrix may be as described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic depiction of the HiTrap used in Example 1 for DBC comparison between the prototype and the commercial product TM Device(Cytiva TM ).

[0024] Figure 2 Fab and trastuzumab chromatograms are shown for a pH gradient ramp of 20 mL.

[0025] Figure 3 Fab and trastuzumab chromatograms are shown for a pH gradient ramp of 40 mL.

[0026] Figure 4 Fab and trastuzumab chromatograms are shown for a pH gradient ramp of 60 mL.

[0027] Figure 5 Fab and trastuzumab chromatograms are shown for a pH gradient ramp of 88 mL.

[0028] definition

[0029] The terms "antibody" and "immunoglobulin" are used interchangeably herein and refer to an antigen binding protein having a basic four polypeptide chain structure consisting of two heavy (H) chains and two light (L) chains, which are 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. The term should be understood to include any antibody, including but not limited to monoclonal antibodies and bispecific antibodies, as well as antibody fragments, fusion proteins comprising antibodies or antibody fragments, and conjugates comprising antibodies or antibody fragments.

[0030] The terms "κ light chain binding polypeptide" and "κ light chain binding protein" are used herein to refer to a κ light chain that is capable of binding to subclass 1, 3, or 4 of an antibody (also known as a V κI 、V κIII and V κIV , such as in BH K Nilson et al.: J. Biol. Chem. 267, 2234-2239, 1992), and includes, for example, protein L and any variants, fragments or fusion proteins thereof that have retained the binding properties.

[0031] The term "κ light chain-containing protein" is used as a synonym for "immunoglobulin κ light chain-containing protein" and refers herein to a protein containing 1, 3 or 4 subclass κ light chains (also called V κI 、V κIII and V κIV , as in BH KNilson et al.: J. Biol. Chem. 267, 10

[0032] 2234-2239, 1992), and includes any whole antibody, antibody fragment, fusion protein, conjugate or recombinant protein containing 1, 3 or 4 subclass kappa light chains.

[0033] The term "mAb" stands for monoclonal antibody.

[0034] The term "Fab" stands for an antigen-binding fragment from an immunoglobulin comprising a kappa light chain or a lambda light chain.

[0035] The term "bispecific antibody" represents an antibody that can bind to two different types of antigens or two different epitopes on the same antigen. Similarly, a trispecific antibody represents an antibody that can bind to three different types of antigens or three different epitopes on the same antigen.

[0036] "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 given flow conditions before significant breakthrough of unbound protein occurs. DBC is determined by loading a sample containing a known concentration of the target protein and monitoring the flow-through. The protein will bind to the resin to a certain inflection point, and then unbound protein will flow through the column.

[0037] DBC can be determined on the breakthrough curve at the loss of, for example, 10% protein. This is referred to as the Qb10% value, or simply Qb10%. The sample is applied to the chromatographic resin column during a specific residence time, and the dynamic binding capacity of each resin is calculated at 10% protein breakthrough (i.e., the amount of the target sample loaded onto the column until the concentration of the target sample in the column effluent is 10% of the target sample concentration in the liquid sample). If the dynamic binding capacity of each resin is calculated at 80% breakthrough capacity, this is referred to as the Qb80% value.

[0038] As used herein, the term "liquid sample" refers to a liquid containing at least one target substance that is sought to be purified from other substances that also exist.Liquid samples can be, for example, aqueous solutions, organic solvent systems, or aqueous / organic solvent mixtures or solutions.Source liquids are typically complex mixtures or solutions containing many biomolecules (such as proteins, antibodies, hormones, and viruses), small molecules (such as salts, sugars, lipids, etc.) and even particulate matter.Although typical source liquids of biological origin can start as aqueous solutions or suspensions, they can also contain organic solvents for earlier separation steps such as solvent precipitation, extraction, etc.The example of a liquid sample that can contain valuable biological substances that are suitable for purification by various embodiments of the present invention includes, but is not limited to, culture supernatants from bioreactors, homogenized cell suspensions, blood plasma, plasma fractions, and milk.Alternately, liquid samples can be referred to as "clarified cell culture feed" or "CCF."

[0039] "Buffer" is a substance that, due to its presence in a solution, increases the amount of acid or base that must be added to cause a unit pH change. Buffer solutions resist changes in pH through the action of their acid-base conjugate components. Buffer solutions used with biological reagents are generally able to maintain a constant 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). Therefore, the physiological pH range is about 7.2 to 7.6. Traditional buffer components include, but are not limited to, organic and inorganic salts, acids and bases. Exemplary buffers for purifying biomolecules (e.g., protein molecules) include zwitterions or "Good" buffers, see, for example, Good et al. (1966) Biochemistry 5:467 and Good and Izawa (1972) Methods Enzymol.24:62.

[0040] "Equilibration buffer" is a buffer used to prepare the binding reagent, solid phase, or both, for loading the source liquid containing the target protein. The equilibrium 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 the source liquid or liquid sample containing the protein containing the binding region and impurities onto the solid phase on which the binding agent is immobilized. Typically, the equilibrium buffer and the loading buffer are the same.

[0041] As used herein, "washing liquid" or "washing buffer" refers herein to a liquid used to remove impurities from a chromatography resin bound to a target substance. More than one washing liquid may be used sequentially, for example, successive washing liquids having different properties such as pH, conductivity, solvent concentration, etc., which are designed to dissociate and remove different types of impurities that are non-specifically bound to the chromatography resin.

[0042] "Eluent" or "elution buffer" are used interchangeably herein and refer herein to a liquid used to dissociate the target substance from the chromatography resin, thereby eluting the protein containing the binding zone from the immobilized binding agent after washing with one or more washing liquids. The eluent is used to dissociate the target substance without irreversibly denaturing it. Typical eluents are well known in the field of chromatography and may have different pHs (typically, lower pH), higher concentrations of salts, free affinity ligands or the like, or other substances that promote the dissociation of the target substance from the chromatography resin. "Elution conditions" refer to the process conditions applied to the chromatography resin bound to the target substance to cause the target substance to dissociate from the chromatography resin, such as contacting the chromatography resin bound to the target substance with an eluent or elution buffer to produce such dissociation.

[0043] Preferably, the elution buffer has a low pH, thereby disrupting the interaction between the kappa light chain binding separation matrix and the target protein. Preferably, the pH of the low pH elution buffer is in the range of about 2 to about 5, most preferably in the range of about 3 to about 4. Examples of buffers that control the pH within this range include glycine, phosphate, acetate and citrate buffers, and combinations of these. Preferred such buffers are citrate and acetate buffers, most preferably sodium citrate or sodium acetate buffers.

[0044] The cleaning solution can be an acidic solution or an alkaline solution for removing resin residues after eluting the target substance. For example, precipitated proteins, hydrophobic proteins, nucleic acids, endotoxins and viruses can be removed by the cleaning solution. Most commonly, alkaline solutions are used for this purpose.

[0045] Cleaning in place (CIP) is an important process for the efficient use of chromatography columns. In order to maximize the number of cycles that a column can be reused, a cleaning procedure is required that effectively removes impurities without damaging the chromatography resin.

[0046] As used herein, the terms "comprises", "comprising", "containing", "having" and the like may mean "includes", "including", and the like; "consisting essentially of" or "consists essentially" is an open term, allowing for the existence of content other than the content, as long as the basic or novel features of the content are not changed by the existence of the content other than the content, but excluding prior art implementations. DETAILED DESCRIPTION

[0047] The inventors aimed to invent a separation matrix containing a ligand derived from protein L which has an improved stability towards alkaline cleaning procedures while maintaining satisfactory binding capacity and efficiency in terms of flow properties in a chromatographic setting and which is preferably superior to currently commercially available separation matrices.

[0048] According to one aspect, this object has been achieved by providing a separation matrix comprising a κ light chain binding ligand covalently coupled to a porous support, wherein the κ light chain binding ligand comprises, consists essentially of, or consists of a polymer of an alkali-stable Feingoldia (formerly known as Peptostreptococcus) protein L domain; and the porous support is a convection-based chromatography matrix.

[0049] The κ light chain binding ligand included in the separation matrix of the present invention comprises a polymer of the Fingoldeella protein L domain that is stable to alkali, is basically composed of it, or is composed of it. The protein L domain can be any functional protein L derived domain, as long as it is alkali-stable. The protein L domain is selected from the functional variants of B1 domain, B2 domain, B3 domain, B4 domain, B5 domain, C2 domain, C3 domain, C4 domain and D1 domain, wherein the position corresponding to the position 10 and 45 in the B2 domain (SEQ ID NO 1) in the comparison is histidine, and the position corresponding to the position 60 in the B2 wt domain (SEQ ID NO 1) in the comparison is tyrosine or glutamine. Therefore, the above-mentioned position corresponding to the position 10,45 and 60 in the B2 wt domain (SEQ ID NO 1) is immutable in the functional protein L domain.

[0050] SEQ ID NO:1 (wtB2)

[0051] PKEEVTIKANLIYADGKTQTAEFKGTFEEAAAEAYRYADALKKDNGEYTVDVADKGYTLNIKFAGKEKTPEE

[0052] Examples of such functional protein L domains may be:

[0053] SEQ ID NO: 2 (B2: N10H, N45H, N60Y mutations)

[0054] PKEEVTIKAHLIYADGKTQTAEFKGTFEEAAAEAYRYADALKKDHGEYTVDVADKGYTLYIKFAGKEKTPEE

[0055] SEQ ID NO: 3 (B2: N10H, N45H, N60Q mutations)

[0056] PKEEVTIKAHLIYADGKTQTAEFKGTFEEAAAEAYRYADALKKDHGEYTVDVADKGYTLQIKFAGKEKTPEE

[0057] SEQ ID NO:4 (B3: N10H, N45H, N60Y mutation)

[0058] PKEEVTIKAH LIYADGKTQT AEFKGTFEEA TAEAYRYADLLAKEHGKYTV DVADKGYTLYIKFAGKEKTP EE

[0059] SEQ ID NO:5 (B3: N10H, N45H, N60Q mutation)

[0060] PKEEVTIKAH LIYADGKTQT AEFKGTFEEA TAEAYRYADLLAKEHGKYTV DVADKGYTLQIKFAGKEKTP EE

[0061] SEQ ID NO:6 (B1: N10H, N45H, N60Y mutation)

[0062] SEEEVTIKAHLIFANGSTQTAEFKGTFEKATSEAYAYADTLKKDHGEYTVDVADKGYTLYIKFAGKEKTPEE

[0063] SEQ ID NO:7 (B1: N10H, N45H, N60Q mutation)

[0064] SEEEVTIKAHLIFANGSTQTAEFKGTFEKATSEAYAYADTLKKDHGEYTVDVADKGYTLQIKFAGKEKTPEE

[0065] SEQ ID NO:8 (B4: N10H, N45H, N60Y mutation)

[0066] PKEEVTIKAHLIYADGKTQTAEFKGTFAEATAEAYRYADLLAKEHGKYTADLEDGGYTIYIRFAGKKVDEKPEE

[0067] SEQ ID NO:9 (B4: N10H, N45H, N60Q mutation)

[0068] PKEEVTIKAHLIYADGKTQTAEFKGTFAEATAEAYRYADLLAKEHGKYTADLEDGGYTIQIRFAGKKVDEKPEE

[0069] SEQ ID NO 10 (B5: N9H, N44H, N59Y mutation)

[0070] KEQVTIKEHIYFEDGTVQTATFKGTFAEATAEAYRYADLLSKEHGKYTADLEDGGYTIQIRFAGKEEPEE

[0071] SEQ ID NO 11 (B5: N9H, N44H, N59Q mutation)

[0072] KEQVTIKEHIYFEDGTVQTATFKGTFAEATAEAYRYADLLSKEHGKYTADLEDGGYTIQIRFAGKEEPEE

[0073] SEQ ID NO 12 (C2b N57Y: N10H, N45H, N60Y mutation)

[0074] PKEEVTIKVHLIFADGKTQTAEFKGTFEEATAKAYAYADLLAKEHGEYTADLEDGGYTIYIKFAGKETPETPEE

[0075] SEQ ID NO 13 (C2b N57Y: N10H, N45H, N60Q mutation)

[0076] PKEEVTIKVHLIFADGKTQTAEFKGTFEEATAKAYAYADLLAKEHGEYTADLEDGGYTIQIKFAGKETPETPEE

[0077] SEQ ID NO 14 (C3b N39D, N57Y: N10H, N45H, N60Y mutation)

[0078] PKEEVTIKVHLIFADGKIQTAEFKGTFEEATAKAYAYADLLAKEHGEYTADLEDGGYTIYIKFAGKETPETPEE

[0079] SEQ ID NO 15 (C3b N39D, N57Y: N10H, N45H, N60Q mutation)

[0080] PKEEVTIKVHLIFADGKIQTAEFKGTFEEATAKAYAYADLLAKEHGEYTADLEDGGYTIQIKFAGKETPETPEE

[0081] SEQ ID NO: 16 (C4: N10H, N45H, N60Y mutations)

[0082] PKEEVTIKVHLIFADGKTQTAEFKGTFEEATAEAYRYADLLAKVHGEYTADLEDGGYTIYIKFAGKEQPGENPG

[0083] SEQ ID NO: 17 (C4: N10H, N45H, N60Q mutations)

[0084] PKEEVTIKVHLIFADGKTQTAEFKGTFEEATAEAYRYADLLAKVHGEYTADLEDGGYTIQIKFAGKEQPGENPG

[0085] SEQ ID NO: 18 (D1: N10H, N45H, N60Y mutations)

[0086] PKEEVTIKAHLIFADGKTQTAEFKGTFEEATAEAYRYADLLAKVHGEYTADLEDGGYTIYIKFAGKEQPGEN

[0087] SEQ ID NO: 19 (D1: N10H, N45H, N60Q mutations)

[0088] PKEEVTIKAHLIFADGKTQTAEFKGTFEEATAEAYRYADLLAKVHGEYTADLEDGGYTIQIKFAGKEQPGEN

[0089] Preferably, the protein L domain is selected from the group consisting of a B3 domain, a C2 domain, a C3 domain and a D domain, wherein the positions corresponding to positions 10 and 45 in the B2 wt domain (SEQ ID NO 1) in the alignment are histidines, and the position corresponding to position 60 in the B2 wt domain (SEQ ID NO 1) in the alignment is tyrosine or glutamine. The above positions corresponding to positions 10, 45 and 60 in the B2 wt domain (SEQ ID NO 1) are invariable within the functional protein L domain.

[0090] The remaining positions in the L domain of this functional protein can be changed, as long as the three-dimensional structure is not changed compared to the B2 wt domain (SEQ ID NO 1), and at least the kappa light chain binding capacity is retained and it is alkali stable compared to the B2 wt domain (SEQ ID NO 1). The change can be a conservative amino acid substitution for an amino acid with similar or identical charge, hydrophobicity, etc., and a technician can determine what this change of amino acid can be.

[0091] The protein L domain may have at least 90%, 95% or 98% sequence identity or 77.5% sequence similarity to any of the amino acid sequences 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, as determined by a BLOSUM matrix of 75, with a gap open penalty of 12 and a gap extension penalty of 3.

[0092] The functional protein L domain can be a truncated sequence. For example, the position corresponding to position 1-4 in the B2 wt domain (SEQ ID NO 1) can be deleted. For example, the position corresponding to the position after position 65 in the B2 wt domain (SEQ ID NO 1) can be deleted.

[0093] SEQ ID NO:20 (B1_trunc, N6H, N41H, N56Y mutations)

[0094] VTIKAHLIFANGSTQTAEFKGTFEKATSEAYAYADTLKKDHGEYTVDVADKGYTLYIKFAG

[0095] SEQ ID NO:21 (B1_trunc, N6H, N41H, N56Q mutations)

[0096] VTIKAHLIFANGSTQTAEFKGTFEKATSEAYAYADTLKKDHGEYTVDVADKGYTLQIKFAG

[0097] SEQ ID NO: 22 (B2_trunc, N6H, N41H, N56Y mutations)

[0098] VTIKAHLIYADGKTQTAEFKGTFEEAAAEAYRYADALKKDHGEYTVDVADKGYTLYIKFAG

[0099] SEQ ID NO:23 (B2_trunc, N6H, N41H, N56Q mutation)

[0100] VTIKAHLIYADGKTQTAEFKGTFEEAAAEAYRYADALKKDHGEYTVDVADKGYTLQIKFAG

[0101] SEQ ID NO:24 (B3_trunc, N6H, N41H, N56Y mutation)

[0102] VTIKAHLIYADGKTQTAEFKGTFEEATAEAYRYADLLAKEHGKYTVDVADKGYTLYIKFAG

[0103] SEQ ID NO:25 (B3_trunc, N6H, N41H, N56Q mutation)

[0104] VTIKAHLIYADGKTQTAEFKGTFEEATAEAYRYADLLAKEHGKYTVDVADKGYTLQIKFAG

[0105] SEQ ID NO:26 (B4_trunc, N6H, N41H, N56Y mutation)

[0106] VTIKAHLIYADGKTQTAEFKGTFAEATAEAYRYADLLAKEHGKYTADLEDGGYTIYIRFAG

[0107] SEQ ID NO:27 (B4_trunc, N6H, N41H, N56Q mutation)

[0108] VTIKAHLIYADGKTQTAEFKGTFAEATAEAYRYADLLAKEHGKYTADLEDGGYTIQIRFAG

[0109] SEQ ID NO:28 (B5_trunc, N6H, N41H, N56Y mutation)

[0110] VTIKEHIYFEDGTVQTATFKGTFAEATAEAYRYADLLSKEHGKYTADLEDGGYTIYIRFAG

[0111] SEQ ID NO:29 (B5_trunc, N6H, N41H, N56Q mutation)

[0112] VTIKEHIYFEDGTVQTATFKGTFAEATAEAYRYADLLSKEHGKYTADLEDGGYTIQIRFAG

[0113] SEQ ID NO:30 (C2b N57Y_trunc, N6H, N41H, N56Y mutation)

[0114] VTIKVHLIFADGKTQTAEFKGTFEEATAKAYAYADLLAKEHGEYTADLEDGGYTIYIKFAG

[0115] SEQ ID NO:31 (C2b N57Y_trunc, N6H, N41H, N56Q mutation)

[0116] VTIKVHLIFADGKTQTAEFKGTFEEATAKAYAYADLLAKEHGEYTADLEDGGYTIQIKFAG

[0117] SEQ ID NO:32 (C3b N39D, N57Y trunc, N6H, N41H, N56Y mutation)

[0118] VTIKVHLIFADGKIQTAEFKGTFEEATAKAYAYADLLAKEHGEYTADLEDGGYTIYIKFAG

[0119] SEQ ID NO:33 (C3b N39D, N57Y_trunc, N6H, N41H, N56Q mutation)

[0120] VTIKVHLIFADGKIQTAEFKGTFEEATAKAYAYADLLAKEHGEYTADLEDGGYTIQIKFAG

[0121] SEQ ID NO:34 (C4_trunc, N6H, N41H, N56Y mutation)

[0122] VTIKVHLIFADGKTQTAEFKGTFEEATAEAYRYADLLAKVHGEYTADLEDGGYTIYIKFAG

[0123] SEQ ID NO:35 (C4_trunc, N6H, N41H, N56Q mutations)

[0124] VTIKVHLIFADGKTQTAEFKGTFEEATAEAYRYADLLAKVHGEYTADLEDGGYTIQIKFAG

[0125] SEQ ID NO:36 (D1_trunc, N6H, N41H, N56Y mutations)

[0126] VTIKAHLIFADGKTQTAEFKGTFEEATAEAYRYADLLAKVHGEYTADLEDGGYTIYIKFAG

[0127] SEQ ID NO:37 (D1_trunc, N6H, N41H, N56Q mutations)

[0128] VTIKAHLIFADGKTQTAEFKGTFEEATAEAYRYADLLAKVHGEYTADLEDGGYTIQIKFAG

[0129] The protein L domain can have at least 90%, 95% or 98% sequence identity or 77.5% sequence similarity to any of the amino acid sequences 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, as determined by a BLOSUM matrix of 75, with a gap opening penalty of 12 and a gap extension penalty of 3.

[0130] As can be seen above, the C2 domain and 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.

[0131] As specified above, the κ light chain binding ligand included in the separation matrix comprises a polymer of an alkali-stabilized protein L domain, is essentially composed of it, or is composed of it. The polymer can include two, three, four, five, six, seven, eight or nine alkali-stabilized protein L domains. In alternative statements, the polymer can be a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer or nonamer. Preferably, the part comprises four, five, six or seven alkali-stabilized protein L domains, such as five or six alkali-stabilized protein L domains.

[0132] The polymer may further comprise a linker, a spacer or additional amino acids. Additional amino acids may, for example, be derived from the cloning process and expression of the ligand, or constitute residues from a cleavage signal transduction sequence. The technician will appreciate and understand that this additional amino acid may be changed without affecting the function of the kappa light chain binding function of the polymer.

[0133] The porous support comprises a convection-based chromatography matrix. The convection-based chromatography matrix can be a fibrous substrate. The fibrous substrate can be based on electrospun polymer fibers or cellulose fibers, optionally based on non-woven fibers, which in use form a stationary phase comprising a plurality of pores through which the mobile phase can penetrate. The fibrous substrate can therefore be a fibrous non-woven polymer matrix. Such a fibrous substrate can be obtained from Cytiva TM HiTrap Fibro TM Found in parts.

[0134] Convection-based chromatography support materials enable a combination of high flow rates and high binding capacity and therefore have benefits for use in, for example, mAb purification.

[0135] The polymer fibers may be nonwoven fibers. The use of randomly deposited fiber mat (nonwoven) structures may promote blocked flow, thereby preventing channeling.

[0136] The polymer fibers can be electrospun. Electrospinning provides fibers of consistent size and can be easily adjusted (e.g., by changing the properties of the atmosphere while spinning) to prepare fibers of different proportions. Electrospinning is a technique that also exhibits excellent distribution properties, particularly useful in producing layered membranes. Fiber mass transfer properties have been shown to be similar to those of monolithic structures, which allow flow rate-independent separations.

[0137] The polymer used in the present invention is not limited to any specific polymer and can be customized for a specific purpose. The polymer can be, for example, nylon, poly(acrylic acid), polyacrylonitrile, polystyrene, polysulfone, polyacrylonitrile, polycaprolactone, collagen, chitosan, agarose and polyethylene oxide and combinations thereof. In order to improve its suitability for electrospinning, the polymer can be derivatized to improve the solubility and / or other properties of the polymer. Derivatized polymers (e.g., polyethersulfone, cellulose acetate or poly(acrylonitrile-co-acrylic acid) can be treated after electrospinning to produce the original polymer or further derivatized to produce new functionality. The polymer used in the present invention is typically cellulose. Cellulose is typically used because it is readily available, cheap, biodegradable, biologically compatible, and has a hydrophilic surface, resulting in low nonspecific binding.

[0138] The polymer fibers may be covalently cross-linked. Once an electrospun network of fibers is formed, the fibers may be fused together at points where the fibers intersect each other by thermal, chemical or other methods. This results in improved manual handling characteristics.

[0139] 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 may even have a diameter of 300 nm to 400 nm. Fibers of this size give improved pore size consistency and size distribution.

[0140] The fiber may have an average length greater than 10 cm. The fiber produced by electrospinning is generally much longer than the fiber present in conventional chromatographic media. Longer fibers provide improved layering properties. In some cases, when electrospinning comprises fibers from a single source, a single continuous fiber and a membrane formed separately from the fiber, or a membrane formed from a small amount (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) of long fibers can be produced.

[0141] The diameter of the pores of the stationary phase can be 10nm to 10μm, typically 25nm to 5μm and can be 50nm to 2μm in diameter. Using pore sizes within these size ranges can help minimize fouling of the chromatographic medium and reduce product losses due to polarization, concentration and repulsion at the particle interface. However, the pores are kept small enough to minimize the loss of target components that pass through the membrane without contacting the medium. The selection of these pore sizes ensures good capacity utilization and sharper penetration curves. It has been previously demonstrated that for membrane structures with similar pore sizes, the permeability of the fiber structure to water flow is 1.5-2 times that of traditional membranes produced by phase inversion. This is due to the relatively high surface porosity produced by the electrospinning process. It has been shown previously that nonwoven fiber membranes with an average fiber diameter of 300nm contain an average pore size of about 500nm, but a porosity of 49% is obtained. By electrospinning fiber membranes, it is possible to achieve a high level of surface porosity with high distribution, which is much higher than what can be expected from traditionally formed membranes, because in their case the relationship of decreasing surface porosity with decreasing pore size is dominant.

[0142] The pores may have a narrow size distribution, wherein the standard deviation of the pore diameter is preferably less than or equal to 250 nm. Pore size uniformity is one of several factors (in addition to axial and radial diffusion and adsorption kinetics) that have been shown to have an impact on key performance factors in chromatography (particularly affinity chromatography), such as breakthrough curve (BTC) sharpness.

[0143] The fibers disclosed above are functionalized and the ligands are then immobilized thereon. The preparation of convection-based chromatography matrices is disclosed in, for example, WO / 2019 / 137869 and WO / 2013 / 068741, which are incorporated herein by reference in their entirety.

[0144] In the present disclosure, "membrane" is generally used interchangeably with porous support. It should be clear that whenever "membrane" is used, it refers to the porous support material 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.

[0145] The separation matrix of the present invention is used in 0.4 mL HiTrap TM The separation matrix of the present invention has a dynamic binding capacity (DBC) of 25 g / mL of an antibody containing a kappa light chain, such as trastuzumab, at 10% breakthrough when running at a flow rate of 10 mL / min in a 0.4 mL HiTrap flow cell.TM The device has a dynamic binding capacity (DBC) of 45 g / mL at 10% breakthrough for a kappa light chain-containing antibody, such as trastuzumab, when run at a flow rate of 10 mL / min.

[0146] The present invention also relates to a method for isolating a κ light chain binding protein. The method is preferably performed using a separation matrix as disclosed above. The separation may be from a cell culture or other fluid containing a κ light chain binding protein.

[0147] Therefore, the present disclosure provides a method for isolating a protein containing a kappa light chain, the method comprising the steps of:

[0148] a) contacting a liquid sample comprising a protein containing a kappa light chain with a separation matrix,

[0149] b) washing the separation matrix with a washing liquid or a combination of washing liquids,

[0150] c) eluting the kappa light chain-containing protein from the separation matrix using an eluent, and

[0151] d) cleaning the separation matrix with a cleaning solution.

[0152] The separation matrix was used in 0.4 mL HiTrap TM The device has a dynamic binding capacity (DBC) of 28 g / mL at 10% breakthrough for a protein containing kappa light chains, such as trastuzumab, when run at a flow rate of 10 mL / min. According to one embodiment, the separation matrix disclosed above is used in the method.

[0153] The generation of bispecific antibodies or IgG molecules is difficult because the pairing of light chain and heavy chain and therefore the variable domains (VL; VH) therein may be messy. The pairing of two different light chains and two different heavy chains may cause a large amount of mispairing, because usually only a specific asymmetric combination is desired, and the multiple combinations achieved will be non-functional or unwanted molecules, such as monospecific homodimers. Therefore, there is an increasing need for improved tools and methods for separating bispecific antibodies from monospecific antibodies and separating mispaired bispecific antibodies from correctly matched bispecific antibodies. This can be accomplished by separation based on the different light chains contained in the bispecific antibodies.

[0154] According to yet another aspect, the present invention provides a method for isolating a bispecific antibody, the method comprising the following steps:

[0155] a) contacting a liquid sample comprising a protein containing a kappa light chain with a separation matrix,

[0156] b) washing the separation matrix with a washing liquid or a combination of washing liquids,

[0157] c) eluting the kappa light chain-containing protein from the separation matrix using an eluent, and

[0158] d) cleaning the separation matrix with a cleaning solution.

[0159] The separation matrix was used in 0.4 mL HiTrap TM The device has a dynamic binding capacity (DBC) of 28 g / mL at 10% breakthrough for a protein containing kappa light chains, such as trastuzumab, when run at a flow rate of 10 mL / min.

[0160] According to one embodiment, the separation matrix disclosed above is used in the method.

[0161] The elution step can be performed with a volume gradient. This is shown in Example X. Alternatively, the elution step can be performed with a pH gradient.

[0162] This method enables the separation of bispecific antibodies from monospecific antibodies or mismatched antibodies based on the presence of a kappa light chain. Monoclonal antibodies have two identical kappa light chains. Bispecific antibodies can be designed to contain two different kappa light chains.

[0163] Any antibody that does not contain a kappa light chain, such as an antibody that contains two lambda light chains, will not bind to the separation matrix and will therefore be present in the effluent stream during step a) or washed away during step b). Any antibody that has at least one kappa light chain will bind to the separation matrix. Upon elution, antibodies that have only one kappa light chain will elute before antibodies that have two kappa light chains.

[0164] By using a separation matrix as disclosed above that binds to κ light chains of subclass 1, 3 or 4, it is also possible to separate antibodies or antibody fragments comprising one or two κ light chains of subclass 2 from antibodies or antibody fragments comprising one or two κ light chains of subclass 1, 3 or 4. By the same principle as described above, antibodies comprising two κ light chains of subclass 2, or one κ light chain of subclass 2 and a λ light chain, will not bind to the separation matrix and will therefore be present in the effluent stream during step a) or will be washed away during step b). Antibodies having one κ light chain of subclass 2 and one κ light chain of any of subclasses 1, 3 or 4 will elute before antibodies having two κ light chains of any of subclasses 1, 3 and 4.

[0165] Example

[0166] The general method used to prepare the materials used in the examples below is:

[0167] 1: Obtain a substrate formed of one or more polymer fibers (cellulose acetate (CA))

[0168] 2: Subject CA to glycidol polymerization and saponification

[0169] 3: Functionalize the material from step 2 with divinyl sulfone (DVS)

[0170] 4: Immobilizing a kappa light chain binding ligand on the material obtained in step 3, comprising:

[0171] Reduction and desalting of the kappa light chain-bound ligand;

[0172] Immobilize the reduced kappa light chain binding ligand on the material obtained from step 3 on a shaker;

[0173] Washing of kappa light chain binding ligand-immobilized material

[0174] 5: Deactivation of residual vinyl groups using 1-thioglycerol

[0175] 6: Wash the material to remove as much unbound kappa light chain binding ligand as possible.

[0176] Unless otherwise specified herein, the steps are performed according to the methods disclosed in Example 6 of WO / 2019 / 137869 and WO / 2018 / 011600.

[0177] Protein reduction

[0178] Sodium sulfate (1.4 M) was added to the protein solution, and when everything 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.

[0179] Desalination

[0180] Desalting was performed by using a pre-packed PD10 column containing Sephadex-G-25 medium. The column was equilibrated with 4 column volumes of degassed desalting solution (0.15 NaCl, 1 mM EDTA) and then loaded with protein (maximum 2.5 mL). The eluted fractions were collected and combined.

[0181] The desalted solution was diluted 20 times with desalting solution and the absorbance was measured at 276 nm and corrected by a blank of desalting buffer. By using the determined protein concentration it is then possible to calculate the required amount of desalted protein solution to be used in the fixation process.

[0182] For fixation, a solution with a concentration of 25-30 mg ligand / ml is used and added to the material activated according to the method mentioned above. Depending on the temperature, pH and reaction time used for ligand fixation, 20 mg of ligand up to at least 50 mg of ligand can be fixed per ml of porous support material. Longer reaction times such as in Example 6 of WO / 2019 / 137869 and WO / 2018 / 011600 generally result in higher ligand density than shorter reaction times in Example 5 of the same disclosure.

[0183] Example 1 - Dynamic Binding Capacity

[0184] The separation matrix according to the invention was prepared as described above and analyzed for dynamic binding capacity.

[0185] Sample preparation

[0186] Prepare 500 mL or 250 mL of 0.5 mg / mL IgG in 20 mM Tris-HCl, 150 mM NaCl, pH 7.5 (binding buffer) by diluting 1.515 mL or 0.758 mL of Gammanorm (165 mg / mL) in binding buffer and adjusting the volume to 500 mL or 250 mL in a volumetric flask. Just 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. Calculate the IgG concentration according to Beer's law:

[0187] A=εL c

[0188] Where A is the UV absorbance at 280 nm, ε is the absorption coefficient (for polyclonal IgG, ε is 1.38 ml / (mg×cm). L is the optical path length of the cell holder. c is the concentration of the solution.

[0189] 3.4 mL of purified trastuzumab (30 g / L) was diluted with 20 mM phosphate 150 mM NaCl pH 7.2 in a total volume of 200 mL. Concentration was determined by UV measurement at 280 nm using 96-well UV plates, 200 μL / well. Blank was 20 mM phosphate 150 mM NaCl pH 7.2. Trastuzumab concentration was calculated using an extinction coefficient of 1.48.

[0190] dAB is a domain antibody containing a variable light chain and according to the paper “Recombinant production of aV LThe single domain antibody was produced by the method described in "Protein Expression and Purification, Volume 51, Issue 2, February 2007, pp. 253-259".

[0191] 3.45 mL of dAb (14.5 g / L) was diluted in a total volume of 100 mL with 20 mM phosphate 150 mM NaCl pH 7.2. Concentration was determined by UV measurement at 280 nm using a 96-well UV plate, 200 μL / well. Blank was 20 mM phosphate 150 mM NaCl pH 7.2. dAb concentration was calculated using an extinction coefficient of 1.6.

[0192] As disclosed in Example 6 of WO / 2019 / 137869 and WO / 2013 / 068741, the ligand is coupled to the porous support.

[0193] Table 1: Buffer

[0194] Buffer composition Balance / Combination 20 mM phosphate 150 mM NaCl pH 7.2 Washing of unbound proteins 20mM phosphate 500mM NaCl pH 7 Washing salt 50 mM citrate pH 5 Elution 50 mM citrate pH 2.3 CIP 0.1MNaOH Rebalancing 20 mM phosphate 150 mM NaCl pH 7.2 The pH of the eluate was adjusted to approximately pH 7. 500 mM phosphate pH 8

[0195] The membrane single disk with the porous support coupled to the ligand is installed in the device and connected to the chromatography system 25IP31154, 10mm cell. The membrane was equilibrated with equilibration / binding buffer and then protein sample loaded with gammanorm or trastuzumab, respectively. The capacity at 10% breakthrough (Qb10%) was calculated and reported. The flow rate used during equilibration and washing of the membrane was 20mL / min. The flow rate was reduced to 10mL / min during protein loading. The analysis was performed on duplicate membrane discs and each disc was analyzed twice. The first run was a blank with no protein loading, followed by two frontal analysis runs with protein.

[0196] The penetration capacity is calculated using Extensions-DBC Calculations-Analyze, which is the same as Evaluation tool in Unicorn software used with 25IP31154.

[0197] To calculate the breakthrough capacity at 10% (Qb10), the following equation was used. That is, this is the amount of trastuzumab loaded onto the column until the trastuzumab concentration in the column effluent is 10% of the trastuzumab concentration in the liquid sample.

[0198]

[0199] A 100％ =100% UV signal

[0200] A sub = absorbance contribution from unbound mAb

[0201] A(V) = absorbance at a given applied volume

[0202] V C = column volume

[0203] V app = Volume applied until 10% penetration

[0204] V sys = System dead volume

[0205] C0 = liquid sample concentration

[0206] For all experiments, sample solutions contained 0.5 mg of protein.

[0207] Table 2: Dynamic binding capacity of two prototypes for trastuzumab results

[0208]

[0209] DBC decreases between runs 1 and 2 and increases again for run 3 and then decreases 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.

[0210] Table 3: Dynamic Binding Capacity Trastuzumab Results

[0211]

[0212] In this experiment, a blank run was included between runs 1 and 2. This indicates that a blank run should be performed between each run.

[0213] Without being bound by any theory, the higher DBC achieved in Run 2 appears to be attributable to the fact that the material had been CIP'd with 0.1 M NaOH prior to Run 2, whereas Run 1 was conducted without prior CIP washing.

[0214] Table 4: Dynamic binding capacity of prototype dAb results

[0215]

[0216] Table 5: Dynamic binding capacity dAb results re-run after CIP 0.1M NaOH with prototype

[0217]

[0218] Next, the DBC according to the prototype of the present invention was compared with a commercial product having the same convection-based matrix. The matrix was contained in the HiTrap TM In the device, such as Figure 1 HiTrap TM The prototype device (1) has a volume of 0.4 mL. The device (1) contains two stacked Fibro prototype membranes (2) with a nonwoven layer (3) as filler material between them. The disk diameter is 26.4 mm. Prototype 1 contains a separation matrix with 52 mg ligand / ml membrane. Prototype 2 contains a separation matrix with 34 mg ligand / ml membrane.

[0219] Table 6: In HiTrap TM DBC comparison of commercial products and prototypes in the device

[0220]

[0221]

[0222] As can be seen above, for both Qb5 and Qb10, the prototypes showed at least comparable performance to the commercial product Fibro TM PrismA is as good as DBC.

[0223] Example 2 - Resolution between Fab fragments and trastuzumab based on volume gradient

[0224] It was further investigated whether resolution between Fab fragments and trastuzumab could be achieved on the prototype according to the invention.

[0225] Fab fragment (Fab) is produced by cleavage of trastuzumab by papain. The trastuzumab solution is adjusted to pH 7.4 by adding 0.5M sodium phosphate and then diluted 1+1 in digestion buffer (25mM sodium phosphate, 1mM EDTA, 5mM mercaptoethanol, pH 7.5). The final volume is about 100mL. Papain crystals are added to the solution. The solution is incubated overnight at 37°C. Thereafter, antipapain (papain inhibitor) is added to the digested trastuzumab. The solution is kept at room temperature for 30min and then applied to Capto TM The Fab was collected during elution on a LHiScale 26 column to remove Fc-containing molecules (Fc or partially digested trastuzumab, collected in the flow-through).

[0226] A linear pH gradient from pH 5.0 to 2.3 with 50 mM citrate buffer was used to separate Fab and trastuzumab. Resolution experiments were performed with a linear gradient from 20 mL to 100 mL, such as increasing the gradient from 20 mL to 40, 60, and 100 mL, with run times ranging from 5.5 min to 13.5 min. The results are shown in Figure 2-5 middle.

[0227] The results showed that when the pH gradient was 20 mL, Fab and trastuzumab were co-eluted. Figure 2 .

[0228] For the separation between Fab and trastuzumab on the prototype, the shoulder on the peak increased as the gradient flattened and increased to 40 mL. Figure 3 .

[0229] When the pH gradient was 60 mL, better separation between Fab and trastuzumab was obtained. Figure 4 .

[0230] Even better separation between Fab and trastuzumab was achieved on Fibro at a pH gradient of 100 mL with a resolution of 0.88. Figure 5 , where the Fab peak is located on the left side of the chromatogram and the trastuzumab peak is located on the right side of the chromatogram.

Claims

1. A separation matrix comprising a kappa light chain binding ligand covalently coupled to a porous carrier, wherein The kappa light chain binding ligand comprises, consists essentially of, or consists of a multimer of alkali-stable Peptostreptococcus magna (formerly known as Peptostreptococcus magna) protein L domains; and The porous support is a convection-based chromatography matrix.

2. The separation matrix of claim 1, wherein the convection-based chromatography matrix is ​​a fibrous substrate.

3. The separation matrix according to claim 2, wherein the fibrous substrate is based on electrospun polymer fibers or cellulose fibers, optionally based on nonwoven fibers.

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.

5. The separation matrix according to any one of claims 3 or 4, wherein the fibrous substrate is a fibrous nonwoven polymer matrix.

6. The separation matrix according to any one of claims 2 to 5, wherein the fibers contained in the fibrous substrate have a cross-sectional diameter of 10-1000 nm, such as 200-800 nm, 200-400 nm or 300-400 nm.

7. The separation matrix according to any one of claims 2 to 6, wherein the ligand is bound to a divinyl sulfone functional group coupled to a glycidyl group grafted onto the fiber substrate.

8. The separation matrix according to any one of the preceding claims, wherein the kappa light chain binding ligand comprises at least two alkali-stable protein L domains.

9. The separation matrix according to claim 8, wherein the alkali-stable 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 of Peptostreptococcus magna (formerly known as Peptostreptococcus magna), wherein the positions corresponding to positions 10 and 45 in the B2 domain (SEQ ID NO 1) in the alignment are histidine, and the position corresponding to position 60 in the B2 domain (SEQ ID NO 1) in the alignment is tyrosine or glutamine.

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

11. The separation matrix according to any one of claims 8 to 10, wherein the alkali-stable protein L domain has at least 90%, 95% or 98% sequence identity or 77.5% sequence similarity to any one of the amino acid sequences 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 as determined by a BLOSUM matrix of 75, wherein the gap opening penalty is 12 and the gap extension penalty is 3, wherein the positions corresponding to positions 10 and 45 in the alignment and the position corresponding to position 60 in the alignment are invariant.

12. The separation matrix of any one of claims 8 to 10, wherein the alkali-stable protein L domain has at least 90%, 95% or 98% sequence identity or 77.5% sequence similarity to any one of the amino acid sequences 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 as determined by a BLOSUM matrix of 75, wherein the gap opening penalty is 12 and the gap extension penalty is 3.

13. A separation matrix according to any of the preceding claims, wherein the ligand density is at least 20 mg / ml porous support, or at least 25 mg / ml porous support, or at least 30 mg / ml porous support, or at least 35 mg / ml porous support, or at least 40 mg / ml porous support, or at least 45 mg / ml porous support, or at least 50 mg / ml porous support.

14. A separation matrix according to any one of the preceding claims, when in 0.4 mL HiTrap TM It has a dynamic binding capacity (DBC) of 25 g / mL at 10% breakthrough for a kappa light chain-containing antibody, such as trastuzumab, when run at a flow rate of 10 mL / min in the device.

15. A separation matrix according to any one of the preceding claims, when in 0.4 mL HiTrap TM It has a dynamic binding capacity (DBC) of 45 g / mL at 10% breakthrough for a kappa light chain-containing antibody, such as trastuzumab, when run at a flow rate of 10 mL / min in the device.

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

17. A method for separating a kappa light chain binding protein from a protein containing a lambda light chain, the method comprising the following steps: a) contacting a liquid sample comprising a protein containing a kappa light chain with a separation matrix; b) washing the separation matrix with a washing liquid or a combination of washing liquids; c) eluting the κ light chain-containing protein from the separation matrix using an eluent; and d) cleaning the separation matrix with a cleaning solution; When the 0.4mL HiTrap TM The separation matrix has a dynamic binding capacity (DBC) of 25 g / mL for a kappa light chain-containing antibody, such as trastuzumab, at 10% breakthrough when run at a flow rate of 10 mL / min in the device.

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

19. The method according to any one of claims 17 or 18, wherein the separation is performed by applying a volume gradient or a pH gradient in step c).

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

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