Alkaline stabilized kappa light chain binding separation matrix

Abstract

The present invention relates to a separation matrix for affinity chromatography and the separation of biomolecules based on the presence of kappa light chains. More specifically, the present invention relates to a separation matrix comprising at least 12 mg / ml of a kappa light chain binding ligand covalently coupled to a porous support, said kappa light chain binding ligand comprising, consisting essentially of, or consisting of a multimer of an alkali-stabilized Finegordia magna (formerly Peptostreptococcus magnus) protein L domain, said porous support comprising polymer particles having a dry solids mass (Dw) of 50-200 mg / ml and a volume-weighted median diameter (D50v) of 30-100 μm. The present invention also relates to a method of using said separation 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 the separation of biomolecules based on the presence of kappa light chains, such as immunoglobulins and immunoglobulin fractions. The present invention also relates to a method of using said separation matrix. [Background technology]

[0002] Immunoglobulins and immunoglobulin fragments represent the most widespread biopharmaceuticals in production or development worldwide. The high commercial demand and therefore value of this particular therapeutic market has led pharmaceutical companies to place a strong emphasis on maximizing the productivity of their respective manufacturing processes while controlling the associated costs.

[0003] Affinity chromatography, typically on a matrix containing staphylococcal protein A or a variant thereof, 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 chain of immunoglobulins provides the common process with a very high clearance of impurities and contaminants.

[0004] Immunoglobulin or antibody fragments, such as Fab, single chain variable fragments (scFv), bispecific T cell engagers (BiTEs), domain antibodies, etc., lacking Fc chains but having subclass 1, 3 or 4 kappa light chains, have been synthesized using a matrix containing protein L 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, 12820-12825, 1992). 267, 2234-2239, 1992 and U.S. Pat. No. 6,822,075) show great promise as a purification platform that can provide the required high degree of selectivity.

[0005] Protein L matrices are commercially available, for example as Capto™ L from Cytiva™, and can be used to separate kappa light chain-containing proteins, such as intact antibodies, Fab fragments, scFv fragments, domain antibodies, etc. Approximately 75% of antibodies produced by healthy humans have kappa light chains, and approximately 90% of therapeutic monoclonal antibodies and antibody fragments contain kappa 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 reliable removal of impurities and / or contaminants. Such impurities and / or contaminants can be, for example, uneluted molecules, such as undesired biomolecules or microorganisms, such as proteins, carbohydrates, lipids, bacteria and viruses, that are adsorbed to the stationary phase or matrix in the chromatographic procedure. Removal of such impurities and / or contaminants from the matrix is ​​usually carried out after the first elution of the desired product in order to regenerate the matrix before further use. Such removal usually involves a procedure known as cleaning in place (CIP), in which agents capable of inactivating or eluting the impurities from the stationary phase are used. One such class of agents that is often used with chromatographic media is an alkaline solution that is passed over the matrix. Currently, the most widely used cleaning and sanitizing agent is NaOH, and depending on the degree and nature of the contamination and impurities, it is desirable to use NaOH at concentrations ranging from 0.05 M up to, for example, 1 M. However, Protein L is a fairly alkaline-sensitive protein, compared to, for example, Protein A, and can only tolerate up to about 15 mM NaOH over a large number of cycles. This means that additional, less desirable cleaning solutions such as urea or guanidinium salts may also have to be used to ensure adequate cleaning. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] U.S. Patent No. 6,822,075 [Patent Document 2] US6602990 [Patent Document 3] US7396467 [Non-patent literature]

[0008] [Non-Patent Document 1] B Akerstrom, L Bjorck: J. Biol. Chem. 264, 19740 - 19746, 1989

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[0009] Thus, there remains a need in the art to obtain separation matrices containing Protein L derived ligands that have improved stability towards alkaline cleaning procedures. [Means for solving the problem]

[0010] According to a first aspect, the present disclosure provides a separation matrix comprising at least 12 mg / ml of a kappa light chain binding ligand covalently coupled to a porous support, said kappa light chain binding ligand comprising, consisting essentially of, or consisting of a multimer of an alkali-stabilized Finegordia magna (formerly Peptostreptococcus magnus) protein L domain, said porous support having a dry solids mass (DMS) of 50-200 mg / ml. W ), a separation matrix is ​​provided comprising polymer particles having a volume-weighted median diameter (D50v) of 30 to 100 μm.

[0011] A separation matrix according to the above may comprise at least 14 mg / ml of kappa light chain binding ligand, such as at least 14.5 mg / ml, at least 15 mg / ml, at least 15.5 mg / ml, at least 16 mg / ml, at least 16.5 mg / ml, at least 17 mg / ml, at least 17.5 mg / ml, at least 18 mg / ml, at least 18.5 mg / ml, or at least 19 mg / ml of kappa light chain binding ligand.

[0012] The porous support may have a D of 50 to 150 mg / ml, 50 to 120 mg / ml, 50 to 100 mg / ml, 50 to 90 mg / ml, 60 to 80 mg / ml, or 60 to 75 mg / ml, for example at least 63 mg / ml, or at least 65 mg / ml, or at least 70 mg / ml. W may have.

[0013] The porous support may have a volume weighted median diameter (D50v) of 35 to 90 μm, 40 to 80 μm, 50 to 70 μm, 55 to 70 μm, 55 to 67 μm, 58 to 70 μm, or 58 to 67 μm, for example at least 60 μm, or at least 62 μm.

[0014] The separation matrix as described above may have a Kd value of 0.6 to 0.95, such as a Kd value of 0.7 to 0.9, or a Kd value of 0.6 to 0.8, such as a Kd value of about 0.67, or a Kd value of about 0.72, or a Kd value of about 0.75, as measured by inverse size exclusion chromatography using dextran of Mw 110 kDa as a probe molecule.

[0015] The polymer particles in the separation matrix according to above may be crosslinked.

[0016] In the separation matrix according to the above, at least two of the alkaline stabilized protein L domains may be selected from the group comprising functional variants of the B1, B2, B3, B4, B5, C2, C3, C4 and D1 domains of Finegordia magna (formerly Peptostreptococcus magnus) protein L, wherein the positions corresponding in the alignment to positions 10 and 45 in the B2 domain (SEQ ID NO: 1) are histidines and the position corresponding in the alignment to position 60 in the B2 domain (SEQ ID NO: 1) is tyrosine or glutamine. The at least two alkaline stabilized protein L domains may preferably be selected from the group comprising the B2, B3, B4, C2, C3, C4 and D1 domains.

[0017] At least two alkali stabilized protein L domains may have 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 with a gap open penalty of 12 and a gap extension penalty of 3, wherein 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 are unchanged.

[0018] The separation matrix according to the above may comprise 3, 4, 5, 6, 7, 8 or 9 alkaline stabilized Protein L domains. The separation matrix according to the above may preferably comprise 4, 5 or 6 alkaline stabilized Protein L domains.

[0019] The ligand may further comprise a coupling element, said coupling element being one or more cysteine ​​residues, one or more lysine residues, or one or more histidine residues at the C-terminus of the ligand. Preferably, the ligand comprises one or more cysteine ​​residues at the C-terminus of the ligand.

[0020] Preferably, the separation matrix according to above has a 10% breakthrough dynamic binding capacity for IgG of at least 55 mg / ml at a residence time of 4 minutes. Preferably, the separation matrix according to above has a 10% breakthrough dynamic binding capacity for IgG of at least 70 mg / ml at a residence time of 6 minutes. Preferably, the separation matrix according to above has a 10% breakthrough dynamic binding capacity for IgG of at least 80 mg / ml at a residence time of 10 minutes.

[0021] Preferably, a separation matrix according to the above has an IgG capacity after 24 hours incubation in 0.1 M NaOH at 22+ / -2° C. that is at least 80%, or at least 85%, or at least 90%, or at least 95% of the IgG capacity before incubation. Preferably, a separation matrix according to any of the above has an IgG capacity after 32 hours incubation in 0.3 M NaOH at 22+ / -2° C. that is at least 90% of the IgG capacity before incubation. Preferably, a separation matrix according to the above has an IgG capacity after 12 hours incubation in 0.5 M NaOH at 22+ / -2° C. that is at least 95%, or at least 93% of the IgG capacity before incubation.

[0022] According to another aspect, the present disclosure provides a method for producing a method for manufacturing a semiconductor device comprising: a) contacting a liquid sample containing a kappa light chain-containing protein with a separation matrix; b) washing the separation matrix with one or a combination of several washing solutions; c) eluting the kappa light chain-containing protein from the separation matrix using an elution solution; and d) cleaning the separation matrix with a cleaning solution wherein the IgG capacity of the separation matrix after 24 hours of incubation in 0.1 M NaOH at 22+ / -2°C is at least 80%, or at least 85%, or at least 90%, or at least 95% of the IgG capacity before incubation.

[0023] According to yet another aspect, the present disclosure provides a method for producing a method for manufacturing a semiconductor device comprising: a) contacting a liquid sample containing a kappa light chain-containing protein with a separation matrix; b) washing the separation matrix with one or a combination of several washing solutions; c) eluting the kappa light chain containing protein from the separation matrix with an elution solution at decreasing pH; d) cleaning the separation matrix with a cleaning solution wherein the IgG capacity of the separation matrix after 24 hours of incubation in 0.1 M NaOH at 22+ / -2°C is at least 80%, or at least 85%, or at least 90%, or at least 95% of the IgG capacity before incubation.

[0024] The decreasing pH in any one of the above methods may be performed by using a pH gradient. Alternatively, the decreasing pH in any one of the above methods may be performed in a stepwise manner. In any one of the above methods, the decreasing pH may be from about 5.5 to about 2, such as from about 5 to about 2, from about 4.5 to about 2, or from about 4 to about 2.

[0025] The cleaning liquid may contain 0.01 to 1.0 M NaOH or KOH, for example 0.05 to 1.0 M, or 0.05 to 0.1 M, or 0.05 to 0.3 M, or 0.05 to 0.5 NaOH or KOH.

[0026] The eluent may comprise at least one anionic species selected from the group consisting of acetate, citrate, glycine, succinate, phosphate, and formate.

[0027] Steps a) to d) in any one of the above methods may be repeated at least 10 times, such as at least 50 times or 50 to 200 times.

[0028] The separation matrix used in any one of the above methods may be a separation matrix according to the first aspect or any variation thereof disclosed above.

[0029] Furthermore, in yet another aspect, there is provided herein a chromatography column comprising a separation matrix according to the first aspect or any variation thereof disclosed above.

[0030] According to a further aspect, there is provided herein the use of a chromatography column according to above in any one of the methods disclosed above. [Brief description of the drawings]

[0031] [Figure 1a] 1 is a graph showing the correlation between ligand density and alkaline stability. FIG. 1 is a graph showing Ligand density versus % Remaining capacity after 112 CIP cycles with 0.1 M NaOH using Trastuzumab. [Figure 1b] Figure 13 is a graph showing the correlation between ligand density and alkaline stability.Figure 14 is a graph showing DBC at 10% breakthrough (Qb10%) for different ligand densities using Fab fragments. [Figure 1c] Figure 1 shows the correlation between ligand density and alkaline stability.Figure 2 shows the relative remaining QB10 for a selection of ligands and trastuzumab. [Diagram 2] FIG. 1 is an illustration of a model of QB10 derived in relation to ligand density and Kd and Dw for a porous support. [Figure 3a] Figure 1 shows alkaline stability from an accelerated alkaline stability study comparing different ligand densities for the commercial product and the prototype.Figure 2 shows Qb10% at 4 min residence time with trastuzumab over 100 CIP cycles with 100 mM NaOH for two prototypes with different ligand densities and Capto™ L. [Figure 3b] Figure 1 shows alkaline stability from an accelerated alkaline stability study comparing different ligand densities for a commercial product and a prototype.Figure 2 shows relative remaining Qb10% (Relative DBC) over 100 CIP cycles with 100 mM NaOH for two prototypes with different ligand densities and Capto™ L. [Figure 3c] Figure 13 is a graph showing alkaline stability from an accelerated alkaline stability study comparing different ligand densities for the commercial product and the prototype. Figure 14 is a graph showing the relative remaining QB10 of the prototype for 0.1 M NaOH, 0.3 M NaOH, and 0.5 M NaOH. [Figure 4a] Figure 1 shows the Qb10% versus residence time (min) for the prototype compared to the commercial products: TOYOPEARL® AF-rProtein L-650F (Tosoh), Capto™ L (Cytiva™) and KanCap™ L (Kaneka). Figure 2 shows the Qb10% with trastuzumab at different residence times. [Figure 4b]Graph showing Qb10% versus residence time (min) for the prototype compared to commercial products: TOYOPEARL® AF-rProtein L-650F (Tosoh), Capto™ L (Cytiva™) and KanCap™ L (Kaneka). Graph showing Qb10% with Fab fragments at different residence times. [Figure 4c] Figure 1 shows the Qb10% versus residence time for the prototype compared to commercial products: TOYOPEARL® AF-rProtein L-650F (Tosoh), Capto™ L (Cytiva™) and KanCap™ L (Kaneka). Figure 2 shows the Qb10% with dAbs at different residence times. [Figure 5a] Figure 1 shows results from chromatography using a prototype separation matrix.Figure 2 shows chromatograms for a 50 ml bsAb01 sample, an elution volume of 5 CV, and a step elution with pH 3.4 and pH 3.1 using an elution flow rate of 1 ml / min. [Figure 5b] Figure 1 shows the results from chromatography using the prototype separation matrix. Figure 2 shows an overview of the elution peaks and MS analyzed fractions. Peak 1, peak 2 and peak 3 are marked with numbers 1-3. MS analyzed fractions are marked with an asterisk. [Figure 6a] Figure 5B is a graph showing MS (mass spectrometry) analysis of peaks 1, 2 and 3 in Figure 5b; Figure 5C is a graph showing the results of a non-reducing MS analysis of peak 2 from Figure 5b. [Figure 6b] Figure 5b shows MS (mass spectrometry) analysis of peaks 1, 2 and 3 in Figure 5b. Figure 5c shows the results of a non-reducing MS analysis of peak 3 from Figure 5b. [Figure 7] Figure 1 shows results from chromatography using the prototype separation matrix. Chromatogram for 10 ml BsAb01 sample, pH gradient elution chromatogram from pH 5.5 to 2.5 over 40 CV in 4 min at room temperature. MS analysis fractions are marked with an asterisk. [Figure 8a] 1 shows an overlay of size exclusion chromatograms for the prototype and Capto™ L. FIG. 2 shows an overlay of chromatograms for the eluate. [Figure 8b] Figure 1 shows an overlay of size-exclusion chromatograms for the prototype and Capto™ L. Figure 2 shows an overlay of chromatograms for the flow-through (FT) fraction. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] definition The terms "antibody" and "immunoglobulin", which may be used interchangeably herein, refer to an antigen-binding protein with a basic four-polypeptide chain structure consisting of two heavy (H) chains and two light (L) chains, which are stabilized by inter- or intra-chain disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains, CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (VL) and a light chain constant region. The light chain constant region is composed 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.

[0033] The terms "kappa light chain binding polypeptide" and "kappa light chain binding protein" as used herein refer to an antibody that binds subclass 1, 3 or 4 kappa light chain (V, V, VH ... κI , V κIII and V κIVBy the term "protein" is meant a polypeptide or protein, respectively, capable of binding to the IL-11 receptor IL-1 (also referred to as IL-1), for example Protein L, and includes any variant, fragment or fusion protein thereof which maintains said binding properties.

[0034] The term "kappa light chain-containing protein" is used synonymously with "immunoglobulin kappa light chain-containing protein" and refers herein to an antibody containing a subclass 1, 3 or 4 kappa light chain (V, V, VHK ... κI , V κIII and V κIV By this term is meant a protein comprising a kappa light chain (also referred to as an antibody), including any intact antibody, antibody fragment, fusion protein, conjugate or recombinant protein containing subclass 1, 3 or 4 kappa light chain.

[0035] The term "mAb" refers to a monoclonal antibody.

[0036] The term "Fab" refers to an antigen-binding fragment derived from an immunoglobulin containing a kappa or lambda light chain.

[0037] The term "bispecific antibody" refers to an antibody that can bind to two different types of antigens or two different epitopes on the same antigen. Similarly, a trispecific antibody refers to an antibody that can bind to three different types of antigens or three different epitopes on the same antigen.

[0038] "DBC" means dynamic binding capacity, which is the binding capacity of 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 target protein and monitoring the flow-through. Proteins bind to the resin up to a certain limit before unbound protein flows through the column.

[0039] DBC can be determined, for example, by breakthrough curve at 10% protein loss. This is referred to as Qb10% value, or simply Qb10%. Samples are applied to a chromatography resin column for a specific residence time, and the dynamic binding capacity for each resin is calculated at 10% of protein breakthrough, i.e., the amount of 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 for each resin is calculated at 80% of breakthrough capacity, this is referred to as Qb80% value.

[0040] Kd is a value for the fraction of pore volume available for a probe molecule of a particular size. In this specification, Kd is measured by inverse size exclusion chromatography using dextran of Mw 110 kDa as a probe molecule, for example, according to the method described in Gel Filtration Principles and Methods, Pharmacia LKB Biotechnology 1991, pp. 6-15, 13.

[0041] D50v is the average particle size for greater than 50% by volume of the particle population.

[0042] The term "liquid sample" as used herein refers to a liquid containing at least one target substance that is sought to be purified from other substances also present. A 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 biological molecules (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 may start as an aqueous solution or suspension, but it may also contain organic solvents used in earlier separation steps such as solvent precipitation, extraction, etc. Examples of liquid samples that may contain valuable biological materials 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, a liquid sample may be referred to as a "clarified cell culture feed" or "CCF".

[0043] A "buffer" is a substance whose presence in a solution increases the amount of acid or alkali that must be added to produce 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 is generally capable of maintaining a constant concentration of hydrogen ions such 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, e.g., Good et al. (1966) Biochemistry 5:467 and Good and Izawa (1972) Methods Enzymol. 24:62.

[0044] An "equilibration buffer" is a buffer used to prepare the binding reagent, the solid phase, or both, for loading a stock solution containing the target protein. The equilibration buffer is preferably isotonic and generally has a pH ranging from about 6 to about 8. A "loading buffer" is a buffer used to load a stock solution, or liquid sample, containing the binding domain-containing protein and impurities onto the solid phase on which the binding agent is immobilized. Often, the equilibration buffer and the loading buffer are the same.

[0045] As used herein, "wash solution" or "wash buffer" refers to any liquid used to remove impurities from the chromatography resin to which the target substance is bound. For example, two or more wash solutions can be used sequentially, with successive wash solutions having different properties such as pH, conductivity, solvent concentration, etc., designed to dissociate and remove different types of impurities non-specifically associated with the chromatography resin.

[0046] "Elution fluid" or "elution buffer" are used interchangeably herein and refer to a liquid used to dissociate the target substance from the chromatography resin, thereby eluting the binding domain-containing protein from the immobilized binder after washing with one or more wash fluids. The elution fluid acts to dissociate the target substance without irreversibly denaturing the target substance. Typical elution fluids are well known in the chromatography art and may have a different pH (typically a lower pH), a higher salt concentration, free affinity ligand or analog, or other substances that promote dissociation of the target substance from the chromatography resin. "Elution conditions" refer to process conditions imposed on the target substance-bound chromatography resin that dissociate the target substance from the chromatography resin, for example, contacting the target substance-bound chromatography resin with an elution fluid or elution buffer to effect such dissociation.

[0047] Preferably, the elution buffer has a low pH, thereby disrupting 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 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 thereof. Preferred such buffers are citrate and acetate buffers, most preferably sodium citrate or sodium acetate buffers.

[0048] The cleaning solution can be an acidic or alkaline solution to remove 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.

[0049] Cleaning in place (CIP) is an important process for the efficient use of chromatography columns. To maximize the number of cycles the column can be reused, a cleaning procedure is required that efficiently removes impurities without being harmful to the chromatography resin.

[0050] "HCP" stands for host cell protein.

[0051] As used herein, the terms "comprises," "comprising," "containing," "having," and the like can mean "includes," "including," and the like; "consisting essentially of" or "consists essentially" are open-ended terms that permit the presence of more than what is recited, but exclude prior art embodiments, so long as the basic or novel characteristics of what is recited are not altered by the presence of more than what is recited.

[0052] Detailed Description The inventors aimed to invent a separation matrix containing Protein L derived ligands that has improved stability against alkaline cleaning procedures, while maintaining sufficient efficiency in binding capacity and flow properties in a chromatographic setting, and preferably superior to current commercially available separation matrices.

[0053] According to one aspect, this object has been achieved by providing a separation matrix comprising at least 12 mg / ml of a kappa light chain binding ligand covalently coupled to a porous support, said kappa light chain binding ligand comprising, consisting essentially of or consisting of multimers of alkali-stabilised Finegordia (formerly Peptostreptococcus) protein L domain, said porous support comprising polymer particles having a dry solids mass (Dw) of 50-200 mg / ml and a volume weighted median diameter (D50v) of 30-100 μm.

[0054] The kappa light chain binding ligands contained in the separation matrix of the present invention comprise, consist essentially of, or consist of multimers of alkaline stabilized Finegordia protein L domains. The protein L domains may be any functional protein L derived domains as long as they are alkaline stabilized. The protein L domains are selected from functional variants of B1, B2, B3, B4, B5, C2, C3, C4 and D1 domains, in which the positions corresponding in the alignment to positions 10 and 45 in the B2 domain (SEQ ID NO:1) are histidines, and the positions corresponding in the alignment to position 60 in the B2 wt domain (SEQ ID NO:1) are tyrosines or glutamines. Thus, the above positions corresponding to positions 10, 45 and 60 in the B2 wt domain (SEQ ID NO:1) are unchanged in the functional protein L domains. SEQ ID NO:1 (wt B2) PKEEVTIKANLIYADGKTQTAEFKGTFEEAAAEAYRYADALKKDNGEYTVDVADKGYTLNIKFAGKEKTPEE

[0055] An example of such a functional Protein L domain may be: SEQ ID NO:2 (B2: N10H, N45H, N60Y mutations) PKEEVTIKAHLIYADGKTQTAEFKGTFEEAAAEAYRYADALKKDHGEYTVDVADKGYTLYIKFAGKEKTPEE SEQ ID NO:3 (B2: N10H, N45H, N60Q mutations) PKEEVTIKAHLIYADGKTQTAEFKGTFEEAAAEAYRYADALKKDHGEYTVDVADKGYTLQIKFAGKEKTPEE SEQ ID NO:4 (B3: N10H, N45H, N60Y mutations) PKEEVTIKAH LIYADGKTQT AEFKGTFEEA TAEAYRYADL LAKEHGKYTV DVADKGYTLY IKFAGKEKTP EE SEQ ID NO:5 (B3: N10H, N45H, N60Q mutations) PKEEVTIKAH LIYADGKTQT AEFKGTFEEA TAEAYRYADL LAKEHGKYTV DVADKGYTLQ IKFAGKEKTP EE SEQ ID NO:6 (B1: N10H, N45H, N60Y mutations) SEEEVTIKAHLIFANGSTQTAEFKGTFEKATSEAYAYADTLKKDHGEYTVDVADKGYTLYIKFAGKEKTPEE SEQ ID NO:7 (B1: N10H, N45H, N60Q mutations) SEEEVTIKAHLIFANGSTQTAEFKGTFEKATSEAYAYADTLKKDHGEYTVDVADKGYTLQIKFAGKEKTPEE SEQ ID NO:8 (B4: N10H, N45H, N60Y mutations) PKEEVTIKAHLIYADGKTQTAEFKGTFAEATAEAYRYADLLAKEHGKYTADLEDGGYTIYIRFAGKKVDEKPEE SEQ ID NO:9 (B4: N10H, N45H, N60Q mutations) PKEEVTIKAHLIYADGKTQTAEFKGTFAEATAEAYRYADLLAKEHGKYTADLEDGGYTIQIRFAGKKVDEKPEE SEQ ID NO: 10 (B5: N9H, N44H, N59Y mutations) KEQVTIKEH IYFEDGTVQTATFKGTFAEATAEAYRYADLLSKEHGKYTADLEDGGYTIQIRFAGKEEPEE SEQ ID NO: 11 (B5: N9H, N44H, N59Q mutations) KEQVTIKEHIYFEDGTVQTATFKGTFAEATAEAYRYADLLSKEHGKYTADLEDGGYTIQIRFAGKEEPEE SEQ ID NO: 12 (C2b N57Y: N10H, N45H, N60Y mutations) PKEEVTIKVHLIFADGKTQTAEFKGTFEEATAKAYAYADLLAKEHGEYTADLEDGGYTIYIKFAGKETPETPEE SEQ ID NO: 13 (C2b N57Y:N10H, N45H, N60Q mutations) PKEEVTIKVHLIFADGKTQTAEFKGTFEEATAKAYAYADLLAKEHGEYTADLEDGGYTIQIKFAGKETPETPEE SEQ ID NO: 14 (C3b N39D, N57Y:N10H, N45H, N60Y mutations) PKEEVTIKVHLIFADGKIQTAEFKGTFEEATAKAYAYADLLAKEHGEYTADLEDGGYTIYIKFAGKETPETPEE SEQ ID NO: 15 (C3b N39D, N57Y:N10H, N45H, N60Q mutations) PKEEVTIKVHLIFADGKIQTAEFKGTFEEATAKAYAYADLLAKEHGEYTADLEDGGYTIQIKFAGKETPETPEE SEQ ID NO: 16 (C4: N10H, N45H, N60Y mutations) PKEEVTIKVHLIFADGKTQTAEFKGTFEEATAEAYRYADLLAKVHGEYTADLEDGGYTIYIKFAGKEQPGENPG SEQ ID NO: 17 (C4: N10H, N45H, N60Q mutations) PKEEVTIKVHLIFADGKTQTAEFKGTFEEATAEAYRYADLLAKVHGEYTADLEDGGYTIQIKFAGKEQPGENPG SEQ ID NO: 18 (D1: N10H, N45H, N60Y mutations) PKEEVTIKAHLIFADGKTQTAEFKGTFEEATAEAYRYADLLAKVHGEYTADLEDGGYTIYIKFAGKEQPGEN SEQ ID NO: 19 (D1: N10H, N45H, N60Q mutations) PKEEVTIKAHLIFADGKTQTAEFKGTFEEATAEAYRYADLLAKVHGEYTADLEDGGYTIQIKFAGKEQPGEN

[0056] Preferably, the Protein L domain is selected from the group comprising the B3 domain, the C2 domain, the C3 domain and the D domain, wherein the positions corresponding in the alignment to positions 10 and 45 in the B2 wt domain (SEQ ID NO:1) are histidine and the position corresponding in the alignment to position 60 in the B2 wt domain (SEQ ID NO:1) is tyrosine or glutamine. The above positions corresponding to positions 10, 45 and 60 in the B2 wt domain (SEQ ID NO:1) are unchanged in the functional Protein L domain.

[0057] The remaining positions in such a functional protein L domain may vary as long as the three-dimensional structure is unchanged compared to that of the B2 wt domain (SEQ ID NO: 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 (SEQ ID NO: 1). The variations may be conservative amino acid substitutions for amino acids with similar or identical charge, hydrophobicity, etc., and one of skill in the art can determine what such variations of amino acids may be.

[0058] The protein L domain may have 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, with a gap open penalty of 12 and a gap extension penalty of 3.

[0059] A functional protein L domain may be a truncated sequence, for example positions corresponding to positions 1-4 in the B2 wt domain (SEQ ID NO: 1) may be deleted, for example positions corresponding to the position following position 65 in the B2 wt domain (SEQ ID NO: 1) may be deleted.

[0060] SEQ ID NO: 20 (B1_trunc, N6H, N41H, N56Y mutations) VTIKAHLIFANGSTQTAEFKGTFEKATSEAYAYADTLKKDHGEYTVDVADKGYTLYIKFAG SEQ ID NO: 21 (B1_trunc, N6H, N41H, N56Q mutations) VTIKAHLIFANGSTQTAEFKGTFEKATSEAYAYADTLKKDHGEYTVDVADKGYTLQIKFAG SEQ ID NO: 22 (B2_trunc, N6H, N41H, N56Y mutations) VTIKAHLIYADGKTQTAEFKGTFEEAAAEAYRYADALKKDHGEYTVDVADKGYTLYIKFAG SEQ ID NO: 23 (B2_trunc, N6H, N41H, N56Q mutations) VTIKAHLIYADGKTQTAEFKGTFEEAAAEAYRYADALKKDHGEYTVDVADKGYTLQIKFAG SEQ ID NO: 24 (B3_trunc, N6H, N41H, N56Y mutations) VTIKAHLIYADGKTQTAEFKGTFEEATAEAYRYADLLAKEHGKYTVDVADKGYTLYIKFAG SEQ ID NO: 25 (B3_trunc, N6H, N41H, N56Q mutations) VTIKAHLIYADGKTQTAEFKGTFEEATAEAYRYADLLAKEHGKYTVDVADKGYTLQIKFAG SEQ ID NO: 26 (B4_trunc, N6H, N41H, N56Y mutations) VTIKAHLIYADGKTQTAEFKGTFAEATAEAYRYADLLAKEHGKYTADLEDGGYTIYIRFAG SEQ ID NO: 27 (B4_trunc, N6H, N41H, N56Q mutations) VTIKAHLIYADGKTQTAEFKGTFAEATAEAYRYADLLAKEHGKYTADLEDGGYTIQIRFAG SEQ ID NO: 28 (B5_trunc, N6H, N41H, N56Y mutations) VTIKEHIYFEDGTVQTATFKGTFAEATAEAYRYADLLSKEHGKYTADLEDGGYTIYIRFAG SEQ ID NO: 29 (B5_trunc, N6H, N41H, N56Q mutations) VTIKEHIYFEDGTVQTATFKGTFAEATAEAYRYADLLSKEHGKYTADLEDGGYTIQIRFAG SEQ ID NO: 30 (C2b N57Y _trunc, N6H, N41H, N56Y mutations) VTIKVHLIFADGKTQTAEFKGTFEEATAKAYAYADLLAKEHGEYTADLEDGGYTIYIKFAG SEQ ID NO: 31 (C2b N57Y _trunc, N6H, N41H, N56Q mutations) VTIKVHLIFADGKTQTAEFKGTFEEATAKAYAYADLLAKEHGEYTADLEDGGYTIQIKFAG SEQ ID NO: 32 (C3b N39D, N57Y trunc, N6H, N41H, N56Y mutations) VTIKVHLIFADGKIQTAEFKGTFEEATAKAYAYADLLAKEHGEYTADLEDGGYTIYIKFAG SEQ ID NO: 33 (C3b N39D, N57Y_trunc, N6H, N41H, N56Q mutations) VTIKVHLIFADGKIQTAEFKGTFEEATAKAYAYADLLAKEHGEYTADLEDGGYTIQIKFAG SEQ ID NO: 34 (C4_trunc, N6H, N41H, N56Y mutations) VTIKVHLIFADGKTQTAEFKGTFEEATAEAYRYADLLAKVHGEYTADLEDGGYTIYIKFAG SEQ ID NO: 35 (C4_trunc, N6H, N41H, N56Q mutations) VTIKVHLIFADGKTQTAEFKGTFEEATAEAYRYADLLAKVHGEYTADLEDGGYTIQIKFAG SEQ ID NO: 36 (D1_trunc, N6H, N41H, N56Y mutations) VTIKAHLIFADGKTQTAEFKGTFEEATAEAYRYADLLAKVHGEYTADLEDGGYTIYIKFAG SEQ ID NO: 37 (D1_trunc, N6H, N41H, N56Q mutations) VTIKAHLIFADGKTQTAEFKGTFEEATAEAYRYADLLAKVHGEYTADLEDGGYTIQIKFAG

[0061] The protein L domain may have 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, with a gap open penalty of 12 and a gap extension penalty of 3.

[0062] As can be seen above, the C2 and C3 domains 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.

[0063] As mentioned above, the kappa light chain binding ligand contained in the separation matrix comprises, consists essentially of or consists of a multimer of alkaline stabilized protein L domains. The multimer may comprise 2, 3, 4, 5, 6, 7, 8 or 9 alkaline stabilized protein L domains. In other words, the multimer may be a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer or nonamer. Preferably, the ligand comprises 4, 5, 6 or 7 alkaline stabilized protein L domains, for example 5 or 6 alkaline stabilized protein L domains.

[0064] The multimer may further comprise linkers, spacers, or additional amino acids, which may, for example, result from the cloning process of the ligand or may constitute residues from a truncated signaling sequence.

[0065] As mentioned above, the separation matrix contains at least 12 mg / ml of kappa light chain binding ligand. Preferably, the separation matrix contains at least 14 mg / ml of kappa light chain binding ligand, such as at least 14.5 mg / ml, at least 15 mg / ml, at least 15.5 mg / ml, at least 16 mg / ml, at least 16.5 mg / ml, at least 17 mg / ml, at least 17.5 mg / ml, at least 18 mg / ml, at least 18.5 mg / ml or at least 19 mg / ml of kappa light chain binding ligand. Figure 1a shows the correlation between ligand density and alkaline stability in terms of the % remaining capacity of the separation matrix for trastuzumab. Figure 1b shows the correlation between ligand density and Qb10% for Fab. From both these figures 1a and 1b, it is clear that a ligand density of at least 12 mg / ml is favorable for both alkaline stability and Qb10%.

[0066] Furthermore, as shown in the examples below, these preferred ligand densities provide improved alkaline stability as well as improved Qb10% compared to existing products on the market, which is further illustrated in Figures 3a and 3b.

[0067] The amount of coupled polypeptide / multimer can be controlled by the concentration of polypeptide / multimer used in the coupling process, by the coupling conditions used, and / or by the pore structure of the support used. Typically, the absolute binding capacity of the matrix increases with the amount of coupled polypeptide / multimer, at least up to the point where the pores become significantly compressed by the coupled polypeptide / multimer. The relative binding capacity per mg of coupled polypeptide / multimer decreases at higher coupling levels, resulting in a cost-benefit optimum within the above range.

[0068] The ligand may comprise a coupling element, said coupling element being one or more cysteine ​​residues, one or more lysine residues, or one or more histidine residues at the C-terminus of the ligand. Preferably, the ligand may comprise one or more cysteine ​​residues at the C-terminus of the ligand.

[0069] The coupling element may be linked directly to the C- or N-terminus or it may be linked via a linker comprising up to 15 amino acids, for example 1-5, 1-10 or 5-10 amino acids. This stretch should preferably be sufficiently stable in an alkaline environment so as not to impair the properties of the mutant ligand. To this end, it is advantageous if the stretch does not contain asparagine. It may be even more advantageous if the stretch does not contain glutamine. The advantage of having a C- or N-terminal cysteine ​​is that end-point coupling of the protein can be achieved through reaction of the cysteine ​​thiol with an electrophilic group on the support, as described above. This provides excellent mobility of the coupled protein, which is important for the binding capacity.

[0070] The ligand or multimer may be coupled to the support via a thioether bond. Methods for carrying out such coupling are well known in the art and are readily carried out by those skilled in the art using standard techniques and equipment. Thioether bonds are flexible and stable, and are generally suitable for use in affinity chromatography. The mobility of the coupled ligand / multimer is enhanced, particularly when the thioether bond is via a terminal or near-terminal cysteine ​​residue on the ligand or multimer, which provides improved binding capacity and binding kinetics. In some embodiments, the ligand / multimer is coupled via a C-terminal cysteine. This allows for efficient coupling of the cysteine ​​thiol to electrophilic groups, such as epoxide groups, halohydrin groups, etc., on the support, resulting in thioether cross-coupling. The ligand / multimer may be coupled, for example, via a single point bond, for example via a single cysteine, or by directed multipoint bond, for example using multiple lysines or other coupling groups near the termini of the polypeptide / multimer.

[0071] The solid support of the matrix according to the invention may be of any suitable known type. Conventional affinity separation matrices are often organic and based on polymers exposing hydrophilic surfaces to the aqueous medium used, i.e. exposing hydroxy (-OH), carboxy (-COOH), carboxamide (optionally N-substituted, -CONH2), amino (optionally substituted, -NH2), oligo or polyethyleneoxy groups on their external and, if present, internal surfaces as well. The solid support should be porous. Porosity can be expressed as a Kav or Kd value (fraction of pore volume available for a probe molecule of a certain size), measured by inverse size exclusion chromatography, for example according to the method described in Gel Filtration Principles and Methods, Pharmacia LKB Biotechnology 1991, pages 6-15 13. By definition, both Kd and Kav values ​​are always in the range of 0 to 1. In one embodiment, the porous support has a Kd value of 0.6 to 0.95, for example a Kd value of 0.7 to 0.90, or a Kd value of 0.6 to 0.8, measured using dextran of Mw 110 kDa as a probe molecule. The porous support may preferably have a Kd value of about 0.67. The porous support may preferably have a Kd value of about 0.72. The porous support may preferably have a Kd value of about 0.75.

[0072] The higher the Kd value, the larger the pores in the porous support, and the greater the proportion of the internal volume of the bead that is accessible to solute molecules, such as biomolecules, such as immunoglobulins.Those skilled in the art are aware of this and can calculate the Kd of the porous support as described above.Using larger pores, as mentioned above as preferred, allows a larger amount of ligand to be coupled to the porous support.Furthermore, as mentioned above, using larger pores allows kappa light chain-containing proteins to also access the ligands in the pores.Therefore, a larger binding capacity is achieved.

[0073] Multimeric ligands, such as pentamers or hexamers, provide higher DBCs compared to lesser multimeric ligands, such as tetrameric ligands or less. This effect is especially true for solid supports with high Kds of 0.7-0.9.

[0074] In certain embodiments, the support comprises a polyhydroxypolymer, such as a polysaccharide. Examples of polysaccharides include, for example, dextran, starch, cellulose, pullulan, agar, agarose, etc. Polysaccharides are hydrophilic in nature, have a low degree of non-specific interactions, offer a high content of reactive (activatable) hydroxyl groups, and they are generally stable to alkaline cleaning solutions used in bioprocessing.

[0075] In some embodiments, the support comprises agar or agarose. The support used in the present invention can be easily prepared according to standard methods, such as inverse suspension gelation (S Hjerten: Biochim Biophys Acta 79(2), 393-398 (1964)). Alternatively, the base matrix is ​​a commercially available product, such as cross-linked agarose beads sold under the name SEPHAROSE™ FF (Cytiva™). In a particularly advantageous embodiment for large-scale separations, the support is adapted to increase its rigidity using the methods described in US6602990 or US7396467, the entire contents of which are incorporated herein by reference, thus making the matrix more suitable for high flow rates.

[0076] In certain embodiments, the support, such as a polysaccharide or agarose support, is crosslinked, for example, using a hydroxyalkyl ether crosslink. Crosslinker reagents that generate such crosslinks can be, for example, epihalohydrins, such as epichlorohydrin, diepoxides, such as butanediol diglycidyl ether, allylation reagents, such as allyl halides or allyl glycidyl ether. Crosslinking is beneficial to the rigidity of the support and improves its chemical stability. Hydroxyalkyl ether crosslinks are alkaline stable and do not cause significant nonspecific adsorption.

[0077] Alternatively, the solid support is based on synthetic polymers, such as polyvinyl alcohol, polyhydroxyalkylacrylates, polyhydroxyalkylmethacrylates, polyacrylamides, polymethacrylamides, etc. In the case of matrices based on hydrophobic polymers, such as divinyl and monovinyl substituted benzenes, the surface of the matrix is ​​often hydrophilized to expose hydrophilic groups as defined above to the surrounding aqueous liquid. Such polymers are easily produced according to standard methods, see for example "Styrene based polymer supports developed by suspension polymerization" (R Arshady: Chimica e L'Industria 70(9), 70-75 (1988)). Alternatively, commercially available products are used, such as SOURCE™ (Cytiva™). In another alternative, the solid support according to the invention comprises an inorganic support, such as silica, zirconium oxide, etc. In another alternative, the support particles are magnetic. An example of such support particles is polysaccharide or synthetic polymer beads, including for example magnetite particles, so that the beads can be used in magnetic batch separation.

[0078] The separation matrix is ​​preferably in the form of beads or particles that are porous. The matrix in the form of beads or particles can be used as a packed bed or in a suspended form. Suspension forms include what are known as expanded beds and pure suspensions, in which the particles or beads are free to move. In the case of packed beds and expanded beds, the separation procedure usually follows conventional chromatography using a concentration gradient. In the case of pure suspensions, a batchwise mode is used. Preferably, the separation matrix according to the above is packed into a chromatography column.

[0079] Resins designed for large scale chromatography and bioprocessing generally may have a D50V typically from 30 μm up to 100 μm and a DW from 50 mg / mL to 200 mg / mL.

[0080] The flow rate should preferably be about 250-500 cm / h for a large scale column using a bed height of 20 cm at <3 bar back pressure and a bed volume of >3 L. One skilled in the art can calculate the flow rate for a column of another size, height and volume and then adjust the settings accordingly.

[0081] The porous support in bead or particle form according to the present disclosure has a dry weight (Dw) of 50-200 mg / ml, for example 50-150 mg / ml, 50-120 mg / ml, 50-100 mg / ml, 50-90 mg / ml, 60-80 mg / ml, or 60-75 mg / ml. Preferably, Dw is at least 63 mg / ml, or at least 65 mg / ml. Dw may be at least 70 mg / ml.

[0082] The porous support in bead or particle form may have a volume weighted median diameter (D50v) of 30 to 100. According to the present disclosure, D50v is preferably 35 to 90 μm, 40 to 80 μm, or 50 to 70 μm, for example 55 to 70 μm, 55 to 67 μm, 58 to 70 μm, or 58 to 67 μm. D50v may, for example, be at least 60 μm, or at least 62 μm.

[0083] The combination of the above ranges for Dw and d50v makes it possible to reach a high DBC. In particular, the combination of the above ranges for Dw and d50v, and the above ligand density makes it possible to reach a high Qb10%, as shown in Figure 2. This DBC is achievable using flow rates and back pressures that are still relevant for large-scale chromatography, as disclosed above.

[0084] Figure 2 shows contour plots for the prototype model at three different ligand densities. It is clear from these plots that the upper right corner of the window results in lower binding capacity. This is especially true when high ligand densities are obtained.

[0085] The alkaline stability of the matrix can be assessed by measuring the kappa light chain binding capacity, for example using a specific kappa light chain-containing protein or polyclonal human IgG, before and after incubation in an alkaline solution at a specified temperature, for example 22+ / -2°C. The incubation can be carried out, for example, in 0.1M NaOH for several 15 minute cycles, for example 100, 200 or 300 cycles. The binding capacity of the matrix after 100 15 minute incubation cycles in 0.1M NaOH at 22+ / -2°C can be at least 80%, for example at least 85%, at least 90% or at least 95% of the binding capacity before incubation. Alternatively, the incubation can be carried out in 0.1M NaOH for several 4 hour cycles, for example 6 cycles giving a total incubation time of 24 hours. The binding capacity of the matrix after a total incubation time of 24 hours in 0.1 M NaOH at 22+ / -2°C may be at least 80%, such as at least 85%, at least 90% or at least 95% of the binding capacity before incubation.

[0086] As shown in the examples below, the separation matrix according to the present invention provides improved alkaline stability compared to the commercially available Protein L Ligand resin. This is shown for example in Figure 4a. The separation matrix according to the above has a 10% breakthrough dynamic binding capacity for IgG of at least 55 mg / ml at a residence time of 4 minutes. Furthermore, the separation matrix according to the above has a 10% breakthrough dynamic binding capacity for IgG of at least 70 mg / ml at a residence time of 6 minutes (Qb10%). Even further, the separation matrix according to the above has a 10% breakthrough dynamic binding capacity for IgG of at least 80 mg / ml at a residence time of 10 minutes (Qb10%).

[0087] According to 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 or a combination of several washing solutions; c) eluting the kappa light chain-containing protein from the separation matrix using an elution solution; and d) cleaning the separation matrix with a cleaning solution Provided herein is a method for isolating a kappa light chain-containing protein comprising:

[0088] According to one embodiment, the separation matrix disclosed above is used in the method. As can be seen in Figure 8a, the method of isolating kappa light chain-containing proteins according to the present invention provides complete capture of kappa light chain (VL) from CCF. As shown in Figure 8b, there is no detectable presence of VL in the flow-through fraction.

[0089] Elution may be performed by using any suitable elution solution used for elution from Protein L separation matrix. The elution solution may be, for example, a solution or buffer having a pH of 4 or less, such as a pH of 2.5-4 or 2.8-3.5. The elution buffer or elution buffer gradient may comprise at least one mono-, di- or trifunctional carboxylic acid or a salt of such a carboxylic acid. The elution buffer or elution buffer gradient may comprise at least one anion species selected from the group consisting of acetate, citrate, glycine, succinate, phosphate, and formate.

[0090] The generation of bispecific antibodies or IgG molecules is difficult because the pairing of light and heavy chains, and therefore the pairing of the variable domains (VL; VH) therein, can be indiscriminate. The pairing of two different light chains and two different heavy chains can result in a large number of mispairings, since usually only one specific asymmetric combination is desired and many combinations achieved are non-functional or undesirable molecules, such as monospecific homodimers. Therefore, there is an increasing need for improved tools and methods for separating bispecific antibodies from monospecific antibodies, as well as 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.

[0091] 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 or a combination of several washing solutions; c) eluting the kappa light chain-containing protein from the separation matrix at a decreasing pH with an elution solution; and d) cleaning the separation matrix with a cleaning solution Provided herein is a method for separating bispecific antibodies comprising:

[0092] According to one embodiment, a separation matrix as disclosed above is used in said method.

[0093] The above method may also be used for the isolation of trispecific antibodies.

[0094] The pH may be decreased by using a pH gradient. Alternatively, the pH may be decreased in a stepwise manner using buffer solutions of different pHs, similar to the gradient. The pH range during elution may be from about 5.5 to about 2, for example, from about 5 to about 2, from about 4.5 to about 2, or from about 4 to about 2. Other than pH, the eluent in the separation method is as disclosed for the isolation method above.

[0095] This method allows for the separation of bispecific antibodies from monospecific 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. Figures 5 to 7 show the results of the method disclosed above, Figures 5 and 6 show the results of a stepwise decrease in pH, and Figure 7 shows the results of a pH gradient to decrease the pH.

[0096] Any antibody that does not contain a kappa light chain, such as two lambda light chains, does not bind to the separation matrix and is consequently 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. During elution, antibodies that only have one kappa light chain elute before antibodies that have two kappa light chains. This is illustrated in Figure 5b and Figure 7 by the schematic diagrams of the respective antibodies as they elute or are present in the effluent.

[0097] By using the above disclosed separation matrix that binds subclass 1, 3 or 4 kappa light chains, it is also possible to separate antibodies or antibody fragments that contain one or two kappa light chains of subclass 2 from antibodies or antibody fragments that contain one or two kappa light chains of subclass 1, 3 or 4. By the same principle as above, antibodies that contain two kappa light chains of subclass 2 or antibodies that contain one kappa light chain and a lambda light chain of subclass 2 do not bind to the separation matrix and, as a result, are present in the effluent stream during step a) or are washed away during step b). Antibodies with one kappa light chain of subclass 2 and one kappa light chain of any of subclasses 1, 3 or 4 elute before antibodies with two kappa light chains of any of subclasses 1, 3 and 4.

[0098] As shown in Figure 5b, by decreasing the pH in the elution step in a stepwise manner, three unique peaks are formed. MS analysis of the eluate shows that the eluate from peak 1 contains lambda light chain dimers, peak 2 contains dimers containing kappa light chains and lambda light chains, and peak 3 contains kappa light chain dimers, see Figure 7. By decreasing the pH in the pH gradient, only two peaks were observed. MS analysis of the eluate (data not shown) showed that the first peak contains dimers containing kappa light chains and lambda light chains, and the second peak contains kappa light chain dimers.

[0099] The method may also comprise, prior to step a), the steps of providing an affinity separation matrix as described above and providing as a liquid sample a liquid sample solution comprising a kappa light chain-containing protein and at least one other substance. The method may further comprise the step of equilibrating the separation matrix with an equilibration buffer prior to adding the liquid sample.

[0100] The wash solution is usually a buffer similar to or the same as the equilibration buffer. Usually, two or more wash solutions are used. For example, a first wash solution may have a high salt content and a neutral pH, followed by a second wash solution that is salt-free and has a lower pH. The use of a high salt content in the wash solution improves the removal of impurities.

[0101] The liquid sample comprising the kappa light chain-containing protein and at least one other substance may also contain host cell proteins (HCPs), such as Chinese Hamster Ovary (CHO) cells, Escherichia coli (E. coli) or yeast cell proteins. The content of CHO cells and E. coli proteins can be conveniently determined by immunoassays directed against these proteins, such as the CHO HCP or E. coli HCP ELISA kits from Cygnus Technologies. The host cell proteins or CHO cells / E. coli / yeast proteins may be desorbed during step b).

[0102] The method may also comprise, after step c), recovering the eluate and optionally subjecting it to a further separation step, for example by anion or cation exchange chromatography, multimodal chromatography and / or hydrophobic interaction chromatography. Suitable compositions of liquid samples, washing solutions and elution solutions, as well as the general conditions for carrying out the separation, are well known in the art of affinity chromatography and in particular in the art of Protein L chromatography.

[0103] The cleaning liquid may preferably be alkaline, such as having a pH of 12-14. Such a solution provides efficient cleaning of the matrix, especially at the upper end of the range. The cleaning liquid may comprise 0.01-1.0 M NaOH or KOH, for example 0.05-1.0, or 0.05-0.5, or 0.05-0.3, or 0.05-0.1 M NaOH or KOH. The high stability of the separation matrix of the invention allows the use of such relatively strong alkaline solutions.

[0104] The IgG capacity of the separation matrix according to above after 24 hours of incubation in 0.1 M NaOH at 22+ / -2°C is at least 80%, or at least 85%, or at least 90%, or at least 95% of the IgG capacity before incubation.

[0105] After cleaning with 130 cycles in 0.3 M NaOH at 22 + / - 2°C with a contact time of 15 minutes per cycle, the IgG capacity of the separation matrix is ​​at least about 90% of the IgG capacity before incubation. In other words, after incubation in 0.3 M NaOH for 32 hours at 22 + / - 2°C, the IgG capacity of the separation matrix is ​​at least about 90% of the IgG capacity before incubation.

[0106] After cleaning with 50 cycles in 0.5 M NaOH at 22 + / - 2°C with a contact time of 15 minutes per cycle, the IgG capacity of the separation matrix is ​​at least 95%, or at least 93%, of the IgG capacity before incubation. In other words, after incubation in 0.5 NaOH for 12 hours at 22 + / - 2°C, the IgG capacity of the separation matrix is ​​at least 95%, or at least 93%, of the IgG capacity before incubation.

[0107] Thus, the separation matrix of the present disclosure can withstand cleaning with alkaline solutions at concentrations higher than those typically used for separation matrices based on Protein L ligand.

[0108] The cleaning of the separation matrix is ​​preferably a CIP process (cleaning in place), which is a process well known to those skilled in the art.

[0109] Steps a)-d) may be repeated at least 10 times, such as at least 50 times or 50-200 times. This is important for process economics in that the matrix can be reused many times. This is shown in Figure 1c and Figure 3, where a separation matrix according to the above maintains a high Qb10% even after 100+ cycles of CIP.

[0110] The high capacity for the present separation matrix provides productivity advantages in biomanufacturing. The improved alkaline stability results in a more robust process and longer resin life, which adds to the overall process economics. Thus, the present separation matrix provides significantly improved process economics for the separation step. It also adds value in terms of key process parameters compared to other commercially available products. EXAMPLES

[0111] Ligand characterization Example 1 Affinity Testing for Ligands - Biacore Protein L variants were immobilized on CM5 Chips (Cytiva™, Sweden) using amine coupling (see Biacore Sensor Surface Handbook, https: / / cdn.cytivalifesciences.com / dmm3bwsv3 / AssetStream.aspx?mediaformatid=10061&destinationid=10016&assetid=16475).

[0112] EDC / NHS was injected onto both flow cells for 420 s at a flow rate of 10 μl / min to activate the CM5 surface, followed by washing the system (but not the sensor surface) with ethanolamine. The variants in immobilization buffer were injected onto flow cell 2 and immobilized onto the activated CM5 surface. Ethanolamine was injected onto both flow cells for 420 s at a flow rate of 10 μl / min to inactivate the surfaces.

[0113] solution: EDC 0.4M 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide in water NHS 0.1M N-hydroxysuccinimide in water Ethanolamine 1M Ethanolamine-HCl pH 8.5 Ligand: Typically 10-50 μg / mL in immobilization buffer

[0114] Injection procedure: Flow rate Contact time 1. EDC / NHS (to activate the surface) 10 μL / min 7 min 2. Ethanolamine (does not pass over the surface) Cleaning - 3. Ligand 10μL / min 7 min4. Ethanolamine (to deactivate excess reactive groups) 10 μL / min 7 min

[0115] Protein L variants were then evaluated for their binding and apparent affinity to IgG (Gammanorm). The following assay conditions were used: Analyte: Gammanorm diluted in HBS-EP+ (universal running buffer; 0.1 M HEPES, 1.5 M NaCl, 0.03 M EDTA, and 0.5% v / v surfactant P20; Cytiva™). Analyte concentrations: 0.026, 0.053, 0.106, 0.2125, 0.425, 0.85, 1.7, 3.4 μM Regeneration: 10mM glycine pH 1.5 Assay: 10 min association, 10 dissociation, 2x regeneration; flow rate: 10 μl / min

[0116] Evaluation was performed with Biacore Insight Evaluation Software, in which kinetic and affinity fitting to sensorgrams (not shown) was performed. K for steady-state affinity D and 1:1 kinetics were obtained from the software.

[0117] [Table 1]

[0118] Example 2 Alkaline Stability of Ligands - Biacore The immobilized Protein L ligand was evaluated for NaOH stability using the following assay conditions: Analyte: Gammanorm diluted in HBS-EP+ (universal running buffer; 0.1 M HEPES, 1.5 M NaCl, 0.03 M EDTA, and 0.5% v / v surfactant P20; Cytiva™). Analyte concentration: 3.4 μM Regeneration: 10mM glycine pH 1.5 NaOH concentration: 100mM Assay: 10 min analyte association, 1 min analyte dissociation, 10 min NaOH injection, 1 min NaOH dissociation, wait 1 min, 2x regeneration, wait 1 min -> repeat 100 cycles. Flow rate: 10 μl / min

[0119] The evaluation was carried out with Biacore Insight Evaluation software, where the responses in the report point "Late Binding" were collected. Cycle 1 (start) and cycle 2 (first cycle of analysis) were excluded from the evaluation. The response in the first evaluation cycle (cycle 3) was set to 100%, and the responses in the following 99 cycles were set to the percentage of the response from the first cycle.

[0120] [Table 2]

[0121] The results show that all variants except pAM240 have better NaOH stability compared to pAM114 (wt B2 domain). The HHY variant appears to have slightly better NaOH stability compared to HHQ. However, in general, both variants are shown to function well. NaOH stability is primarily domain dependent, with B5, C1, C2 and C3 showing the lowest alkaline stability. For both variants HHY and HHQ, C3b, B3, C2b, B2, B4, C4 and D1 all show very good alkaline stability.

[0122] From the experiments disclosed above, it is clear that the mutations N10H, N45H, as well as N60Y or N60Q have a positive effect on NaOH stability for Protein L, while the affinity is maintained. It is noteworthy that this effect is shown for a broader range of Protein L domains, in particular for the B2, B3, B4, C2b, C3b, C4 and D1 domains.

[0123] Separation Matrix For the prototype tested, the ligand B2-1 (pAM237) was used, which corresponds to SEQ ID NO: 3. The pentamer of SEQ ID NO: 3 was expressed and purified by conventional means known to those skilled in the art.

[0124] Example 3 Coupling and filling Purified pentamer of SEQ ID NO:3 was immobilized on agarose beads as a base matrix according to the exemplary method below.

[0125] activation The base matrix used was rigid cross-linked agarose beads with the volume weighted median diameter indicated, prepared according to the method of US6602990, and with the indicated pore size corresponding to a reverse gel filtration chromatography Kav value of 0.70 for Mw 110 kDa dextran, according to the method described in Gel Filtration Principles and Methods, Pharmacia LKB Biotechnology 1991, pp. 6-13.

[0126] 25 mL (g) of drained base matrix, 10.0 mL of distilled water, and 2.02 g of NaOH(s) were mixed in a 100 mL flask with mechanical stirring for 10 minutes at 25° C. 4.0 mL of epichlorohydrin was added and the reaction was allowed to proceed for 2 hours. The activated gel was washed with 10 gel settling volumes (GV) of water.

[0127] Coupling To the aqueous protein solution, sodium carbonate (0.01 M), sodium bicarbonate (0.1 M), sodium chloride (0.15 M) and EDTA disodium salt (1 mmol) were added and once everything was dissolved, dithiothreitol (DTT, 0.1 M) was added. The pH was adjusted to pH > 8.0. The reaction mixture was placed on a shaking table (23° C., 500 rpm) and left to reduce for 2 hours.

[0128] Proteins were desalted using a PD10 prepacked gel filtration column (Cytiva™). The column was equilibrated with desalting solution (0.15 NaCl, 1 mM EDTA) before loading the protein (up to 2.5 mL). Eluted fractions were collected and combined.

[0129] The protein concentration of the desalted solution was determined by UV absorbance at 276 nm using a protein extinction coefficient of 1.0.

[0130] The activated gel was washed with 5 GV of 0.1 M trisaminomethane (Tris) buffer pH 8.4. 15 mL of gel, 20 mg of ligand / mL gel ((11.7 mL), 3.3 mL of Tris buffer and 7.0 g of sodium sulfate were mixed in a 50 mL flask and stirred at 33° C. for 4 hours.

[0131] After fixation, the gel was washed with 3 x 1 GV distilled water. Gel and 1 GV (0.1 M phosphate / 1 mM EDTA / 7.5% thioglycerol pH 8.5) were mixed and the flask was left stirring at room temperature for 15-20 hours. The gel was then washed three times alternately with 3 x 1 GV 0.5 M HAc and 3 x 1 GV 0.1 M TRIS / 0.15 M NaCl pH 8.5, then 10 x 1 GV mL distilled water. The gel was conditioned in 20% EtOH in a 50% slurry.

[0132] Amino acid analysis, determining ligand density through AAA All synthesized prototypes were dried and the dry mass was determined. Those skilled in the art are aware of commonly known methods for carrying out such a procedure. The prototypes were then dried and sent for amino acid analysis. Using an Excel spreadsheet containing all the data on the corresponding dry mass, as well as protein size information and primary amino acid sequence of the protein, the ligand density could be obtained in mg ligand / mL resin.

[0133] Packing of separation matrix prototypes 2 ml of the resin was packed into a TRI CORN™ 5 / 100 column (Cytiva™).

[0134] Example 4 Dynamic binding capacity (DBC) Preparation of trastuzumab samples Herceptin (Trastuzumab) solutions were diluted to 2 mg / mL. Sample solution concentrations were determined by triplicate measurements of absorbance in a spectrophotometer (QS High Precision cell, Hellma Analytics) at 280 nm using an absorption coefficient of 1.48 mL / mg*cm. Trastuzumab concentrations were confirmed by in-line UV measurements at 280 nm in a 2 mm UV cell in a Unicorn 7.5.

[0135] Determination of 100% absorbance signal PBS buffer was allowed to flow through the bypass position until a stable baseline was reached. Autozero was performed and Trastuzumab solution was applied through the bypass to obtain a stable 100% signal, 6 minutes at room temperature. Absorbance values ​​were noted. PBS buffer was allowed to flow through the bypass position until a stable baseline was reached.

[0136] DBC measurement Adsorption / equilibration buffer: Phosphate buffer 20 mM + 0.15 M NaCl, pH 7.2 Elution buffer: Citrate buffer 50 mM, pH 2.5 CIP: 0.1M NaOH

[0137] The trastuzumab sample was loaded onto the 2 ml column via an S pump (sample pump) at AKTA™ pure 25 M at the desired flow rate (depending on residence time) until a UV signal of approximately 20% of maximum was reached. The column was then washed with adsorption buffer at a flow rate of 1 mL / min. The protein was eluted with elution buffer at a flow rate of 0.8 mL / min.

[0138] The column was cleaned with 0.1 M NaOH at a flow rate of 0.2 mL / min for 3 CV with a contact time of 15 min using a CIP protocol, followed by re-equilibration with adsorption buffer. The column was manually cleaned with 20% EtOH.

[0139] Breakthrough capacity was calculated using Exctensions-DBC Calculations-Analyze, an evaluation tool in the Unicorn software used with the AKTA™ pure 25 M.

[0140] The following formula was used to calculate the breakthrough capacity at 10% (Qb10), which is the amount of trastuzumab loaded onto the column until the concentration of trastuzumab in the column effluent is 10% of the concentration of trastuzumab in the liquid sample:

[0141]

number

[0142] A 100% = 100% UV signal A sub = absorbance contribution from unbound mAb A(V) = absorbance in a given applied volume V C = column volume V app = Volume applied until 10% breakthrough V sys = Dead volume of the system C 0 = Liquid sample concentration

[0143] Correspondingly, the breakthrough capacity at 80% (Qb80) is the amount of trastuzumab that is loaded onto the column until the concentration of trastuzumab in the column effluent is 80% of the concentration of trastuzumab in the liquid sample.

[0144] [Table 3]

[0145] Selected ligand densities were further tested for alkaline stability.

[0146] [Table 4]

[0147] The results from Table 4 are plotted in FIG. 1a, where a clear correlation between ligand density and alkaline stability can be observed.

[0148] Additionally, a selection of ligand densities were tested for DBC at 10% breakthrough using Fab fragments at a dwell time of 6 minutes.

[0149] Fab fragments were generated from trastuzumab by papain digestion. The trastuzumab solution was adjusted to pH 7.4 by 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. Antipain (papain inhibitor) was then 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.

[0150] [Table 5]

[0151] The results from Table 5 are plotted in FIG. 1b, where again a clear correlation between ligand density and DBC can be observed.

[0152] Figure 2 shows the DBC in relation to ligand density, Kd and Dw. It is clear that the ranges disclosed herein for KD, d50V and ligand density for the alkali-stabilized protein L domains disclosed herein provide favorable DBC.

[0153] Selected ligand densities were then specifically tested for alkaline stability.

[0154] [Table 6]

[0155] As can be seen in Figure 1c, all of the prototypes show a Qb10% of over 80% after 112 CIP cycles. In particular, the prototypes with ligand densities within the range disclosed for this invention show a Qb10% of over 90%. The least functional prototypes have a ligand density of less than 12 mg / ml.

[0156] Example 5 Alkaline Stability DBCs with three different ligand densities (base matrix: KD 0.75, Dw 66.5mg / ml and d50v 62.3μm) were tested and compared with two commercial products during the CIP process. The antibody used in the study was trastuzumab.

[0157] Trastuzumab was diluted to 2 mg / mL. The concentration can be determined according to any method known in the art.

[0158] The prototypes shown were included in an accelerated alkaline stability study to investigate how stability depends on ligand density. Capto™ L was included for reference. DBC Qb at 4 min residence time with trastuzumab measured during incubation in 100 mM NaOH 10% The data are presented in Table 5.

[0159] The stripping step was performed in 50 mM citrate pH 2.3, followed by equilibration (20 mM sodium phosphate + 150 mM NaCl pH 7.2) and then incubation in 100 mM NaOH. Incubation was carried out for 4 hours, corresponding to 16 cycles of CIP with a contact time of 15 minutes. DBC Qb with Trastuzumab at a dwell time of 4 minutes 10% was measured between incubation runs. Dynamic binding capacity was measured with trastuzumab and the relative remaining capacity to the initial DBC was calculated between incubations corresponding to a total of 100 cycles with CIP.

[0160] [Table 7]

[0161] The results are shown in Figure 3a. The same results recalculated as relative DBC are shown in Figure 3b. Starting DBC Qb 10% The values ​​show that the prototype 17.7 mg / ml exhibits a lower capacity compared to the other prototype at 14.2 mg / ml. However, the DBC Qb for the prototype 17.7 mg / ml 10% increases with the number of CIP cycles, i.e., incubation time in NaOH, indicating better alkaline stability compared to the other prototype at 14.2 mg / ml (FIG. 3a).

[0162] To compare the separation matrices with respect to alkaline stability, starting DBC Qb 10% Relative residual DBC Qb value 10% was calculated over the number of CIP cycles. After 100 cycles, i.e., a total of 25 hours of incubation in 100 mM NaOH, all prototypes included in this study achieve more than 80% remaining capacity.

[0163] Thus, it is clear that both prototypes tested, with ligand densities of 14.2 mg / ml and 17.7 mg / ml, have very good DBC after 100 cycles, indicating a higher DBC than Capto™ L.

[0164] Additionally, we tested whether separation matrices according to the present disclosure could withstand cleaning under alkaline concentrations higher than those traditionally used for Protein L separation matrices. A Tricorn 5 / 50 column was packed with the prototype resin containing 17.7 mg / ml ligand, as disclosed above.

[0165] 300mM NaOH The column was subjected to 15 min CIP with 300 mM NaOH / cycle using trastuzumab. DBC Qb10% was determined for a 3 min residence time. Experiments were performed with AKTA™ pure 25 M.

[0166] Equilibration: 20mM Na Phosphate 150mM NaCl pH7.2, 1ml / min, 5CV Sample load: via sample pump, 0.333 ml / min (3 min at room temperature, up to 20% of absorbance maximum) Wash with 10 mM Na phosphate, 150 mM NaCl pH 7.2, 1 ml / min, 7 CV Elution: 50 mM Na citrate pH 2.5, 1 ml / min, 5 CV Wash: Milli-Q water, 1 ml / min, 5 CV CIP: 300 mM NaOH, 0.2 ml / min, 3 CV (15 min contact time) Re-equilibration: 20mM Na Phosphate 150mM NaCl pH7.2, 1ml / min, 5CV

[0167] [Table 8]

[0168] 500mM NaOH The column was run with Trastuzumab at 15 min CIP with 500 mM NaOH / cycle. DBC Qb10% was determined for a 4 min residence time. Experiments were performed with AKTA™ pure 25 M.

[0169] Equilibration: 20mM Na Phosphate 150mM NaCl pH7.2, 1ml / min, 5CV Sample load: via sample pump, 0.25 ml / min (4 min at room temperature, up to 20% of absorbance maximum) Wash with 10 mM Na phosphate, 150 mM NaCl pH 7.2, 1 ml / min, 7 CV Elution: 50 mM Na citrate pH 2.5, 1 ml / min, 5 CV Wash: Milli-Q water, 1 ml / min, 5 CV CIP: 500 mM NaOH, 0.2 ml / min, 3 CV (15 min contact time) Re-equilibration: 20mM Na Phosphate 150mM NaCl pH7.2, 1ml / min, 5CV.

[0170] [Table 9]

[0171] As can be seen in Figure 3c, where the relative remaining capacity for 0.1 M NaOH, 0.3 M NaOH and 0.5 M NaOH are plotted, the separation matrix of the present disclosure has excellent alkaline stability even for higher alkaline concentrations. For 0.1 M NaOH, the separation matrix has 90% remaining capacity after 200 cycles. For 0.3 M NaOH, the separation matrix has approximately 90% remaining capacity after 130 cycles, and for 0.5 M NaOH, the separation matrix has approximately 90% remaining capacity after 50 cycles.

[0172] Example 6 Comparison Test - DBC Next, DBC Qb using adalimumab, Fab, dAb and trastuzumab 10%was measured at residence times of 1, 2.4, 4, 6 and 10 minutes for the prototype (with ligand density of about 14.3 mg / ml, KD of about 0.75, Dw of about 66.5 mg / ml and d50v of about 62.3 μm).

[0173] Fab was produced as disclosed above. dAb was produced as described in the paper "Recombinant production of a V L The antibody was produced according to the method described in "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).

[0174] Data, DBC Qb 10% The results are presented in Table 10 together with the

[0175] [Table 10]

[0176] The results were: DBC Qb 10% reached an optimum at residence times of 6 min or more, whereas DBC Qb 10% can potentially be increased further at higher residence times. The kinetics for dAbs are very fast, whereas the DBC Qb for the smaller fragments is 10% The optimum is already reached at a residence time of 2.4 min for the prototype.

[0177] The prototype resin was then compared to three commercially available Protein L resins. The prototype resin was packed into triplicate Tricorn 5 / 100 columns and DBC was performed according to the method described above. DBC Qb10% was determined at the indicated retention times using antibodies or antibody fragments for the prototype, TOYOPEARL® AF-rProtein L-650F (Tosoh), KanCap™ L (Kaneka) and Capto™ L (Cytiva™). The average of the data from the triplicate columns is presented below.

[0178] [Table 11]

[0179] The results shown in Figure 4a show that for a residence time of 1 minute, the prototype performs slightly better than Capto™ L and KanCap™ L, while TOYOPEARL® AF-rProtein L-650F exhibits a higher capacity. However, for a residence time of 2.4 minutes, the prototype and TOYOPEARL® AF-rProtein L-650F are comparable, and for residence times of 6, 8, and 10 minutes, the prototype exhibits a significantly higher DBC Qb than TOYOPEARL® AF-rProtein L-650F. 10% It presents.

[0180] Compared to Capto™ L, the prototype showed 2x higher DBC Qb for higher residence times (6-10 min). 10% It presents.

[0181] DBC was also determined using Fab fragments at the indicated retention times on duplicate columns with the indicated resins according to the method described above.

[0182] [Table 12]

[0183] The results show that the prototype has significantly better DBC Qb performance at all residence times for Fab compared to TOYOPEARL® AF-r Protein L-650F and KanCap™ L, as well as compared to Capto™ L. 10% See FIG. 4b.

[0184] In addition, DBCs were determined using dAbs at the indicated retention times on duplicate columns with the indicated resins according to the method described above.

[0185] [Table 13]

[0186] Again, the results show that the prototype not only exhibits significantly better DBC Qb activity at all retention times for dAbs compared to TOYOPEARL® AF-rProtein L-650F, but also compared to Capto™ L. 10% See FIG. 4c.

[0187] Example 7 Bispecific antibody isolation The ability to separate bispecific antibodies from monospecific antibodies on the basis of their kappa light chains was tested. The separation matrix as in the previous example, packed in a Tricorn 5 / 100 column, was used in this experiment.

[0188] The Ab-containing liquid sample (bsAb01) tested in this experiment is a commercially available sample from Thermo Fisher containing a) κ light chain, trastuzumab kappa class 1 anti-HER2 light chain (1 and 2); b) λ light chain, abelmab lambda class 2 anti-PDL1 light chain; and c) FC chain, anti-HER2 heavy chain (1 and 2), which are present in the sample in a ratio of 30:30:40, respectively.

[0189] Stepwise decrease in pH during dissolution

[0190] [Table 14]

[0191] Liquid samples were prepared by thawing at room temperature and filtering through a 0.2 μm filter. The concentration of mAb in the liquid samples was estimated to be approximately 0.1 mg / ml based on peak integration.

[0192] After column equilibration with equilibration buffer, 50 ml of liquid sample was applied onto the column at 0.250 ml / min, followed by 10 CV of wash buffer.

[0193] A step elution was performed by first applying 5 CV of pH 3.4 elution buffer 1 followed by 5 CV of pH 3.1 elution buffer 2 at a flow rate of 1.0 ml / min. After elution, a 3 CV pH 2.3 strip step was performed followed by a 3 CV 0.1 M NaOH CIP step. Fractions were collected using peak fractionation. The results are shown in Figure 5a. This was repeated with the elution volume changed to 10 CV. The results are shown in Figure 5b.

[0194] As can be seen in Figure 5a, after the first 5 CV elution volume was run, three distinct peaks could be observed, with some tailing and overlap between the major peaks. An initial minor peak eluting at pH 4.45 was also observed.

[0195] At an elution volume of 10 CV, baseline separation of the κλ-heterodimers and κκ-homodimers is achieved, as can be seen in FIG. 5b.

[0196] Fractions from the 10 CV run were analyzed by mass spectrometry (MS) in full size and reduced form, these fractions are marked with an * in Figure 5b.

[0197] Non-reduced MS results from fraction 1 (peak 1 in Figure 5b) showed that it resembled the full size mAb (data not shown). When the sample was reduced, some peaks corresponding to triplets of light chain peaks were split, and FC and unknown peaks were observed (data not shown). The light chain observed was of the same mass as that observed for the λ light chain of the κλ-dimer. The FC was of the same mass as that observed for the κλ-dimer and κκ-dimer. This λλ-dimer elutes very easily at pH 4.45 and could possibly be removed or isolated by increased washing of the resin after sample application or a less steep initial pH gradient.

[0198] Peak 2 in Figure 5b is a bispecific κλ mAb with a full size of 147406 Da, see Figure 6a. When reduced, two types of light chains could be observed (data not shown).

[0199] Peak 3 in Figure 5b is the κκ-homodimer, with a small percentage of free κ-light chain observed (see Figure 6b). In the reduced MS data (data not shown), only one type of light chain was observed, which corresponded to the size of the κ-light chain observed for the κλ-bispecific mAb.

[0200] Thus, stepwise pH separation of bispecific kappa light chain-containing monoclonal antibodies is possible using the separation matrix according to the invention. By simple adjustment of the elution volume at each pH baseline, separation of bispecific heterodimers and homodimers can be achieved.

[0201] Elution with a pH gradient

[0202] [Table 15]

[0203] Adsorption / equilibration buffer: Phosphate buffer 20 mM + 0.15 M NaCl, pH 7.2 Elution buffer: 50 mM citrate buffer, pH 5.5, 50 mM citrate buffer, pH 2.5 Strip buffer: 50 mM citrate, pH 2.3 CIP: 0.1M NaOH

[0204] After equilibration, approximately 100 ml of the BsAb_mAb01 sample was loaded onto the 1 ml column via the B pump at a desired flow rate of 0.25 ml / min. The column was then washed with wash buffer for 10 CV at a flow rate of 1 mL / min. Protein was eluted using a pH gradient of 50 mM citrate pH 5.5 to 2.5 over 40 CV at a flow rate of 0.25 ml / min.

[0205] The column was cleaned with 50 mM citrate pH 2.3 at a flow rate of 0.4 mL / min, followed by a strip with 0.1 M NaOH, and finally re-equilibrated with 10 CV of adsorption buffer.

[0206] The eluate was collected by peak splitting starting at 50 mAu.

[0207] The bound fraction elutes in two major peaks A and B at pH 3.36 and pH 3.12, see Figure 7. Presumably these peaks correlate with the bispecific κλ-heterodimers eluting first at pH 3.26 and the κκ-homodimers eluting later at pH 3.12. The λλ-homodimers do not bind and do not elute during the gradient.

[0208] As shown in Figure 7, samples from fractions A6 and B4 were also reduced and then analyzed by MS (data not shown). This analysis clearly showed that A6 is composed of κ-light chain (23439 Da), λ-light chain (22785 Da) and FC chain (25297 Da). B4 was shown to be composed only of κ-light chain (23439 Da) and FC chain (25297 Da).

[0209] Thus, the above examples show that the separation matrices according to the appended claims have improved alkaline stability as well as DBC at 10% breakthrough for all antibodies or antibody fragments tested compared to similar commercial products.

[0210] The written description uses examples to disclose the invention and also enables one skilled in the art to practice the invention. The patentable scope of the invention is defined by the claims and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal words of the claims, or if they include equivalent structural elements that have minor differences from the literal words of the claims. Any patents or patent applications mentioned herein are incorporated by reference in their entirety into this specification as if they were individually incorporated.

Claims

1. A separation matrix comprising at least 12 mg / ml of a kappa light chain binding ligand covalently coupled to a porous support, the kappa light chain binding ligand comprises, consists essentially of, or consists of a multimer of an alkali-stabilized Finegordia magna (formerly Peptostreptococcus magnus) protein L domain; A separation matrix, wherein said porous support comprises polymer particles having a dry solids weight (Dw) of 50-200 mg / ml and a volume weighted median diameter (D50v) of 30-100 μm.

2. 2. The separation matrix of claim 1, wherein the matrix comprises at least 14 mg / ml of kappa light chain binding ligand, such as at least 14.5 mg / ml, at least 15 mg / ml, at least 15.5 mg / ml, at least 16 mg / ml, at least 16.5 mg / ml, at least 17 mg / ml, at least 17.5 mg / ml, at least 18 mg / ml, at least 18.5 mg / ml, or at least 19 mg / ml of kappa light chain binding ligand.

3. The porous support has a D of 50 to 150 mg / ml, 50 to 120 mg / ml, 50 to 100 mg / ml, 50 to 90 mg / ml, 60 to 80 mg / ml, or 60 to 75 mg / ml, for example at least 63 mg / ml, or at least 65 mg / ml, or at least 70 mg / ml. W 3. The separation matrix according to claim 1, comprising:

4. 4. The separation matrix according to any one of claims 1 to 3, wherein the porous support has a volume weighted median diameter (D50v) of 35 to 90 μm, 40 to 80 μm, 50 to 70 μm, 55 to 70 μm, 55 to 67 μm, 58 to 70 μm, or 58 to 67 μm, such as at least 60 μm, or at least 62 μm.

5. 5. The separation matrix according to claim 4, having a Kd value of 0.6 to 0.95, such as a Kd value of 0.7 to 0.9, or a Kd value of 0.6 to 0.8, such as a Kd value of about 0.67, or a Kd value of about 0.72, or a Kd value of about 0.75, measured by inverse size exclusion chromatography using dextran of Mw 110 kDa as a probe molecule.

6. 6. The separation matrix according to claim 1, wherein the polymer particles are crosslinked.

7. 7. A separation matrix according to any one of claims 1 to 6, wherein at least two of the alkaline-stabilized protein L domains are selected from the group comprising functional variants of the B1, B2, B3, B4, B5, C2, C3, C4 and D1 domains of Finegordia magna (formerly Peptostreptococcus magnus) protein L, wherein the positions corresponding in the alignment to positions 10 and 45 in the B2 domain (SEQ ID NO: 1) are histidine and the position corresponding in the alignment to position 60 in the B2 domain (SEQ ID NO: 1) is tyrosine or glutamine.

8. 8. The separation matrix of claim 7, wherein the at least two alkali-stabilized protein L domains are selected from the group comprising B2 domain, B3 domain, B4 domain, C2 domain, C3 domain, C4 domain and D1 domain.

9. 9. The separation matrix of claim 7 or 8, wherein at least two alkali-stabilized Protein L domains have at least 90%, 95% or 98% sequence identity, or 77.5% sequence similarity, with 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 with a gap open penalty of 12 and a gap extension penalty of 3, and wherein 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 are unchanged.

10. 10. A separation matrix according to claim 1, comprising 3, 4, 5, 6, 7, 8 or 9 alkali-stabilised protein L domains.

11. 11. The separation matrix of claim 10, comprising 4, 5 or 6 alkali-stabilized protein L domains.

12. 12. The separation matrix according to claim 1, wherein the ligand comprises a coupling element, said coupling element being one or more cysteine ​​residues, one or more lysine residues, or one or more histidine residues at the C-terminus of the ligand.

13. 13. The separation matrix according to claim 12, wherein the ligand comprises one or more cysteine ​​residues at the C-terminus of the ligand.

14. 14. The separation matrix according to claim 1, having a 10% breakthrough dynamic binding capacity for IgG of at least 55 mg / ml at a residence time of 4 minutes.

15. 15. The separation matrix according to any one of claims 1 to 14, having a 10% breakthrough dynamic binding capacity for IgG of at least 70 mg / ml at a residence time of 6 minutes.

16. 16. The separation matrix according to any one of claims 1 to 15, having a 10% breakthrough dynamic binding capacity for IgG of at least 80 mg / ml at a residence time of 10 minutes.

17. 17. The separation matrix according to any one of claims 1 to 16, wherein the IgG capacity of the separation matrix after incubation for 24 hours in 0.1 M NaOH at 22 + / - 2°C is at least 80%, or at least 85%, or at least 90%, or at least 95% of the IgG capacity before incubation.

18. 17. The separation matrix according to any one of claims 1 to 16, wherein the IgG capacity of the separation matrix after incubation for 32 hours in 0.3 M NaOH at 22 + / - 2°C is at least 90% of the IgG capacity before incubation.

19. 17. A separation matrix according to any one of claims 1 to 16, wherein the IgG capacity of the separation matrix after incubation for 12 hours in 0.5 M NaOH at 22 + / - 2°C is at least 95%, or at least 93%, of the IgG capacity before incubation.

20. a) contacting a liquid sample containing a kappa light chain-containing protein with a separation matrix; b) washing the separation matrix with one or a combination of several washing solutions; c) eluting the kappa light chain-containing protein from the separation matrix using an elution solution; and d) cleaning the separation matrix with a cleaning solution 1. A method for isolating a kappa light chain-containing protein comprising:

21. a) contacting a liquid sample containing a kappa light chain-containing protein with a separation matrix; b) washing the separation matrix with one or a combination of several washing solutions; c) eluting the kappa light chain containing protein from the separation matrix with an elution solution at decreasing pH; d) cleaning the separation matrix with a cleaning solution wherein the IgG capacity of the separation matrix after 24 hours of incubation in 0.1 M NaOH at 22+ / -2°C is at least 80%, or at least 85%, or at least 90%, or at least 95% of the IgG capacity before incubation.

22. 22. The method of claim 21 , wherein reducing the pH is performed by using a pH gradient.

23. 22. The method of claim 21 , wherein the decreasing of the pH is carried out in a stepwise manner.

24. 24. The method of any one of claims 21 to 23, wherein the pH is decreased from about 5.5 to about 2, such as from about 5 to about 2, from about 4.5 to about 2, or from about 4 to about 2.

25. 25. The method of any one of claims 20 to 24, wherein the cleaning liquid comprises 0.01 to 1.0 M NaOH or KOH, such as 0.05 to 1.0 M, or 0.05 to 0.1 M, or 0.05 to 0.3 M, or 0.05 to 0.5 NaOH or KOH.

26. 26. The method of any one of claims 20 to 25, wherein the eluent comprises at least one anionic species selected from the group consisting of acetate, citrate, glycine, succinate, phosphate, and formate.

27. 27. The method according to any one of claims 20 to 26, wherein steps a) to d) are repeated at least 10 times, such as at least 50 times or from 50 to 200 times.

28. 28. The method according to any one of claims 20 to 27, wherein the separation matrix is ​​as defined in any one of claims 1 to 19.

29. A chromatography column comprising a separation matrix according to any one of claims 1 to 19.

30. 30. Use of a chromatography column according to claim 29 in a method according to any one of claims 20 to 28.

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