Modified κ-light chain linked polypeptide

Abstract

One objective is to provide polypeptides with improved alkali stability. [Solution] The present invention discloses a κ light chain-linked polypeptide comprising a Peptostreptococcus protein L mutant-binding domain, wherein one or more asparagine residues of a parent domain defined by a specific sequence, or having 95% or more or 98% sequence homology thereto, are mutated to another amino acid residue that is neither asparagine, proline, nor cysteine.

Description

Technical Field

[0001] Technical field of the present invention The present invention relates to the field of affinity chromatography, and specifically to a polypeptide comprising the kappa light chain binding domain of protein L, which is useful for affinity chromatography of various types of immunoglobulins and immunoglobulin fragments. The present invention also relates to a separation matrix comprising such a polypeptide and a separation method using such a separation matrix.

Background Art

[0002] Background of the present invention Immunoglobulins and immunoglobulin fragments are the most common biopharmaceutical products manufactured or developed worldwide. Due to the high commercial demand and value of this therapeutic drug market, pharmaceutical companies focus on maximizing the productivity of their production processes while keeping associated costs under control.

[0003] Typically, affinity chromatography using a matrix comprising 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 a general step with a very high removal rate of impurities and contaminants.

[0004] For antibody fragments lacking an Fc chain but possessing κ light chain subclass 1, 3, or 4, such as Fab, single-chain variable fragments (scFv), bispecific T-cell engagers (BiTEs), and domain antibodies, a matrix containing protein L derived from Peptostreptococcus magnus (B Akerstrom, L Bjorck: J. Biol. Chem. 264, 19740-19746, 1989; W Kastern et al: J. Biol. Chem. 267, 12820-12825, 1992; BHK Nilson et al: J. Biol. Chem. 267, 2234-2239, 1992 and U.S. Patent No. 6822075) shows great potential as a purification platform that provides the required high selectivity. Protein L disclosed in U.S. Patent No. 6,822,075 contains the amino acid sequence of Sequence ID No. 1 with an additional AVEN sequence added to the N-terminus.

[0005] Sequence ID 1 (Protein L) KEETPETPETD SEEEVTIKAN LIFANGSTQT AEFKGTFEKA TSEAYAYADT LKKDNGEYTV DVADKGYTLN IKFAGKEKTPEE PKEEVTIKAN LIYADGKTQT AEFKGTFEEA TAEAYRYADA LKKDNGEYTV DVADKGYTLN IKFAGKEKTPEE PKEEVTIKAN LIYADGKTQT AEFKGTFEEA TAEAYRYADL LAKENGKYTV DVADKGYTLN IKFAGKEKTPEE PKEEVTIKAN LIYADGKTQT AEFKGTFAEA TAEAYRYADL LAKENGKYTA DLEDGGYTIN IRFAGKKVDEKPEE.

[0006] Protein L matrix is ​​commercially available from GE Healthcare Bio-Sciences (Sweden) as Capto® L (Capto L data file 29-0100-08 AC, 2014) and can be used to isolate κ light chain-containing proteins, such as intact antibodies, Fab fragments, scFv fragments, and domain antibodies. Approximately 75% of antibodies produced by healthy humans contain κ light chains, and many therapeutic monoclonal antibodies and antibody fragments also contain κ light chains.

[0007] In any bioprocess chromatography application, comprehensive care is required to ensure the reliable removal of contaminants. Such contaminants may be non-eluting molecules adsorbed to the stationary phase or matrix during the chromatography procedure, such as unwanted biomolecules or microorganisms including proteins, carbohydrates, lipids, bacteria, and viruses. Removal of such contaminants from the matrix is ​​usually performed after the initial elution of the desired product, in order to regenerate the matrix before subsequent use. This removal typically involves a procedure known as clean-in-place (CIP), using reagents capable of eluting contaminants from the stationary phase. Among such reagents, alkaline solutions are commonly used in chromatography media. The most widely used cleaning and disinfecting agent today is NaOH, and it is desirable to use it at concentrations of approximately 0.05 M to 1 M, depending on the degree and nature of the contaminants. However, protein L is a protein that is more alkali-sensitive than protein A, and can only tolerate NaOH below approximately 15 mM for many cycles. This means that in order to ensure sufficient cleaning, it may be necessary to use additional cleaning solutions that are less desirable (e.g., urea or guanidinium salts).

[0008] Extensive research has been conducted to develop modified protein A ligands that exhibit improved properties for tolerance to alkaline pH values. For example, International Publication 2003 / 080655 discloses that protein A domains with specific asparagine mutations are considerably more alkaline stable than the natural protein.

[0009] Therefore, there is still a need in the field to obtain a separation matrix containing a protein L-derived ligand that has improved stability against alkaline washing procedures. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] International Publication No. 2005 / 033130 [Overview of the project]

[0011] Summary of the present invention One aspect of the present invention is to provide a polypeptide with improved alkali stability. This is achieved by the polypeptide described in claim 1.

[0012] One advantage is improved alkali stability in protein L and the parent polypeptide. An additional advantage is that the highly selective binding to κ light chain-containing proteins, as exemplified by protein L, is maintained in the polypeptide of the present invention.

[0013] A second aspect of the present invention is to provide a nucleic acid or vector encoding a polypeptide or polymer with improved alkali stability. This is achieved by the nucleic acid or vector described in the claims.

[0014] A third aspect of the present invention is to provide an expression system capable of expressing polypeptides or polymers with improved alkali stability. This is achieved by the expression system described in the claims. It can be done.

[0015] A fourth aspect of the present invention is to provide a separation matrix that can selectively bind to κ-light chain-containing proteins and exhibits improved alkaline stability. This is achieved by the separation matrix described in the claims.

[0016] A fifth aspect of the present invention is to provide an efficient and economical method for isolating κ-light chain-containing proteins. This is achieved by the method described in the claims.

[0017] Other preferred embodiments of the present invention are described in the dependent claims.

[0018] definition The terms “antibody” and “immunoglobulin” are used interchangeably herein and include antibody fragments, fusion proteins containing antibodies or antibody fragments, and conjugates containing antibodies or antibody fragments.

[0019] In this specification, the terms "κ light chain binding polypeptide" and "κ light chain binding protein" refer to the κ light chain subclass 1, 3, or 4 of an antibody (BHK, as in Nilson et al: J. Biol. Chem. 267, 2234-2239, 1992, etc.). κI , V κIII and V κIV This refers to polypeptides or proteins that can bind to (also known as) protein L, and includes, for example, protein L, as well as variants, fragments, or fusion proteins that retain its binding properties.

[0020] The term "κ light chain-containing protein" is used as a synonym for "immunoglobulin κ light chain-containing protein," and in this specification, it refers to antibody-derived κ light chain subclass 1, 3, or 4 (BHK, as in Nilson et al: J. Biol. Chem. 267, 2234-2239, 1992, etc., V). κI , V κIII and V κIVmeans a protein comprising (also referred to as), and includes any intact antibody, antibody fragment, fusion protein, conjugate or recombinant protein comprising kappa light chain subclass 1, 3 or 4. [BRIEF DESCRIPTION OF THE DRAWINGS]

[0021] Brief explanation of the drawing [Figure 1] Fig. 1 shows an alignment of the five kappa light chain binding domains of protein L described in US Patent No. 6822075 and W Kastern et al: J Biol. Chem. 267, 12820-12825, 1992. [Figure 2] Fig. 2 shows the alkaline stability of various kappa light chain binding domains of protein L. [Figure 3] Fig. 3 shows the alkaline stability of a mutant kappa light chain binding domain of protein L. [Figure 4] Fig. 4 shows the alkaline stability of a protein L ligand comprising four domains. [Figure 5] Fig. 5 shows the alkaline stability of mutant dimeric, tetrameric and hexameric kappa light chain binding domains of protein L, in comparison with native protein L. [MODE FOR CARRYING OUT THE INVENTION]

[0022] Detailed description of the embodiment In one embodiment, the present invention discloses a κ light chain binding polypeptide comprising one or more binding domains of protein L of Peptostreptococcus magnus, each comprising or substantially comprising one or more binding domains selected from the group consisting of domain 2, domain 3, and domain 4. Domain 2 may have the amino acid sequence defined by SEQ ID NO: 3 or SEQ ID NO: 12, or may have 90% or more, for example 95% or more, sequence homology with SEQ ID NO: 3 or SEQ ID NO: 12. SEQ ID NO: 12 is a variant of SEQ ID NO: 3 having alanine at position 31. Domain 3 may have the amino acid sequence defined by SEQ ID NO: 4, or may have 90% or more, for example 95% or more, sequence homology with SEQ ID NO: 4. Domain 4 is a variant of SEQ ID NO: 5 It may have a mino acid sequence, or it may have 90% or more, for example 95% or more, sequence homology with SEQ ID NO: 5.

[0023] In some embodiments of the polypeptide, each domain is selected from the group consisting of domains 3 and 4, or each domain is domain 3. Specifically, the polypeptide may contain or substantially consist of polymers of domain 3.

[0024] In certain embodiments, two or more of these domains are selected from the group consisting of domain 2, domain 3, and domain 4, or from the group consisting of domain 3 and domain 4.

[0025] In one embodiment, the polypeptide does not contain any domain 1 of Peptostreptococcus protein L. Domain 1 may have the amino acid sequence defined by SEQ ID NO: 2, or it may have 90% or more, for example 95% or more, sequence homology with SEQ ID NO: 2.

[0026] In certain embodiments of the polypeptide, in one or more binding domains, for example, all binding domains, at least the amino acid at the position corresponding to position 45 of SEQ ID NOs. 2-5 (for example, the amino acid at position 45 of SEQ ID NOs. 2-5 or 12) is mutated to an amino acid that is neither asparagine, proline, nor cysteine. The amino acid at position 45 can be mutated to, for example, alanine.

[0027] In one embodiment of the polypeptide, in one or more binding domains, for example, all binding domains, at least the amino acid at the position corresponding to position 10 of SEQ ID NOs. 2-5 (for example, the amino acid at position 10 of SEQ ID NOs. 2-5 or 12) is mutated to an amino acid other than asparagine, proline, or cysteine. The amino acid at position 10 can be mutated to, for example, glutamine.

[0028] In certain embodiments of the polypeptide, in one or more binding domains, for example, all binding domains, at least the amino acid at the position corresponding to position 60 of SEQ ID NOs. 2-5 (for example, the amino acid at position 60 of SEQ ID NOs. 2-5 or 12) is mutated to an amino acid that is neither asparagine, proline, nor cysteine. The amino acid at position 60 can be mutated to, for example, glutamine.

[0029] Specifically, one or more binding domains, for example all binding domains, may have a mutation selected from the group consisting of N10Q;N45A;N60Q;N10Q,N45A;N45A,N60Q,N10Q,N60Q and N10Q,N45A,N60Q, or a mutation selected from the group consisting of N45A;N10Q,N45A;N45A,N60Q and N10Q,N45A,N60Q.

[0030] In one embodiment of the polypeptide, in one or more binding domains, for example all of the binding domains, at least the amino acid at the position corresponding to position 19 of SEQ ID NOs: 2-5 (for example, the amino acid at position 19 of SEQ ID NOs: 2-5 or 12) is mutated to an amino acid that is neither glutamine, asparagine, proline, nor cysteine. The amino acid at position 19 can be mutated to, for example, glutamic acid or alanine. Specifically, one or more binding domains, for example all of the binding domains, may have mutations selected from the group consisting of Q19E and Q19A.

[0031] In certain embodiments of the polypeptide, one or more binding domains, for example, all binding domains, are sequence numbers 7, 8, 9, 10, 11, The polypeptide has an amino acid sequence selected from the group consisting of sequences defined in Sequence ID 13 and Sequence ID 14. Alternatively, one or more binding domains, for example all binding domains, may have an amino acid sequence selected from the group consisting of sequences defined in Sequence ID 7, Sequence ID 8, Sequence ID 9, Sequence ID 10 and Sequence ID 11. The polypeptide may further contain at its N-terminus a plurality of amino acid residues that constitute residues derived from the cloning process or cleaved signal sequences. The number of additional amino acid residues may be, for example, 15 or less, for example, 10 or less or 5 or less. Specifically, the polypeptide may contain an AQV sequence at its N-terminus.

[0032] Sequence ID 7 (Domain 3, N45A mutation) PKEEVTIKAN LIYADGKTQT AEFKGTFEEA TAEAYRYADL LAKEAGKYTV DVADKGYTLN IKFAGKEKTPEE.

[0033] Sequence ID No. 8 (Domain 3, N10Q, N45A mutation) PKEEVTIKAQ LIYADGKTQT AEFKGTFEEA TAEAYRYADL LAKEAGKYTV DVADKGYTLN IKFAGKEKTPEE.

[0034] Sequence ID No. 9 (Domain 3, N45A, N60Q mutation) PKEEVTIKAN LIYADGKTQT AEFKGTFEEA TAEAYRYADL LAKEAGKYTV DVADKGYTLQ IKFAGKEKTPEE.

[0035] Sequence ID No. 10 (Domain 3, N10Q, N60Q mutation) PKEEVTIKAQ LIYADGKTQT AEFKGTFEEA TAEAYRYADL LAKENGKYTV DVADKGYTLQ IKFAGKEKTPEE.

[0036] Sequence ID No. 11 (Domain 3, N10Q, N45A, N60Q mutations) PKEEVTIKAQ LIYADGKTQT AEFKGTFEEA TAEAYRYADL LAKEAGKYTV DVADKGYTLQ IKFAGKEKTPEE.

[0037] Sequence ID 12 (a variant of domain 2) PKEEVTIKAN LIYADGKTQT AEFKGTFEEA AAEAYRYADA LKKDNGEYTV DVADKGYTLN IKFAGKEKTPEE.

[0038] Sequence ID 13 (Domain 3, Q19A mutation) PKEEVTIKAN LIYADGKTAT AEFKGTFEEA TAEAYRYADL LAKENGKYTV DVADKGYTLN IKFAGKEKTPEE.

[0039] Sequence ID No. 14 (Domain 3, Q19E mutation) PKEEVTIKAN LIYADGKTET AEFKGTFEEA TAEAYRYADL LAKENGKYTV DVADKGYTLN IKFAGKEKTPEE.

[0040] In some embodiments, the polypeptide is a polymer containing or substantially composed of multiple mutant or non-mutant domains as defined in the embodiments described above. The polymer may be, for example, a dimer, trimer, tetramer, pentamer, or hexamer. The polymer may be a homopolymer in which all units of the polymer are identical, or it may be a heteropolymer in which one or more units differ from the others. Advantageously, all units in the polymer are alkali-stable, for example, by containing the mutants described above. The domains can be directly linked to each other by peptide bonds between the C-terminus and N-terminus of these domains. Alternatively, two or more units in the polymer can be linked by elements containing oligomers or polymer species, for example, elements containing 15 or 30 or fewer amino acids, for example, elements containing 1-5, 1-10, or 5-10 amino acids. The nature of such linkages is preferably such that they do not destabilize the spatial conformation of the domains. This can be achieved, for example, by avoiding the presence of cysteine ​​in the linkages. Furthermore, the ligation must preferably be sufficiently stable in an alkaline environment so as not to impair the domain's properties. For this purpose, it is advantageous if the ligation does not contain asparagine. It may be even more advantageous if the ligation does not contain glutamine. The multimer may further contain at the N-terminus several amino acid residues constituting residues derived from the cloning process or from the cleaved signal sequence. The number of additional amino acid residues may be, for example, 15 or less. The number may be 10 or less, or 5 or less. Specifically, the polymer may contain an AQV sequence at its N-terminus.

[0041] In certain embodiments, the polymer contains or substantially comprises sequences selected from the group consisting of SEQ ID NOs: 15, 16, 17, and 18, for example, sequences selected from the group consisting of SEQ ID NOs: 16, 17, and 18.

[0042] Sequence ID No. 15 (Domain 3, tetramer) PKEEVTIKAN LIYADGKTQT AEFKGTFEEA TAEAYRYADL LAKENGKYTV DVADKGYTLN IKFAGKEKTPEE PKEEVTIKAN LIYADGKTQT AEFKGTFEEA TAEAYRYADL LAKENGKYTV DVADKGYTLN IKFAGKEKTPEE PKEEVTIKAN LIYADGKTQT AEFKGTFEEA TAEAYRYADL LAKENGKYTV DVADKGYTLN IKFAGKEKTPEE PKEEVTIKAN LIYADGKTQT AEFKGTFEEA TAEAYRYADL LAKENGKYTV DVADKGYTLN IKFAGKEKTPEE.

[0043] Sequence ID 16 Domain 3 (N10Q, N45A, N60Q) 2 PKEEVTIKAQ LIYADGKTQT AEFKGTFEEA TAEAYRYADL LAKEAGKYTV DVADKGYTLQ IKFAGKEKTPEE PKEEVTIKAQ LIYADGKTQT AEFKGTFEEA TAEAYRYADL LAKEAGKYTV DVADKGYTLQ IKFAGKEKTPEE.

[0044] Sequence ID 17 Domain 3 (N10Q, N45A, N60Q) 4 PKEEVTIKAQ LIYADGKTQT AEFKGTFEEA TAEAYRYADL LAKEAGKYTV DVADKGYTLQ IKFAGKEKTPEE PKEEVTIKAQ LIYADGKTQT AEFKGTFEEA TAEAYRYADL LAKEAGKYTV DVADKGYTLQ IKFAGKEKTPEE PKEEVTIKAQ LIYADGKTQT AEFKGTFEEA TAEAYRYADL LAKEAGKYTV DVADKGYTLQ IKFAGKEKTPEE PKEEVTIKAQ LIYADGKTQT AEFKGTFEEA TAEAYRYADL LAKEAGKYTV DVADKGYTLQ IKFAGKEKTPEE.

[0045] Sequence ID 18 Domain 3 (N10Q, N45A, N60Q) 6 PKEEVTIKAQ LIYADGKTQT AEFKGTFEEA TAEAYRYADL LAKEAGKYTV DVADKGYTLQ IKFAGKEKTPEE PKEEVTIKAQ LIYADGKTQT AEFKGTFEEA TAEAYRYADL LAKEAGKYTV DVADKGYTLQ IKFAGKEKTPEE PKEEVTIKAQ LIYADGKTQT AEFKGTFEEA TAEAYRYADL LAKEAGKYTV DVADKGYTLQ IKFAGKEKTPEE PKEEVTIKAQ LIYADGKTQT AEFKGTFEEA TAEAYRYADL LAKEAGKYTV DVADKGYTLQ IKFAGKEKTPEE PKEEVTIKAQ LIYADGKTQT AEFKGTFEEA TAEAYRYADL LAKEAGKYTV DVADKGYTLQ IKFAGKEKTPEE PKEEVTIKAQ LIYADGKTQT AEFKGTFEEA TAEAYRYADL LAKEAGKYTV DVADKGYTLQ IKFAGKEKTPEE.

[0046] In some embodiments, the polypeptide and / or polymer described above further comprises one or more coupling elements selected from the group consisting of cysteine ​​residues, multiple lysine residues, and multiple histidine residues at its C-terminus or N-terminus. The coupling element may, for example, be a single cysteine ​​at the C-terminus. The coupling element may be directly linked to the C-terminus or N-terminus, or linked via a linker containing 15 or fewer amino acids, e.g., 1-5, 1-10, or 5-10 amino acids. This extension must preferably be sufficiently stable in an alkaline environment so as not to impair the properties of the mutant protein. For this purpose, it is advantageous if the extension does not contain asparagine. It may be even more advantageous if the extension does not contain glutamine. The advantage of having a C- or N-terminal cysteine ​​is that terminal coupling of the protein can be achieved via the reaction of cysteine ​​thiol with an electrophile on the support. This provides excellent mobility of the coupling protein, which is important for binding capacity.

[0047] The alkali stability of a polypeptide or polymer can be evaluated by coupling it to an SPR chip, for example, the Biacore CM5 sensor chip described in the examples, and measuring the κ light chain binding capacity of the chip before and after incubation in an alkaline solution at a predetermined temperature, for example, 22±2°C, using, for example, a specific κ light chain-containing protein or human polyclonal IgG (where the majority of the IgG molecule has κ light chains). Incubation can be performed, for example, in 0.1 M NaOH, with numerous 10-minute cycles, for example The process can be carried out for 50, 96, or 100 cycles. After 96 to 100 cycles of incubation in 0.1 M NaOH at 22 ± 2°C for 10 minutes each, the binding capacity of the matrix may be 40% or more of the binding capacity before incubation, e.g., 50% or more or 55% or more. Alternatively, the residual binding capacity of a particular mutant after 96 to 100 cycles, as measured above, can be compared to the residual binding capacity of the parent polypeptide / multimer. In this case, the residual binding capacity of the mutant may be 105% or more of the parent polypeptide / multimer, e.g., 110% or more, 125% or more, 150% or more or 200% or more.

[0048] The present invention also discloses a κ-light chain-linked polypeptide comprising one or more mutant-binding domains of Peptostreptococcus protein L, wherein one or more asparagine residues of the parent domain defined by SEQ ID NOs. 2-6 or 12, or having 95% or more or 98% sequence homology with SEQ ID NOs. 2-6 or 12, are mutated to another amino acid residue that is neither asparagine, proline, nor cysteine. The polypeptide may contain at least mutant N45A and / or mutant N60Q. In certain embodiments, the mutations are selected from the group consisting of N45A;N10Q, N45A;N45A,N60Q, N10Q, N60Q, and N10Q, N45A,N60Q, or from the group consisting of N45A;N10Q, N45A;N45A,N60Q, and N10Q, N45A,N60Q. Alkali stability is improved compared to the parent polypeptide and can be measured as described above.

[0049] In one embodiment, the polypeptide comprises or essentially consists of multiple mutant-binding domains, e.g., 2, 3, 4, 5, or 6 domains, each domain containing one or more mutants N10Q, N45A, and N60Q, e.g., N45A and / or N60Q. Specifically, the mutants in each domain can be selected from the group consisting of N45A;N10Q, N45A;N45A,N60Q, N10Q, N60Q, and N10Q, N45A, N60Q, or from the group consisting of N45A;N10Q, N45A;N45A,N60Q, and N10Q, N45A, N60Q. The domains may optionally be linked to each other by elements containing 15 or fewer amino acids.

[0050] In a second aspect, the present invention discloses nucleic acids encoding polypeptides or polymers according to the embodiments described above. Accordingly, the present invention encompasses all forms of the nucleic acid sequence, such as RNA and DNA, that encode polypeptides or polymers. The present invention also encompasses vectors, such as plasmids, which, in addition to the coding sequence, include signal sequences necessary for the expression of the polypeptide or polymer according to the present invention. In one embodiment, the vector comprises nucleic acids encoding the polymer according to the present invention, and the individual nucleic acids encoding each unit may have homologous or heterologous DNA sequences.

[0051] In a third aspect, the present invention discloses an expression system comprising the nucleic acid or vector described above. The expression system may be, for example, a Gram-positive or Gram-negative prokaryotic host cell line, such as a Bacillus or Escherichia coli, modified to express the polypeptide or macromer. In an alternative embodiment, the expression system is a eukaryotic host cell line, such as yeast, such as Pichea pastoris or Saccharomyces cerevisiae.

[0052] In a fourth aspect, the present invention discloses a separation matrix in which a plurality of polypeptides or polymers according to any of the above embodiments are coupled to a solid support. Such a matrix is ​​useful for the separation of κ-light chain-containing proteins, and due to the improved alkaline stability of the polypeptides / polymers, the matrix can withstand highly alkaline conditions during washing, which is essential for long-term repeated use in bioprocess separation settings. The alkaline stability of the matrix is ​​obtained by immersing it in an alkaline solution at a given temperature, for example, 22±2°C. The κ light chain binding capacity can be evaluated before and after incubation by measuring it using, for example, a specific κ light chain-containing protein or human polyclonal IgG. Incubation can be performed, for example, in 0.1 M NaOH, in a large number of 15-minute cycles, e.g., 100, 200, or 300 cycles. The binding capacity of the matrix after 100 repetitions of 15-minute incubation in 0.1 M NaOH at 22 ± 2 °C may be 80% or more of the binding capacity before incubation, e.g., 85% or more, 90% or more, or 95% or more. Alternatively, incubation may be performed in a large number of 4-hour cycles in 0.1 M NaOH, e.g., 6 cycles, for 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 80% or more of the binding capacity before incubation, e.g., 85% or more, 90% or more, or 95% or more.

[0053] As those skilled in the art will understand, expressed polypeptides or polymers must be moderately purified before immobilization on a support. Such purification methods are well known in the art, and immobilization of protein-based ligands on supports is readily carried out using standard methods. Suitable methods and supports are described below in detail.

[0054] The solid support of the matrix according to the present invention may be any suitable known type. Conventional affinity separation matrices are often organic and are exposed to aqueous media using hydrophilic surfaces, i.e., based on polymers that expose hydroxy(-OH), carboxy(-COOH), carboxyamide (-CONH2, which may be N-substituted), amino (-NH2, which may be substituted), oligo-, or polyethyleneoxy groups to their exteriors and, if present, to their interiors as well. The solid support may be appropriately porous. Porosity can be expressed as a Kav or Kd value (the percentage of pore volume available to a probe molecule of a particular size) measured by reverse-size exclusion chromatography as described in Gel Filtration Principles and Methods, Pharmacia LKB Biotechnology 1991, pp6-13. By definition, both Kd and Kav values ​​are always in the range of 0 to 1. Advantageously, when measured using dextran with a molecular weight of 110 kDa as a probe molecule, the Kav value can be 0.6 to 0.95, for example, 0.7 to 0.90 or 0.6 to 0.8. The advantage of this is that the support has a large proportion of pores that can accommodate both the polypeptide / multimer of the present invention and the immunoglobulin bound to the polypeptide / multimer, and can provide mass transport of the immunoglobulin to and from the binding site.

[0055] Polypeptides or polymers can be conjugated to a support via conventional coupling techniques that utilize thiol, amino, and / or carboxyl groups present in the ligand, for example. Well-known coupling reagents include bicepoxide, epichlorohydrin, CNBr, and N-hydroxysuccinimide (NHS). A molecule known as a spacer can be introduced between the support and the polypeptide / polymer, thereby improving the availability of the polypeptide / polymer and facilitating its chemical coupling to the support. Suitable spacers can be introduced, for example, by activating the support using epichlorohydrin, butanediol diepoxide, or allyl glycidyl ether. Alternatively, the polypeptide / polymer may be conjugated to the support by non-covalent bonding, such as physicoadsorption or biospecific adsorption.

[0056] In one embodiment, the matrix comprises 5 to 20 polypeptides or polymers coupled to a support, for example, 5 to 15 mg / ml, 5 to 11 mg / ml, or 8 to 11 mg / ml. The amount of polypeptide / polymer to be coupled depends on the concentration of polypeptide / polymer used in the coupling process, the coupling conditions used, and / or the support used. This can be controlled by the pore structure of the host organism. Generally, the absolute binding capacity of the matrix increases with the amount of coupled polypeptide / multimer, at least up to the point where the pores are significantly narrowed by the coupled polypeptide / multimer. The relative binding capacity per mg of coupled polypeptide / multimer decreases as the coupling level increases, providing optimal cost-benefit within the above range.

[0057] In some embodiments, a polypeptide is coupled to a support via multipoint coupling. This can be adequately achieved by using coupling conditions such that multiple reactive groups in the polypeptide react with reactive groups in the support. Typically, multipoint coupling can involve the reaction of several intrinsic reactive groups of amino acid residues in the sequence, e.g., amines in lysine, with reactive groups on the support, e.g., epoxides, cyanate esters (e.g., derived from CNBr activation), succinimidyl esters (e.g., derived from NHS activation). However, it is also possible to influence the coupling properties by deliberately introducing reactive groups at different positions in the polypeptide. To provide lysine-mediated multipoint coupling, the coupling reaction is adequately carried out at a pH where a substantial portion of the primary amine of lysine is in a non-protonated nucleophilic state, for example, above 8.0, e.g., above 10.

[0058] In certain embodiments, polypeptides or polymers are coupled to a support via thioether bonds. Methods for carrying out such coupling are well known in the art and can be easily performed by those skilled in the art using standard techniques and apparatus. Thioether bonds are flexible and stable and generally suitable for use in affinity chromatography. In particular, when the thioether bond is via terminal or near-terminal cysteine ​​residues on the polypeptide or polymer, the mobility of the coupled polypeptide / polymer is improved, and the binding capacity and reaction rate are improved. In some embodiments, polypeptides / polymers are coupled via a C-terminal cysteine ​​provided to the protein. This allows for efficient coupling of the cysteine ​​thiol with electrophiles on the support, such as epoxide groups or halohydrin groups, resulting in thioether crosslinking coupling. Polypeptides / polymers may be coupled, for example, via a single-point bond, for example, via a single cysteine, or by a directional multi-point bond using multiple lysine or other coupling groups near the near terminus of the polypeptide / polymer.

[0059] In certain embodiments, the support comprises a polyhydroxy polymer, such as a polysaccharide. Examples of polysaccharides include, for example, dextran, starch, cellulose, pullulan, agar, and agarose. Polysaccharides are inherently hydrophilic, have a low degree of nonspecific interaction, have a high content of reactive (activatable) hydroxyl groups, and are generally stable to alkaline cleaning solutions used in bioprocesses.

[0060] In one embodiment, the support comprises agar or agarose. The support used in the present invention can be readily prepared by standard methods, for example, by reverse suspension gelation (S. Hjerten: Biochim Biophys Acta 79(2), 393-398 (1964)). Alternatively, the basic matrix is ​​a commercially available product, such as cross-linked agarose beads sold under the name SEPHAROSE® FF (GE Healthcare). In one embodiment particularly advantageous for large-scale separation, the support is adapted to increase its rigidity using the method described in U.S. Patent No. 6,602,990 or U.S. Patent No. 7,396,467 (the said patents are incorporated herein by reference in their entirety), thereby making the matrix suitable for high flow rates.

[0061] In certain embodiments, a support such as a polysaccharide or agarose support is crosslinked by hydroxyalkyl ether crosslinking or the like. The crosslinking agent reagent that generates such crosslinking is, for example, epic The crosslinking agent may be an epihalohydrin such as lorhohydrin, a diepoxide such as butanediol diglycidyl ether, or an allylation agent such as an allyl halide or allyl glycidyl ether. Crosslinking is beneficial to the rigidity of the support and improves its chemical stability. Hydroxyalkyl ether crosslinking is alkali-stable and does not cause significant nonspecific adsorption.

[0062] Alternatively, the solid support may be based on synthetic polymers, such as polyvinyl alcohol, polyhydroxyalkyl acrylate, polyhydroxyalkyl methacrylate, polyacrylamide, polymethacrylamide, etc. In the case of hydrophobic polymers such as divinyl and monovinyl-substituted benzene-based matrices, the matrix surface is often hydrophilized to expose the hydrophilic groups defined above to the surrounding aqueous liquid. Such polymers are readily manufactured 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 such as SOURCE® (GE Healthcare) may be used. Another alternative is that the solid support according to the present invention may include inorganic supports, such as silica, zirconium oxide, etc.

[0063] In yet another embodiment, the solid support may be a surface, a chip, a capillary, or a filter (e.g., a membrane or a deep filter matrix).

[0064] Regarding the shape of the matrix according to the present invention, in one embodiment, the matrix is ​​in the form of a porous monolith. In another embodiment, the matrix is ​​in the form of beads or particles, which may be porous or non-porous. The matrix in the form of beads or particles can be used as a packed bed or in suspension form. Suspension forms include forms known as expansion beds and pure suspensions, in which the particles or beads move freely. In the case of monoliths, packed beds and expansion beds, the separation procedure generally follows conventional chromatography using a concentration gradient. In the case of pure suspensions, a batch method is used.

[0065] In a sixth aspect, the present invention discloses a method for isolating κ-light chain-containing proteins using the isolation matrix described above.

[0066] In certain embodiments, this method a) A step of bringing a liquid sample containing κ light chain-containing protein into contact with the separation matrix described above, b) A step of washing the separation matrix with a washing solution, c) A step of eluting the κ light chain-containing protein from the separation matrix using the elution solution. d) A step of washing the separation matrix with a washing solution, Includes.

[0067] The method further comprises the steps of: before step a), preparing an affinity separation matrix according to any of the embodiments described above, and preparing a solution containing the κ light chain-containing protein and one or more other substances as a liquid sample; and after step c), recovering the eluate and optionally subjecting this eluate to an additional separation step, for example, anion or cation exchange chromatography, multimodal chromatography and / or hydrophobic interaction chromatography. Appropriate compositions of the liquid sample, washing solution and eluate, and general conditions for carrying out the separation are well known in the field of affinity chromatography, particularly in the field of protein L chromatography. The liquid sample containing the κ light chain-containing protein and one or more other substances may contain host cell proteins (HCPs), such as proteins from Chinese hamster ovary (CHO) cells, Escherichia coli, or yeast cells. The protein content of CHO cells and Escherichia coli can be determined by immunoassays against these proteins, for example, C HO HCP or E. coli HCP can be easily determined using an ELISA kit from Cygnus Technologies. Host cell proteins or CHO cell / E. coli / yeast proteins can be desorbed during step b).

[0068] Elution can be performed using any suitable solution used for elution from the protein L medium. This may be, for example, a solution or buffer with a pH of 4 or less, e.g., pH 2.5-4 or 2.8-3.5. In some embodiments, the elution buffer or elution buffer gradient comprises at least one mono-, di-, or tri-functional carboxylic acid or a salt of such carboxylic acid. In specific embodiments, the elution buffer or elution buffer gradient comprises at least one anionic species selected from the group consisting of acetates, citrates, glycine, succinates, phosphates, and formates.

[0069] In one embodiment, the cleaning solution is alkaline, having a pH of 12-14. Such a solution provides efficient cleaning of the matrix, particularly at the upper end of the spacing.

[0070] In certain embodiments, the washing solution contains 0.01 to 1.0 M NaOH or KOH, for example, 0.05 to 1.0 or 0.05 to 0.1 M NaOH or KOH. The high stability of the polypeptide of the present invention allows for the use of such relatively strong alkaline solutions.

[0071] In some embodiments, steps a) to d) are repeated 10 or more times, for example, 50 or more times, or 50 to 200 times. This is important for process economics in that the matrix can be reused many times. [Examples]

[0072] Examples Protein mutation introduction Monomeric constructs were designed from protein L containing four κ-light chain binding domains disclosed in U.S. Patent No. 6,822,075 (SEQ ID NO: 1). These were numbered 1, 2, 3, and 4 starting from the N-terminus (Figure 1). The DNA fragments were purchased from DNA Synthesis Company (DNA2.0). Four monomeric constructs, each with an N-terminal cysteine, were prepared in the pJexpress201 cloning vector. See SEQ ID NOs: 2, 4, 5, and 12 for a summary of the constructs. Constructs containing the E. coli GAP promoter and OmpA signal peptide sequences for localizing the target protein to the periplasm were subcloned into the expression vector pGO. Sequences encoding the four domains were prepared by amplification with oligonucleotides containing the restriction enzyme recognition sites of FspI and PstI at the 5' and 3' ends, respectively. The prepared DNA fragments encoding each domain were digested using FspI and PstI (New England Biolabs). Separately, expression vectors were prepared by digestion using FspI and PstI, purified by agarose gel electrophoresis, and recovered. Both were mixed and ligated using the Quick Ligation Kit (New England Biolabs). Chemically competent E. coli K12 strain was transformed using the ligated plasmids expressing each domain by the heat shock method.

[0073] Additional mutations in amino acids N10, N45, Q19, and N60 of domain 3 were prepared in the expression vector pJexpress401 (DNA2.0) containing the T5 promoter under the control of the lac operon (SEQ ID NOs. 7-11, 13-14). Constructs were designed with and without the OmpA signal peptide, and without the C-terminal cysteine.

[0074] The tetramer of domain 3, and the dimer, tetramer, and hexamer of domain 3 with mutations N45, N10, and N60, also have a C-terminal cysteine ​​(SEQ ID NOs: 15-18), and The untreated material was prepared using pJexpress401.

[0075] Expression and purification of constructs Recombinant E. coli K12 cells were cultured at 37°C in a shaking flask containing LB-broth (10 g peptone, 5 g yeast extract, 5 g NaCl) supplemented with 25 mg / l kanamycin until the optical density at 600 nm reached 0.8. At this point, protein expression was induced using isopropyl β-D-1-thiogalactopyranoside (VWR International) at a final concentration of 1 mM. After induction, the temperature was reduced to 30°C and the culture was incubated for 5 hours. The culture was stopped, the cells were centrifuged at 4000xg for 15 minutes, and the supernatant was discarded. The cells were resuspended in phosphate-buffered saline (PBS) to 1 / 10 of the culture volume and sonicated using pulsed ultrasound for an activation time of 2 minutes. The sonicated sample was clarified from cell debris by centrifugation at 6000xg for 30 minutes, followed by microfiltration through a membrane with a pore size of 0.2 μm.

[0076] The purified ligand was analyzed using LC-MS to determine its purity and confirm that its molecular weight (based on the amino acid sequence) matched the prediction.

[0077] Example 1 In the case of non-mutant single-domain ligands, the purified monomeric ligands listed in Table 1, further comprising a C-terminal cysteine ​​and an N-terminal AQV sequence, were immobilized on a Biacore CM5 sensor chip (GE Healthcare, Sweden) in sufficient quantities to obtain a signal intensity of approximately 1000 RU using a GE Healthcare amine coupling kit (for carbodiimide coupling of amines of carboxymethyl groups on the chip) in a Biacore instrument (GE Healthcare, Sweden). To track the IgG binding capacity of the immobilized surface, 1 mg / ml of human polyclonal IgG (Gammanorm) was flowed onto the chip and the signal intensity was recorded. The surface was then subjected to a clean-in-place (CIP) wash, i.e., washed with 100 mM NaOH at room temperature (22±2°C) for 10 minutes. This was repeated for 96 cycles, and the alkaline stability of the immobilized ligand was tracked as the relative loss of IgG binding capacity (signal intensity) after each cycle. The results for the non-mutant domains are shown in Figure 2, which indicate that domain 1 has significantly lower alkaline stability than the other domains, while domain 3 has the highest alkaline stability. The results for the single-domain asparagine mutant of domain 3 are shown in Figure 3, which show improved alkaline stability for all mutants compared to the parent domain 3 used as a reference in parallel with the mutation.

[0078] [Table 1]

[0079] Example 2 The purified multidomain ligands listed in Table 2 are used in GE Healthcare's amine coupling (for amine carbodiimide coupling of carboxymethyl groups on the chip). Using a pulling kit, sufficient amounts were immobilized onto Biacore CM5 sensor chips (GE Healthcare, Sweden) to obtain a signal intensity of approximately 1000 RU in a Biacore instrument (GE Healthcare, Sweden). Protein L had an additional AIHNRA sequence at its N-terminus. To track the IgG binding capacity of the immobilized surface, 1 mg / ml of human polyclonal IgG (Gammanorm) was flowed onto the chip and the signal intensity was recorded. The surface was then subjected to clean-in-place (CIP), i.e., washed with 100 mM NaOH at room temperature (22±2°C) for 10 minutes. This was repeated for 96 cycles, and the alkaline stability of the immobilized ligand was tracked as the relative loss of IgG binding capacity (signal intensity) after each cycle. The results are shown in Table 2 and Figure 4, and these results indicate that tetramer domain 3 showed improved alkaline stability compared to protein L, which was performed in parallel as a reference.

[0080] [Table 2]

[0081] Example 3 The purified multidomain ligands listed in Table 3 were immobilized on a Biacore CM5 sensor chip and evaluated using the method used in Example 2. The -cys at the end of the ligand symbol indicates that the ligand has a C-terminal cysteine ​​in addition to the sequence defined in SEQ ID NOs. 16-18. The results are shown in Table 3 and Figure 5, and these results indicate that all dimers, tetramers, and hexamers of mutant domain 3 showed improved alkaline stability compared to protein L, which was tested in parallel as a reference.

[0082] [Table 3]

[0083] Example 4 The purified dimer, tetramer, and hexamer ligands shown in Table 4 were immobilized on agarose beads using the method described below, and their volume was evaluated. The results are shown in Table 4.

[0084] [Table 4]

[0085] activation The base matrix used was prepared according to the method of U.S. Patent No. 6602990, with a median diameter of 85 μm (volume-weighted), as described in Gel Filtration Principles and Methods, Pharmacia LKB Biotechno. According to the method described in Logy 1991, pp. 6-13, the Kav value of the reverse gel filtration chromatography is a hard crosslinked agarose bead having a pore size corresponding to 0.70 for dextran with a molecular weight of 110 kDa.

[0086] 25 mL (g) of strained base matrix, 10.0 mL of distilled water, and 2.02 g of NaOH were mixed in a 100 mL flask with mechanical stirring at 25°C for 10 minutes. 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 precipitate volumes (GV) of water.

[0087] Coupling The activated gel was washed with 5 GV 0.2 M phosphate / 1 mM EDTA pH 11.5 (coupling buffer). 15 ml of gel + 13 mg ligand / gel (5.0 ml) + 5.5 ml of coupling buffer + 4.7 g of sodium sulfate were mixed in a 50 ml flask and stirred at 30°C for 18.5 hours. The measured pH was 10.8.

[0088] After fixation, the gel was washed with 3xGV of distilled water, then with 5xGV of 0.1M phosphate / 1mM EDTA pH 8.5. The gel was mixed with 1GV of {0.1M phosphate / 1mM EDTA / 7.5% thioglycerol pH 8.5}, and the flask was stirred at 45°C for 2 hours and 20 minutes. The gel was then alternately washed with 1xGV of 0.1M HAc and 1xGV of {0.1M TRIS / 0.15M NaCl pH 8.5}, and then washed with 6xGV of distilled water. The gel sample was sent to an external laboratory for amino acid analysis, and the ligand content (mg / ml gel) was calculated from the total amino acid content. The coupling protocol used provided multi-point coupling, with several lysines from each domain bound to the gel.

[0089] 2 ml of resin was packed into a TRICORN(trademark) 5 100 column.

[0090] protein a) Purified Fab prepared from papain-digested IgG mAb was diluted to 1 mg / ml in equilibration buffer. b) Purified Dab prepared from the supernatant of heat-treated E. coli was diluted to 1 mg / ml in equilibrium buffer. The Dab contained only the κ light chain and no antigen-binding sites at all.

[0091] Equilibrium buffer APB phosphate buffer 20 mM + 0.15 M NaCl, pH 7.4 (Medicago).

[0092] Adsorption buffer APB phosphate buffer 20 mM + 0.15 M NaCl, pH 7.4 (Medicago).

[0093] Elution buffer 25 mM citrate, pH 2.5.

[0094] Breakthrough volume was measured using the AKTAExplorer10 system with a residence time of 4 minutes. Equilibrium buffer was flowed through the bypass column until a stable baseline was obtained. This was done before automatic zeroing. The sample was applied to the column until a 100% UV signal was obtained. Then, equilibrium buffer was applied again until a stable baseline was obtained.

[0095] The sample was loaded onto the column until the UV signal reached 85% of the maximum absorbance. Next, the color The sample was washed with equilibration buffer and eluted at a flow rate of 0.5 ml / min at pH 2.5.

[0096] The following formula was used to calculate the 10% breakthrough capacity. This is the amount of Fab / Dab loaded onto the column until the concentration of Fab / Dab in the column effluent becomes 10% of the concentration of Fab / Dab in the feed solution.

[0097]

number

[0098] The dynamic binding capacity (DBC) at the 10% breakthrough was calculated, and the shape of the curve was studied. This curve was further studied with respect to binding, elution, and CIP peaks. The dynamic binding capacity (DBC) was calculated for both the 10% and 80% breakthroughs.

[0099] This specification discloses the present invention in the best mode and has described it with examples to enable those skilled in the art to implement the invention, including the manufacture, use, and method of manufacturing the apparatus or system. The patentable scope of the present invention is defined by the claims and includes other examples that are obvious to those skilled in the art. Such other examples fall within the technical scope of the claims if they have components that are identical in wording to the claims or equivalent components that are substantially identical to the claims. [Embodiment 1] A κ-light chain-linked polypeptide containing one or more mutant-binding domains of Peptostreptococcus protein L, wherein one or more asparagine residues in the parent domain defined by SEQ ID NOs. 2-6 or 12, or having 95% or more or 98% sequence homology thereto, are mutated to another amino acid residue that is neither asparagine, proline, nor cysteine. [Embodiment 2] The polypeptide according to Embodiment 1, comprising at least mutant N45A. [Embodiment 3] The polypeptide according to Embodiment 1 or 2, comprising at least the mutant N60Q. [Embodiment 4] The polypeptide according to any one of Embodiments 1 to 3, wherein the mutation is selected from the group consisting of N10Q;N45A;N60Q;N10Q, N45A;N45A, N60Q, N10Q, N60Q and N10Q, N45A, N60Q, or selected from the group consisting of N45A;N10Q, N45A;N45A, N60Q, N10Q, N60Q and N10Q, N45A, N60Q. [Embodiment 5] The polypeptide according to any one of Embodiments 1 to 4, comprising or essentially consisting of multiple mutant-binding domains, e.g., 2, 3, 4, 5, or 6 domains, each domain containing mutant N45A and / or N60Q. [Embodiment 6] The mutations in each domain are selected from the group consisting of N10Q;N45A;N60Q;N10Q, N45A;N45A, N60Q, N10Q, N60Q and N10Q, N45A, N60Q, or selected from the group consisting of N45A;N10Q, N45A;N45A, N60Q, N10Q, N60Q and N10Q, N45A, N60Q, as described in Embodiment 5. Polypeptide. [Embodiment 7] The polypeptide according to embodiment 5 or 6, wherein the domains are linked by elements containing 15 or fewer amino acids. [Embodiment 8] The polypeptide according to any one of Embodiments 1 to 7, wherein the alkaline stability, as measured by the remaining binding capacity to κ light chain-containing proteins after 96 to 100 incubations of 10 minutes each in a 0.1 M NaOH aqueous solution at 22 ± 2 °C, is improved compared to the parent polypeptide. [Embodiment 9] The polypeptide according to any one of Embodiments 1 to 8, wherein the parent domain is defined by an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 12, or has 95% or more or 98% sequence homology thereto. [Embodiment 10] A polypeptide according to any one of Embodiments 1 to 9, wherein the parent domain is defined by Sequence ID No. 4 or has 95% or more or 98% sequence homology thereto. [Embodiment 11] A nucleic acid or vector encoding a polypeptide or polymer according to any one of Embodiments 1 to 10. [Embodiment 12] An expression system comprising nucleic acid or vector as described in Embodiment 11. [Embodiment 13] A separation matrix in which a plurality of polypeptides according to any one of Embodiments 1 to 10 are coupled to a solid support. [Embodiment 14] The separation matrix according to embodiment 13, wherein polypeptides are coupled to a solid support by multi-point bonding. [Embodiment 15] The isolation matrix according to any one of embodiments 13 to 14, wherein the binding capacity of the matrix to κ light chain-containing proteins after 100 incubations in 0.1 M NaOH at 22 ± 2 °C for 10 minutes is 40% or more, for example, 50% or more or 55% or more, of the binding capacity before incubation. [Embodiment 16] A method for isolating a κ-light chain-containing protein using the isolation matrix described in any one of Embodiments 13 to 15. [Embodiment 17] a) A step of bringing a liquid sample containing a κ light chain-containing protein into contact with a separation matrix according to any one of Embodiments 13 to 15, b) A step of washing the separation matrix with a washing solution, c) A step of eluting the κ light chain-containing protein from the separation matrix using the elution solution. d) A step of washing the separation matrix with a washing solution, The method according to embodiment 16, including the method described in embodiment 16. [Embodiment 18] The method according to Embodiment 17, wherein the cleaning solution is alkaline, having a pH of 12 to 14. [Embodiment 19] The method according to Embodiment 17 or 18, wherein the washing solution contains 0.01 to 1.0 M NaOH or KOH, for example, 0.05 to 1.0 M or 0.05 to 0.1 M NaOH or KOH. [Embodiment 20] Steps a) to d) are repeated 10 or more times, for example, 50 or more times, or 50 to 200 times. , the method according to any one of embodiments 17 to 19.

Claims

1. A κ-light chain-linked polypeptide comprising a polymer of the mutant-binding domain of Peptostreptococcus protein L, wherein in the domain, at least one asparagine residue of the parent domain defined by SEQ ID NO: 4 or having at least 95% sequence identity with SEQ ID NO: 4 is mutated to another amino acid residue that is neither asparagine, proline, nor cysteine. At least one of the domains includes mutations N45A and N60Q, Alkali stability is improved compared to the polypeptide defined by Sequence ID No.

1. The polypeptide comprises or consists of multiple mutation-binding domains, wherein the mutation in at least one of the domains is one of the following (i) to (v): (i) N45A, (ii) N60Q, (iii) N10Q and N45A, (iv) N10Q and N60Q, and (v) N10Q, N45A, and N60Q Selected from the group consisting of, Polypeptide.

2. The polypeptide according to claim 1, wherein the mutation in each of the domains is selected from the group consisting of (i) to (v).

3. The polypeptide according to claim 1 or 2, comprising or consisting of multiple mutant-binding domains, each domain comprising mutants N45A and N60Q.

4. The polypeptide according to claim 3, comprising or consisting of 2, 3, 4, 5, or 6 mutant-binding domains.

5. The polypeptide according to claim 3 or 4, wherein the domains are linked by elements containing up to 15 amino acids.

6. A separation matrix in which a plurality of polypeptides according to any one of claims 1 to 5 are coupled to a solid support.

7. The separation matrix according to claim 6, wherein the polypeptide is coupled to the solid support by multi-point bonding.

8. The isolation matrix according to claim 6 or 7, wherein the binding capacity of the matrix to κ light chain-containing proteins after 100 cycles of 10-minute incubation in 0.1 M NaOH at 22 ± 2°C is at least 40% of the binding capacity before incubation.

9. The separation matrix according to claim 8, wherein the binding capacity after incubation is at least 50% of the binding capacity before incubation.

10. The separation matrix according to claim 9, wherein the binding capacity after incubation is at least 55% of the binding capacity before incubation.

11. A method for isolating a κ-light chain-containing protein using the isolation matrix described in any one of claims 6 to 10.

12. a) A step of contacting a liquid sample containing a κ light chain-containing protein with the separation matrix described in any one of claims 6 to 10. b) Washing the separation matrix with a washing solution, c) The step of eluting the κ light chain-containing protein from the separation matrix using the elution solution, and d) Washing the separation matrix with a washing solution. The method according to claim 11, including the method described in claim 11.

13. The method according to claim 12, wherein steps a) to d) are repeated at least 10 times.

14. The method according to claim 13, wherein steps a) to d) are repeated at least 50 times.

15. The method according to claim 14, wherein steps a) to d) are repeated at least 50 to 200 times.

16. The method according to any one of claims 12 to 15, wherein the cleaning solution is alkaline, and / or the cleaning solution contains 0.01 to 1.0 M of NaOH or KOH.

17. The method according to claim 16, wherein the cleaning solution is an alkali having a pH of 12 to 14.

18. The method according to claim 16 or 17, wherein the washing solution contains 0.05 to 1.0 M NaOH or KOH.

19. The method according to claim 18, wherein the washing solution contains 0.05 to 0.1 M NaOH or KOH.

Images