New ligands for affinity chromatography

Abstract

The present invention relates to novel ligands for affinity chromatography, particularly for the purification of antibodies. The novel ligands are immunoglobulin (Ig)-binding proteins with excellent properties for highly efficient purification methods for antibodies (immunoglobulins). The invention further relates to affinity matrices comprising the ligands of the invention. The invention also relates to the use of these Ig-binding proteins or affinity matrices for the affinity purification of immunoglobulins, and to methods of affinity purification using the Ig-binding proteins of the invention.

Description

[Technical Field]

[0001] The present invention relates to novel ligands for affinity chromatography, particularly for the purification of antibodies. The novel ligands are immunoglobulin (Ig)-binding proteins with excellent properties for highly efficient purification methods for antibodies (immunoglobulins). The invention further relates to affinity matrices comprising the ligands of the invention. The invention also relates to the use of these Ig-binding proteins or affinity matrices for the affinity purification of immunoglobulins, and to methods of affinity purification using the Ig-binding proteins of the invention. [Background technology]

[0002] Many biotechnology and pharmaceutical applications require the removal of contaminants from antibody-containing samples. A well-established procedure for capturing and purifying antibodies and molecules containing the Fc domain is affinity chromatography, using bacterial cell surface protein A from Staphylococcus aureus as a selective ligand for immunoglobulins (see, for example, the review by Huse et al., J. Biochem. Biophys. Methods 51, 2002:217-231). Wild-type protein A binds with high affinity and selectivity to the Fc region of IgG molecules. Protein A variants with improved properties, such as alkaline stability, are available for antibody purification, and various chromatography matrices containing protein A ligands are commercially available. However, currently available protein A-based chromatography matrices exhibit a loss of immunoglobulin binding capacity after exposure to alkaline conditions and require elution conditions at a pH below 4. Summary of the Invention [Problem to be solved by the invention]

[0003] Most large-scale production processes for antibodies or Fc-containing (fusion) proteins use protein A for affinity purification. However, due to limitations in the application of protein A in affinity chromatography, there is a need in the art to provide novel Ig-binding proteins with improved properties for specific binding to immunoglobulins to facilitate immunoglobulin affinity purification. To maximize the value of a chromatography matrix containing an Ig-binding protein, it is desirable to use the affinity ligand matrix multiple times. Between chromatography cycles, a thorough cleaning procedure is required to disinfect and remove residual contaminants on the matrix. For this procedure, it is common practice to apply an alkaline solution containing a high concentration of NaOH, such as 0.5 M NaOH, to the affinity ligand matrix. Wild-type protein A domains cannot withstand such harsh alkaline conditions for extended periods of time and rapidly lose their immunoglobulin-binding capacity. Furthermore, a cleaning step under acidic conditions is usually required for repeated use of the affinity ligand matrix.

[0004] Thus, there is a continuing need in the art to obtain novel proteins that are capable of binding to immunoglobulins, e.g., proteins containing Ig sequences or Fc regions of antibodies, and that better withstand the harsh conditions applied in affinity purification of immunoglobulins.

[0005] The present invention provides Ig-binding proteins that are particularly suited for affinity purification of immunoglobulins. In particular, the Ig-binding proteins of the present invention have several advantages. One significant advantage of the binding proteins of the present invention is their improved stability over extended periods at high pH (alkaline conditions) (e.g., 1.5-2 days in 1 M NaOH), without a significant decrease in Ig-binding capacity, combined with a high dynamic binding capacity. Furthermore, the novel proteins of the present invention are particularly useful for affinity purification of antibodies, which require weakly acidic elution conditions at pH 4.0 or higher.

[0006] The above summary does not necessarily describe all of the problems solved by the present invention. [Means for solving the problem]

[0007] One aspect of the present invention is to provide Ig-binding proteins suitable for affinity purification.

[0008] [1] This is achieved in an immunoglobulin (Ig)-binding protein comprising the amino acid sequence of SEQ ID NO: 13, where the amino acid corresponding to position 5 is phenylalanine (F), the amino acid corresponding to position 8 is isoleucine (I), the amino acid corresponding to position 28 is histidine (H), and the amino acid corresponding to position 42 is lysine (K), or an Ig-binding protein comprising an amino acid sequence having at least 89.5% amino acid identity to SEQ ID NO: 13. In various embodiments, the amino acid corresponding to position 4 is glutamine (Q) or lysine (K), and / or the amino acid corresponding to position 7 is lysine (K) or glutamic acid (E). In various embodiments, the amino acid corresponding to position 58 is proline (P).

[0009] [2] The Ig-binding protein according to item [1], wherein the amino acid corresponding to position 9 is alanine (A) or glutamine (Q).

[0010] [3] The Ig-binding protein according to item [1] or [2], wherein the amino acid corresponding to position 11 is alanine (A) or isoleucine (I).

[0011] [4] The Ig-binding protein according to any one of items [1] to [3], wherein the amino acid corresponding to position 15 is alanine (A) or glutamic acid (E).

[0012] [5] The Ig-binding protein according to any one of items [1] to [4], wherein the Ig-binding protein is a multimer and contains at least three Ig-binding proteins.

[0013] [6] The Ig-binding protein according to item [5], wherein the multimeric Ig-binding protein is a pentamer.

[0014] [7] The Ig-binding protein according to any one of items [1] to [6], wherein the Ig-binding protein comprises any amino acid sequence having at least 89.5% of any of SEQ ID NOs: 1 to 18.

[0015] [8] The Ig-binding protein according to any one of items [1] to [7], wherein the protein binds to one or more of IgG1, IgG2, IgG4, IgM, IgA, an Ig fragment, an Fc fragment, a Fab fragment, a fusion protein comprising an Ig region, and a conjugate comprising an Ig region. In various embodiments, the Ig-binding protein is bound to a protein comprising an Fc region or is bound to an Fc fragment.

[0016] In various embodiments, the Ig binding protein binds to a protein comprising an Fc region, or binds to an Fc fragment, optionally with a binding affinity of less than 100 nM, as determined via SPR.

[0017] [9] An Ig-binding protein according to any one of items [1] to [8], wherein the protein is immobilized on a solid support.

[0018]

[10] The Ig-binding protein according to any one of items [1] to [9], wherein the Ig-binding protein is stable under alkaline conditions, preferably in 1 M NaOH, for at least 37 hours, and optionally has about 80% residual IgG-binding activity after incubation in 1 M NaOH for at least 37 hours.

[0019]

[11] An affinity separation matrix comprising any one of the Ig-binding proteins of items [1] to

[10] coupled to the affinity separation matrix.

[0020] [9] Use of any one of the Ig-binding proteins of items [1] to [7] or the affinity separation matrix of item [8] for affinity purification, particularly for affinity purification of any protein having affinity for Ig-binding proteins.

[0021]

[12] Use of any one of the Ig-binding proteins of items [1] to

[10] or the affinity separation matrix of item

[11] for affinity purification, particularly for affinity purification of any protein having affinity for Ig-binding proteins.

[0022]

[13] A method for affinity purifying a protein comprising an Fc region of immunoglobulin (Ig), comprising: a) providing a sample, preferably a liquid sample, containing a protein comprising the Fc region of an Ig; b) Providing an affinity separation matrix according to item

[11] ; c) contacting the affinity separation matrix with the (liquid) sample under conditions that allow binding of at least one Ig-binding protein of the affinity separation matrix to proteins containing the Fc region of Ig; and d) recovering, preferably eluting, said protein comprising the Fc region of an Ig from said affinity separation matrix, thereby obtaining (affinity purified) protein comprising the Fc region of an Ig, preferably obtaining an eluate containing said protein comprising the Fc region of an Ig; A method comprising:

[0023]

[14] The method according to item

[13] , wherein in step (d), more than about 90%, preferably more than about 95%, of the proteins comprising the Fc region of Ig are eluted from the affinity separation matrix at pH 4.0.

[0024]

[15] The method according to any of items

[13] to

[14] , optionally or preferably comprising an additional step (e) of washing the affinity purification matrix with an alkaline cleaning solution, wherein the Ig-binding protein retains at least about 80% of its Ig-binding activity after incubation with 1 M NaOH for at least 37 hours.

[0025] This summary of the invention does not necessarily describe all features of the invention, other embodiments will become apparent from review of the following detailed description. DETAILED DESCRIPTION OF THE INVENTION

[0026] Before the present invention is described in detail below, it is to be understood that the present invention is not limited to the particular methodology, protocols, and reagents described herein, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which may be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0027] Preferably, the terms used herein correspond to the definitions provided in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)" Leuenberger, H.G.W., Nagel, B. and Kolbl, H. (eds.) (1995), Helvetica Chimica Acta, Basel CH-4010, Switzerland.

[0028] Throughout this specification and the claims that follow, unless the context requires otherwise, it will be understood that the words "comprise" and variations such as "comprises" and "comprising" imply the inclusion of a stated element, integer or step or group of elements, integers or steps, and not the exclusion of any other element, integer or step or group of elements, integers or steps.

[0029] As used in the description of this invention and the appended claims, the singular forms "a," "an," and "the" are used interchangeably and include the plural forms and are intended to be applicable in each sense, unless the context clearly indicates otherwise. Similarly, as used herein, "and / or" refers to and includes all possible combinations of one or more of the listed items, as well as the lack of combination when interpreted as an alternative ("or").

[0030] As used herein, the term "about" encompasses the explicitly recited amount as well as a deviation thereof of ±10%. More preferably, a deviation of 5% is encompassed by the term "about."

[0031] Numerous documents (e.g., patents, patent applications, scientific publications, manufacturer's specifications, etc.) are cited throughout the teachings of this specification. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. Some of the documents cited herein are characterized as being "incorporated by reference." In the event of a conflict between a definition or teaching of such incorporated reference and a definition or teaching set forth herein, the teachings of this specification shall control.

[0032] All sequences referred to herein are disclosed in the accompanying Sequence Listing, which together with the entire contents and disclosure thereof, forms part of this specification.

[0033] In the context of the present invention, the term "Ig-binding protein" or "immunoglobulin-binding protein" is used to describe a protein capable of specifically binding to an immunoglobulin. Furthermore, in the context of the present invention, the term "Ig-binding domain" or "immunoglobulin-binding domain" is used to describe a protein capable of specifically binding to an immunoglobulin. The Ig-binding protein or Ig-binding domain of the present invention may be referred to herein as a ligand of the present invention. "Immunoglobulin" or "Ig" as understood herein may include, but is not necessarily limited to, for example, human IgG1, human IgG2, human IgG4, mouse IgG, rat IgG, goat IgG, bovine IgG, guinea pig IgG, rabbit IgG; human IgM, human IgA; and immunoglobulins or immunoglobulin fragments comprising an Fc region (also referred to as "Fc fragment" or "Fc") and / or immunoglobulin fragments comprising a Fab region (also referred to as "Fab fragment" or "Fab"). Ig-binding proteins are capable of binding to whole immunoglobulins as well as to Ig fragments comprising an Fc region and / or Ig fragments comprising a Fab region. The definition "immunoglobulin" as understood herein encompasses fusion proteins comprising an immunoglobulin, a fragment of an immunoglobulin comprising an Fc region (Fc fragment), a fragment of an immunoglobulin comprising a Fab region (Fab fragment), a fusion protein comprising a fragment of an immunoglobulin comprising an Fc region, a fusion protein comprising a fragment of an immunoglobulin comprising a Fab region, a conjugate comprising an Ig or an Ig fragment comprising an Fc region (Fc fragment), and a conjugate comprising an Ig fragment comprising a Fab region (Fab fragment).

[0034] As will be recognized by those skilled in the art, the terms "immunoglobulin" and "antibody" may be used interchangeably herein. Any definition disclosed herein for the term "immunoglobulin" applies accordingly to the term "antibody."

[0035] The term "binding" according to the present invention preferably relates to specific binding, meaning that an Ig-binding protein or Ig-binding domain binds to a specific immunoglobulin more strongly than it binds to another non-immunoglobulin target.

[0036] The term "binding activity" refers to the ability of an Ig-binding protein or Ig-binding domain of the present invention to bind to an immunoglobulin. For example, binding activity can be determined before and / or after alkaline treatment. The terms (immunoglobulin) "binding activity" and "binding capacity" are sometimes used interchangeably herein. Binding activity can be determined for an Ig-binding protein or for an Ig-binding protein coupled to a matrix, i.e., for an immobilized Ig-binding protein. Similarly, binding activity can be determined for an Ig-binding domain or for an Ig-binding domain coupled to a matrix, i.e., for an immobilized Ig-binding domain. The term "artificial" refers to an object that is not naturally occurring, i.e., an object that has been generated or modified by humans. For example, a polypeptide or polynucleotide sequence that has been created by humans (e.g., by genetic engineering, shuffling, or in a laboratory by chemical reaction, etc.) or intentionally modified is artificial.

[0037] The term "dissociation constant" or "K D " defines the specific binding affinity. As used herein, the term "K D (usually measured in "mol / L", sometimes abbreviated as "M") is intended to refer to the dissociation equilibrium constant of a particular interaction between a first protein and a second protein. In the context of the present invention, the term K D is used to describe the binding affinity between an Ig-binding protein or Ig-binding domain and an immunoglobulin, in particular. The Ig-binding proteins or Ig-binding domains of the invention have a dissociation constant K for immunoglobulins of at least 500 nM or less, or preferably 100 nM or less, more preferably 50 nM or less, more preferably 10 nM or less. D is considered to bind to immunoglobulin if it has

[0038] The terms "protein" and "polypeptide" refer to any linear molecular chain of two or more amino acids linked by peptide bonds, and do not refer to a specific length of the product. Thus, "peptide," "protein," "amino acid chain," or any other term used to refer to a chain of two or more amino acids is included in the definition of "polypeptide," and the term "polypeptide" may be used in place of, or interchangeably with, any of these terms. The term "polypeptide" also refers to products of post-translational modifications of polypeptides, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, proteolytic cleavage, modifications with non-naturally occurring amino acids, and similar modifications known in the art. Thus, Ig binding proteins comprising two or more protein domains also fall within the definition of the term "protein" or "polypeptide."

[0039] The terms "alkaline-stable" or "alkaline stability" or "caustic-stable" or "caustic stability" (also abbreviated herein as "cs") may be used interchangeably herein and refer to the ability of an Ig-binding protein or Ig-binding domain of the invention to withstand alkaline conditions without significant loss of its ability to bind immunoglobulin. One skilled in the art can easily test alkaline stability by incubating the Ig-binding protein or Ig-binding domain with, for example, sodium hydroxide solution, as described in the Examples, and then testing the binding capacity or activity to immunoglobulin by routine experiments known to those skilled in the art, for example, by chromatographic approaches. Alkaline stability may also be determined by coupling the Ig-binding protein or Ig-binding domain of the invention to a surface plasmon resonance (SPR) sensor chip and assaying the binding capacity or activity to immunoglobulin before and after exposure to alkaline solution. High-alkaline treatment may be carried out, for example, in 1 M NaOH for an extended period of time, for example, for at least 30 hours, and in various embodiments, for at least 36 hours or 48 hours.

[0040] Ig-binding proteins or Ig-binding domains of the invention, as well as matrices comprising Ig-binding proteins or Ig-binding domains of the invention, exhibit "high" or "improved" alkaline stability, meaning that molecules and matrices incorporating the Ig-binding proteins or Ig-binding domains are stable under alkaline conditions for extended periods of time compared to a reference. In various embodiments, the reference can be an Ig-binding protein or Ig-binding domain, or a matrix comprising an Ig-binding protein or Ig-binding domain, that does not bear the specific amino acid residues at positions 5, 8, 28, and 42, and / or does not bear the specific amino acid substitutions at positions 9, 11, and 15 described elsewhere herein, and / or does not bear the specific amino acid substitutions at positions 4 and / or 7 described elsewhere herein. In various embodiments, the reference Ig-binding protein or Ig-binding domain, or a matrix comprising an Ig-binding protein or Ig-binding domain, does not bear the specific amino acid substitution (K to P) at the position corresponding to position 58 of SEQ ID NO: 19.

[0041] As used herein, the term "variant" encompasses an amino acid sequence of an Ig binding protein or Ig binding domain that differs from another amino acid sequence by at least one amino acid substitution, deletion, or insertion. These modifications may be made by genetic manipulation performed by a human or by chemical synthesis or reaction.

[0042] The term "conjugate" as used herein relates to a molecule comprising or consisting essentially of at least one first protein chemically attached to another substance, such as a second protein or a non-proteinaceous moiety.

[0043] The term "modification" or "amino acid modification" refers to the replacement, deletion, or insertion of an amino acid at a particular position within a polypeptide sequence with another amino acid. Given the known genetic code and recombinant and synthetic DNA techniques, skilled scientists can readily construct DNA encoding amino acid variants.

[0044] The term "substitution" or "amino acid substitution" refers to the replacement of an amino acid at a particular position in a polypeptide sequence with another amino acid. The term "deletion" or "amino acid deletion" refers to the removal of an amino acid at a particular position in a polypeptide sequence.

[0045] The term "insertion" or "amino acid insertion" refers to the addition of an amino acid to a polypeptide sequence.

[0046] Throughout this specification, amino acid residue position numbers are designated as corresponding to their position numbers, e.g., in SEQ ID NO: 1. Thus, for example, the amino acid corresponding to position 5 refers to the amino acid residue at the position corresponding to position 5 in SEQ ID NO: 1. In embodiments of the invention related to SEQ ID NOs: 1-6, 13, 15, 17, and 19, the amino acid corresponding to position 5 is phenylalanine (F), the amino acid corresponding to position 8 is isoleucine (I), the amino acid corresponding to position 28 is histidine (H), and the amino acid corresponding to position 42 is lysine (K). Furthermore, in embodiments of the invention related to SEQ ID NOs: 1-6, 13, 15, 17, and 19, the amino acid corresponding to position 9 is alanine (A) or glutamine (Q), the amino acid corresponding to position 11 is alanine (A) or isoleucine (I), and / or the amino acid corresponding to position 15 is alanine (A) or glutamic acid (E). Furthermore, in embodiments of the invention related to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 13, 15, 17, and 19, the amino acid corresponding to position 9 is alanine (A) or glutamine (Q), the amino acid corresponding to position 11 is alanine (A) or isoleucine (I), the amino acid corresponding to position 15 is alanine (A) or glutamic acid (E), the amino acid corresponding to position 4 is glutamine (Q) or lysine (K), and / or the amino acid corresponding to position 7 is lysine (K) or glutamic acid (E). For corresponding positions in multimers, particularly pentamers, of the invention, reference is made to Table 1 below. The term "amino acid sequence identity" refers to a quantitative comparison of the identities (or differences) of the amino acid sequences of two or more proteins. "Percent (%) amino acid sequence identity" or "percent identical" or "percent identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a sequence that are identical with the amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.In various embodiments, the term "sequence identity" means that two (nucleotide or) amino acid sequences share at least 85% sequence identity, or at least 89.5% sequence identity, or at least 91% sequence identity, or at least 95% sequence identity, or more, when aligned, such as by the programs GAP or BESTFIT, optionally using default gap weights.

[0047] To determine sequence identity, the sequence of a query protein is aligned and compared to the sequence of a reference protein. Methods for sequence alignment and sequence comparison algorithms are well known in the art. For example, to determine the degree of amino acid sequence identity of any polypeptide compared to a reference amino acid sequence, the SIM local similarity program is preferably used. For multiple alignment analysis, ClustalW, known to those skilled in the art, is preferably used.

[0048] The degree of sequence identity is generally calculated over the entire length of the unmodified sequence. As used herein, the phrase "percent identical" or "percent (%) amino acid sequence identity" or "percent identity" in the context of two polypeptide sequences refers to two or more sequences or subsequences that, when compared and aligned for maximum correspondence, have in some embodiments at least 89.5%, in some embodiments at least 91%, in some embodiments at least 92%, in some embodiments at least 93%, in some embodiments at least 94%, in some embodiments at least 95%, in some embodiments at least 96%, in some embodiments at least 97%, in some embodiments at least 98%, and in some embodiments 100% amino acid residue identity, as measured using one of the following sequence comparison algorithms or by visual inspection. For clarity, for example, a sequence having at least 89.5% identity includes all sequences identifying greater than 89.5% identity, e.g., embodiments having at least 89.6%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid identity.

[0049] The percent identity exists over a region of at least 52 residues in some embodiments, over a region of at least 53 residues in some embodiments, over a region of at least 54 residues in some embodiments, over a region of at least 55 residues in some embodiments, over a region of at least 56 residues in some embodiments, over a region of at least 57 residues in some embodiments, and over a region of at least 58 residues in some embodiments. In certain embodiments, the percent identity exists over a region of (at least) 56 residues, e.g., with respect to an Ig binding protein described herein having a deletion of amino acid positions corresponding to positions 1 and 2 of any of SEQ ID NOs: 1-6, 13, 15, 17, and 19.

[0050] The term "fused" means that polypeptide components or units are linked by peptide bonds, either directly or via a peptide linker. In various embodiments, the term "fused" can also mean that polypeptide components or units are linked by a non-peptide linker, for example, through chemical conjugation.

[0051] The term "fusion protein" refers to a protein comprising at least one first protein genetically joined to at least one second protein. Fusion proteins are created through the joining of two or more genes that originally encoded separate proteins. Thus, a fusion protein may comprise multimers of the same or different proteins expressed as a single linear polypeptide. In various embodiments, fusion proteins are created through the joining of two or more polypeptides via non-peptide linkers, for example, through chemical conjugation. In various embodiments, dimers of Ig-binding proteins or Ig-binding domains of the present invention may be considered "fusion proteins."

[0052] The term "linker" as used herein refers in its broadest sense to a molecule that covalently joins at least two other molecules. In typical embodiments of the present invention, a "linker" should be understood as a moiety that connects an Ig-binding protein or Ig-binding domain to at least one additional Ig-binding protein or Ig-binding domain, i.e., a moiety that links two protein domains together to create a dimer or multimer. In preferred embodiments, the "linker" is a peptide linker, i.e., the moiety that links two binding proteins or binding domains is a single amino acid or a peptide comprising two or more amino acids. In various embodiments, dimers or multimers of the present invention may comprise a linker that joins two or more Ig-binding proteins or Ig-binding domains together.

[0053] The term "chromatography" refers to a separation technique that uses a mobile phase and a stationary phase to separate one type of molecule (e.g., immunoglobulins) from other molecules (e.g., contaminants or other immunoglobulins) in a sample. The liquid mobile phase contains a mixture of molecules and transports them across or through a stationary phase (such as a solid matrix). Differential interactions of different molecules in the mobile phase with the stationary phase allow the molecules in the mobile phase to be separated.

[0054] The term "affinity chromatography" refers to a specific mode of chromatography in which a ligand coupled to a stationary phase interacts with molecules (i.e., immunoglobulins) in a mobile phase (sample), i.e., the ligand has a specific binding affinity or capacity that allows the molecules to be purified. As understood in the context of the present invention, affinity chromatography involves the addition of a (liquid) sample containing immunoglobulins to a stationary phase that comprises a chromatographic ligand, such as an Ig-binding protein or Ig-binding domain of the present invention.

[0055] The terms "solid support" or "solid matrix" are used interchangeably herein and in various embodiments refer to the stationary phase.

[0056] The terms "affinity matrix" or "affinity separation matrix" or "affinity chromatography matrix," used interchangeably herein, refer to a matrix, e.g., a chromatography matrix, to which an affinity ligand, e.g., an Ig-binding protein or Ig-binding domain of the present invention, is attached. The ligand (e.g., an Ig-binding protein or Ig-binding domain) is capable of specifically binding to a molecule of interest (e.g., an immunoglobulin, as defined above) to be purified or removed from a mixture (in a liquid sample). As will be recognized by those skilled in the art, the terms "affinity matrix" or "affinity separation matrix" or "affinity chromatography matrix" describe the separation of molecules of interest (particularly immunoglobulins) by using an Ig-binding protein or Ig-binding domain of the present invention. Thus, the terms "affinity matrix" or "affinity separation matrix" or "affinity chromatography matrix" or "separation matrix" may be used interchangeably herein.

[0057] The term "affinity purification" as used herein refers to a method of purifying an immunoglobulin of interest, as defined above, from a liquid (sample) by binding the immunoglobulin of interest to an Ig-binding protein or Ig-binding domain immobilized on a matrix. This removes all other components of the mixture other than the immunoglobulin of interest. In various embodiments, said other components of the mixture may include, for example, other immunoglobulins not of interest. In a further step, the immunoglobulin of interest is eluted in purified form. The terms "affinity purification" or "affinity chromatography purification" or "affinity separation" or "affinity chromatography separation" may be used interchangeably herein.

[0058] Embodiments of the invention The present invention will now be further described. In the following passages, different embodiments of the present invention are defined in more detail. Each embodiment defined below may be combined with any other embodiment, unless expressly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0059] The present invention provides an immunoglobulin (Ig)-binding protein comprising the amino acid sequence of SEQ ID NO: 13, wherein the amino acid corresponding to position 5 of SEQ ID NO: 13 is phenylalanine (F), the amino acid corresponding to position 8 of SEQ ID NO: 13 is isoleucine (I), the amino acid corresponding to position 28 of SEQ ID NO: 13 is histidine (H), and the amino acid corresponding to position 42 of SEQ ID NO: 13 is lysine (K); or an Ig-binding protein comprising an amino acid sequence with at least 89.5% amino acid identity to SEQ ID NO: 13.

[0060] In various embodiments, the amino acid corresponding to position 9 of SEQ ID NO: 13 is alanine (A) or glutamine (Q).

[0061] In various embodiments, the amino acid corresponding to position 11 of SEQ ID NO: 13 is alanine (A) or isoleucine (I).

[0062] In various embodiments, the amino acid corresponding to position 15 of SEQ ID NO: 13 is alanine (A) or glutamic acid (E).

[0063] In various embodiments, the amino acid corresponding to position 4 of SEQ ID NO: 13 is lysine (K) or glutamine (Q).

[0064] In various embodiments, the amino acid corresponding to position 7 of SEQ ID NO: 13 is lysine (K) or glutamic acid (E).

[0065] In various embodiments, the amino acid corresponding to position 58 of SEQ ID NO: 13 is lysine (K) or proline (P).

[0066] In some embodiments, the Ig-binding protein or Ig-binding domain of the present invention is selected from the following (1) to (2): (1) a protein comprising an amino acid sequence corresponding to SEQ ID NO: 13; (2) A protein containing an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids at positions 5, 8, 28, and 42 of SEQ ID NO: 13 specified in (1) are not mutated.

[0067] In some embodiments, the Ig-binding protein or Ig-binding domain of the present invention is selected from the following (1) to (6): (1) A protein containing amino acids corresponding to SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 17 or SEQ ID NO: 19, in which the amino acid residue corresponding to position 5 is Phe (F), the amino acid residue corresponding to position 8 is Ile (I), the amino acid residue corresponding to position 28 is His (H), and the amino acid residue corresponding to position 42 is Lys (K); (2) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids at positions 5, 8, 28, and 42 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 17, and 19 specified in (1) are not mutated; (3) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), in which the amino acid corresponding to position 4 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, or 17 is lysine (K) and the amino acid corresponding to position 7 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, or 17 is glutamic acid (E); (4) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), in which the amino acid corresponding to position 4 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 17, or 19 is glutamine (Q) and the amino acid corresponding to position 7 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, or 17 is lysine (K); (5) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence set forth in (1), in which the amino acid corresponding to position 4 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, or 17 is glutamine (Q) and the amino acid corresponding to position 7 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, or 17 is glutamic acid (E); (6) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), in which the amino acid corresponding to position 4 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, or 17 is lysine (K) and the amino acid corresponding to position 7 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, or 17 is lysine (K).

[0068] In some embodiments, the Ig-binding protein or Ig-binding domain of the present invention is selected from the following (1) to (4): (1) a protein comprising the amino acid sequence of SEQ ID NO: 13; (2) A protein comprising the amino acid sequence specified in (1), in which the amino acid residue corresponding to position 9 of SEQ ID NO: 13 is Ala (A) or Gln (Q), and / or the amino acid residue corresponding to position 11 of SEQ ID NO: 13 is Ile (I) or Ala (A), and / or the amino acid residue corresponding to position 15 of SEQ ID NO: 13 is Glu (E) or Ala (A); (3) A protein comprising the amino acid sequence specified in (1), wherein the amino acid residue corresponding to position 4 of SEQ ID NO: 13 is lysine (K) or glutamine (Q), and / or the amino acid residue corresponding to position 7 of SEQ ID NO: 13 is lysine (K) or glutamic acid (E), and / or the amino acid residue corresponding to position 9 of SEQ ID NO: 13 is Ala (A) or Gln (Q), and / or the amino acid residue corresponding to position 11 of SEQ ID NO: 13 is Ile (I) or Ala (A), and / or the amino acid residue corresponding to position 15 of SEQ ID NO: 13 is Glu (E) or Ala (A); (4) A protein containing an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids at positions 5, 8, 28, and 42 specified in (1) are not mutated.

[0069] The present invention further provides immunoglobulin (Ig)-binding proteins comprising the amino acid sequence of SEQ ID NO: 15, wherein the amino acid corresponding to position 5 of SEQ ID NO: 15 is phenylalanine (F), the amino acid corresponding to position 8 of SEQ ID NO: 15 is isoleucine (I), the amino acid corresponding to position 28 of SEQ ID NO: 15 is histidine (H), and the amino acid corresponding to position 42 of SEQ ID NO: 15 is lysine (K), or Ig-binding proteins comprising an amino acid sequence having at least 89.5% amino acid identity to SEQ ID NO: 15. In various preferred embodiments, the amino acid corresponding to position 4 of SEQ ID NO: 15 is lysine (K) and the amino acid corresponding to position 7 of SEQ ID NO: 15 is glutamic acid (E). In various other embodiments, the amino acid corresponding to position 4 of SEQ ID NO: 15 is glutamine (Q) and the amino acid corresponding to position 7 of SEQ ID NO: 15 is lysine (K). In various further embodiments, the amino acid corresponding to position 4 of SEQ ID NO: 15 is glutamine (Q) and the amino acid corresponding to position 7 of SEQ ID NO: 15 is glutamic acid (E). Further, in various embodiments, the amino acid corresponding to position 4 of SEQ ID NO:15 is lysine (K), and the amino acid corresponding to position 7 of SEQ ID NO:15 is lysine (K). In various further embodiments, the amino acid corresponding to position 58 of SEQ ID NO:15 is proline (P). In various further embodiments, the amino acid corresponding to position 4 of SEQ ID NO:15 is glutamine (Q), the amino acid corresponding to position 7 of SEQ ID NO:15 is lysine (K), the amino acids corresponding to positions 9 and 11 of SEQ ID NO:15 are alanine (A), the amino acid corresponding to position 15 of SEQ ID NO:15 is glutamic acid (E), and the amino acid corresponding to position 58 of SEQ ID NO:15 is proline (P).

[0070] In some embodiments, the Ig-binding protein or Ig-binding domain of the present invention is selected from the following (1) to (4): (1) a protein comprising an amino acid sequence corresponding to SEQ ID NO: 15; (2) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids corresponding to the amino acids at positions 5, 8, 28, and 42 of SEQ ID NO: 15 specified in (1) are not mutated; (3) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids corresponding to the amino acids at positions 5, 8, 28, and 42 of SEQ ID NO: 15 specified in (1) are not mutated, the amino acid corresponding to the amino acid at position 9 of SEQ ID NO: 15 is Ala (A) or Gln (Q), and / or the amino acid corresponding to the amino acid at position 11 of SEQ ID NO: 15 is Ile (I) or Ala (A), and / or the amino acid corresponding to the amino acid at position 15 of SEQ ID NO: 15 is Glu (E) or Ala (A); (4) The amino acids corresponding to the amino acids at positions 5, 8, 28, and 42 of SEQ ID NO: 15 as specified in (1) are not mutated, the amino acid corresponding to the amino acid at position 9 of SEQ ID NO: 15 is Ala (A) or Gln (Q), and / or the amino acid corresponding to the amino acid at position 11 of SEQ ID NO: 15 is Ile (I) or Ala (A), and / or the amino acid corresponding to the amino acid at position 15 of SEQ ID NO: 15 is Glu (E) or Ala (A), the amino acid corresponding to the amino acid at position 4 of SEQ ID NO: 15 is lysine (K) or glutamine (Q), and / or the amino acid corresponding to the amino acid at position 7 of SEQ ID NO: 15 is lysine (K) or glutamic acid (E); in various preferred embodiments, In various other embodiments, the amino acid corresponding to position 4 of SEQ ID NO: 15 is glutamine (Q) and the amino acid corresponding to position 7 of SEQ ID NO: 15 is lysine (K); in various further embodiments, the amino acid corresponding to position 4 of SEQ ID NO: 15 is glutamine (Q) and the amino acid corresponding to position 7 of SEQ ID NO: 15 is glutamic acid (E); and further, in various embodiments, the amino acid corresponding to position 4 of SEQ ID NO: 15 is lysine (K) and the amino acid corresponding to position 7 of SEQ ID NO: 15 is lysine (K).

[0071] In some embodiments, the Ig-binding protein or Ig-binding domain of the present invention is selected from the following (1) to (6): (1) a protein comprising an amino acid sequence corresponding to SEQ ID NO: 1; (2) A protein comprising the amino acid sequence specified in (1), which further has one or more amino acid substitutions at positions other than positions 5, 8, 28, and 42 of SEQ ID NO: 1, preferably further has one or more amino acid substitutions at any of positions 4, 7, 9, 11, and 15 of SEQ ID NO: 1 specified in (1); (3) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids at positions 5, 8, 28, and 42 of SEQ ID NO: 1 specified in (1) are not mutated; (4) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids at positions 5, 8, 28, and 42 of SEQ ID NO: 1 specified in (1) are not mutated, and the amino acids at positions 9, 11, and 15 of SEQ ID NO: 1 specified in (1) are not mutated; (5) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids at positions 5, 8, 28, and 42 of SEQ ID NO: 1 specified in (1) are not mutated, and the amino acids at positions 9, 11, and 15 of SEQ ID NO: 1 specified in (1) are not mutated; (6) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids at positions 5, 8, 28, and 42 of SEQ ID NO: 1 specified in (1) are not mutated, the amino acids at positions 9, 11, and 15 of SEQ ID NO: 1 specified in (1) are not mutated, and the amino acids at positions 4 and 7 of SEQ ID NO: 1 specified in (1) are not mutated.

[0072] In some embodiments, the Ig-binding protein or Ig-binding domain of the present invention is selected from the following (1) to (6): (1) a protein comprising an amino acid sequence corresponding to SEQ ID NO: 2; (2) A protein comprising the amino acid sequence specified in (1), further comprising one or more amino acid substitutions at one or more positions other than positions 5, 8, 28, and 42 of SEQ ID NO: 2, preferably at any one of positions 4, 7, 9, 11, and 15 of SEQ ID NO: 2 specified in (1); (3) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids at positions 5, 8, 28, and 42 of SEQ ID NO: 2 specified in (1) are not mutated; (4) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids at positions 5, 8, 28, and 42 of SEQ ID NO: 2 specified in (1) are not mutated, and the amino acids at positions 9, 11, and 15 of SEQ ID NO: 2 specified in (1) are not mutated; (5) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids at positions 5, 8, 28, and 42 of SEQ ID NO: 2 specified in (1) are not mutated, and the amino acids at positions 9, 11, and 15 of SEQ ID NO: 2 specified in (1) are not mutated; (6) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids at positions 5, 8, 28, and 42 of SEQ ID NO: 2 specified in (1) are not mutated, the amino acids at positions 9, 11, and 15 of SEQ ID NO: 2 specified in (1) are not mutated, and the amino acids at positions 4 and 7 of SEQ ID NO: 2 specified in (1) are not mutated.

[0073] In some embodiments, the Ig-binding protein or Ig-binding domain of the present invention is selected from the following (1) to (6): (1) a protein comprising an amino acid sequence corresponding to SEQ ID NO: 3; (2) A protein comprising the amino acid sequence specified in (1), further comprising one or more amino acid substitutions at one or more positions other than positions 5, 8, 28, and 42 of SEQ ID NO: 3, preferably at any one of positions 4, 7, 9, 11, and 15 of SEQ ID NO: 3 specified in (1); (3) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids at positions 5, 8, 28, and 42 of SEQ ID NO: 3 specified in (1) are not mutated; (4) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids at positions 5, 8, 28, and 42 of SEQ ID NO: 3 specified in (1) are not mutated, and the amino acids at positions 9, 11, and 15 of SEQ ID NO: 3 specified in (1) are not mutated; (5) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids at positions 5, 8, 28, and 42 of SEQ ID NO: 3 specified in (1) are not mutated, and the amino acids at positions 9, 11, and 15 of SEQ ID NO: 3 specified in (1) are not mutated; (6) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids at positions 5, 8, 28, and 42 of SEQ ID NO: 3 specified in (1) are not mutated, the amino acids at positions 9, 11, and 15 of SEQ ID NO: 3 specified in (1) are not mutated, and the amino acids at positions 4 and 7 of SEQ ID NO: 3 specified in (1) are not mutated.

[0074] In various embodiments related to SEQ ID NO:3 described in (1) and (6) above and elsewhere herein, the amino acid corresponding to position 58 of SEQ ID NO:15 is proline (P).

[0075] In some embodiments, the Ig-binding protein or Ig-binding domain of the present invention is selected from the following (1) to (6): (1) a protein comprising an amino acid sequence corresponding to SEQ ID NO: 4; (2) A protein comprising the amino acid sequence specified in (1), further comprising one or more amino acid substitutions at one or more positions other than positions 5, 8, 28, and 42 of SEQ ID NO: 4, preferably at any one of positions 4, 7, 9, 11, and 15 of SEQ ID NO: 4 specified in (1); (3) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids at positions 5, 8, 28, and 42 of SEQ ID NO: 4 specified in (1) are not mutated; (4) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids at positions 5, 8, 28, and 42 of SEQ ID NO: 4 specified in (1) are not mutated, and the amino acids at positions 9, 11, and 15 of SEQ ID NO: 4 specified in (1) are not mutated; (5) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids at positions 5, 8, 28, and 42 of SEQ ID NO: 4 specified in (1) are not mutated, and the amino acids at positions 9, 11, and 15 of SEQ ID NO: 4 specified in (1) are not mutated; (6) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids at positions 5, 8, 28, and 42 of SEQ ID NO: 4 specified in (1) are not mutated, the amino acids at positions 9, 11, and 15 of SEQ ID NO: 4 specified in (1) are not mutated, and the amino acids at positions 4 and 7 of SEQ ID NO: 4 specified in (1) are not mutated.

[0076] In some embodiments, the Ig-binding protein or Ig-binding domain of the present invention is selected from the following (1) to (6): (1) a protein comprising an amino acid sequence corresponding to SEQ ID NO: 5; (2) A protein comprising the amino acid sequence specified in (1), further comprising one or more amino acid substitutions at one or more positions other than positions 5, 8, 28, and 42 of SEQ ID NO: 5, preferably at any one of positions 4, 7, 9, 11, and 15 of SEQ ID NO: 5 specified in (1); (3) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids at positions 5, 8, 28, and 42 of SEQ ID NO: 5 specified in (1) are not mutated; (4) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids at positions 5, 8, 28, and 42 of SEQ ID NO: 5 specified in (1) are not mutated, and the amino acids at positions 9, 11, and 15 of SEQ ID NO: 5 specified in (1) are not mutated; (5) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids at positions 5, 8, 28, and 42 of SEQ ID NO: 5 specified in (1) are not mutated, and the amino acids at positions 9, 11, and 15 of SEQ ID NO: 5 specified in (1) are not mutated; (6) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids at positions 5, 8, 28, and 42 of SEQ ID NO: 5 specified in (1) are not mutated, the amino acids at positions 9, 11, and 15 of SEQ ID NO: 5 specified in (1) are not mutated, and the amino acids at positions 4 and 7 of SEQ ID NO: 5 specified in (1) are not mutated.

[0077] In some embodiments, the Ig-binding protein or Ig-binding domain of the present invention is selected from the following (1) to (6): (1) a protein comprising an amino acid sequence corresponding to SEQ ID NO: 6; (2) A protein comprising the amino acid sequence specified in (1), further comprising one or more amino acid substitutions at one or more positions other than positions 5, 8, 28, and 42 of SEQ ID NO: 6, preferably at any one of positions 4, 7, 9, 11, and 15 of SEQ ID NO: 6 specified in (1); (3) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids at positions 5, 8, 28, and 42 of SEQ ID NO: 6 specified in (1) are not mutated; (4) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids at positions 5, 8, 28, and 42 of SEQ ID NO: 6 specified in (1) are not mutated, and the amino acids at positions 9, 11, and 15 of SEQ ID NO: 6 specified in (1) are not mutated; (5) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids at positions 5, 8, 28, and 42 of SEQ ID NO: 6 specified in (1) are not mutated, and the amino acids at positions 9, 11, and 15 of SEQ ID NO: 6 specified in (1) are not mutated; (6) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids at positions 5, 8, 28, and 42 of SEQ ID NO: 6 specified in (1) are not mutated, the amino acids at positions 9, 11, and 15 of SEQ ID NO: 6 specified in (1) are not mutated, and the amino acids at positions 4 and 7 of SEQ ID NO: 6 specified in (1) are not mutated.

[0078] In some embodiments, the Ig-binding protein or Ig-binding domain of the present invention is selected from the following (1) to (6): (1) a protein comprising an amino acid sequence corresponding to SEQ ID NO: 17; (2) A protein comprising the amino acid sequence set forth in (1), further comprising one or more amino acid substitutions at one or more positions other than positions 5, 8, 28, and 42 of SEQ ID NO: 17, preferably at any one of positions 4, 7, 9, 11, and 15 of SEQ ID NO: 17; (3) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids at positions 5, 8, 28, and 42 of SEQ ID NO: 17 specified in (1) are not mutated; (4) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids at positions 5, 8, 28, and 42 of SEQ ID NO: 17 specified in (1) are not mutated, and the amino acids at positions 9, 11, and 15 of SEQ ID NO: 17 specified in (1) are not mutated; (5) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids at positions 5, 8, 28, and 42 of SEQ ID NO: 17 specified in (1) are not mutated, and the amino acids at positions 9, 11, and 15 of SEQ ID NO: 17 specified in (1) are not mutated; (6) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids at positions 5, 8, 28, and 42 of SEQ ID NO: 17 specified in (1) are not mutated, the amino acids at positions 9, 11, and 15 of SEQ ID NO: 17 specified in (1) are not mutated, and the amino acids at positions 4 and 7 of SEQ ID NO: 17 specified in (1) are not mutated.

[0079] The present invention encompasses immunoglobulin (Ig)-binding proteins comprising the amino acid sequence of SEQ ID NO: 19, wherein the amino acid corresponding to position 5 of SEQ ID NO: 19 is phenylalanine (F), the amino acid corresponding to position 8 of SEQ ID NO: 19 is isoleucine (I), the amino acid corresponding to position 28 of SEQ ID NO: 19 is histidine (H), and the amino acid corresponding to position 42 of SEQ ID NO: 19 is lysine (K), or Ig-binding proteins comprising an amino acid sequence with at least 89.5% amino acid identity to SEQ ID NO: 19. In a preferred embodiment of SEQ ID NO: 19, the amino acid corresponding to position 5 is F, the amino acid corresponding to position 8 is I, the amino acid corresponding to position 28 is H, the amino acid corresponding to position 42 is K, the amino acid corresponding to position 4 is glutamine (Q), the amino acid corresponding to position 7 is lysine (K), the amino acids corresponding to positions 9 and 11 are alanine (A), the amino acid corresponding to position 15 is glutamic acid (E), and the amino acid corresponding to position 58 is proline (P).

[0080] In some embodiments, the Ig-binding protein or Ig-binding domain of the present invention is selected from the following (1) to (7): (1) a protein comprising an amino acid sequence corresponding to SEQ ID NO: 19; (2) A protein comprising the amino acid sequence set forth in (1), further comprising one or more amino acid substitutions at one or more positions other than positions 5, 8, 28, and 42 of SEQ ID NO: 19, preferably at any one of positions 4, 7, 9, 11, and 15 of SEQ ID NO: 19; (3) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids at positions 5, 8, 28, and 42 of SEQ ID NO: 19 specified in (1) are not mutated; (4) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids at positions 5, 8, 28, and 42 of SEQ ID NO: 19 specified in (1) are not mutated, and the amino acids at positions 9, 11, and 15 of SEQ ID NO: 19 specified in (1) are not mutated; (5) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids at positions 5, 8, 28, and 42 of SEQ ID NO: 19 specified in (1) are not mutated, and the amino acids at positions 9, 11, and 15 of SEQ ID NO: 19 specified in (1) are not mutated; (6) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids at positions 5, 8, 28, and 42 of SEQ ID NO: 19 specified in (1) are not mutated, the amino acids at positions 9, 11, and 15 of SEQ ID NO: 19 specified in (1) are not mutated, and the amino acids at positions 4 and 7 of SEQ ID NO: 19 specified in (1) are not mutated; (7) A protein comprising an amino acid sequence having at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids at positions 5, 8, 28, and 42 of SEQ ID NO: 19 specified in (1) are not mutated, the amino acids at positions 9, 11, and 15 of SEQ ID NO: 19 specified in (1) are not mutated, the amino acids at positions 4 and 7 of SEQ ID NO: 19 specified in (1) are not mutated, and the amino acid at position 58 of SEQ ID NO: 19 specified in (1) is not mutated.

[0081] A surprising advantage of the Ig-binding proteins and Ig-binding domains of the present invention is their stability under extreme conditions, such as high pH (pH 13 or higher), without loss of Ig-binding properties. The Ig-binding proteins and Ig-binding domains described herein demonstrate high alkaline stability for extended periods (at least 37 hours) without loss of Ig-binding properties (see Examples). Furthermore, they are stable at low pH without significant loss of Ig-binding properties. The feature of extremely high alkaline stability is particularly important for chromatographic approaches that involve harsh cleanup procedures using alkaline solutions with NaOH concentrations as high as 1 M to remove contaminants on the matrix so that the matrix can be used multiple times. In addition to extremely high caustic stability, the Ig-binding proteins also exhibit high coupling efficiencies, as shown in the Examples.

[0082] Furthermore, a critical step in affinity chromatography is the elution of the protein of interest, particularly the immunoglobulin of interest, bound to the Ig-binding protein or Ig-binding domain of the present invention. This step is usually carried out at a low pH. The affinity ligands of the present invention do not lose their Ig-binding properties after this treatment, while elution of the protein of interest is possible at a low pH. In some situations, it is important to have conditions for elution of antibodies (immunoglobulins) from the ligand at a pH higher than 3.7, such as pH 4.0 or 4.5, and higher. The ligands of the present invention have a residual IgG-binding capacity of about 80% after at least 37 hours with 1 M NaOH, and elution of at least about 92%, at least about 95%, preferably at least 97%, and more preferably 100% of the bound IgG from the matrix at a mild pH of 4.0 or higher.

[0083] In a preferred embodiment of the invention, the ligands of the invention that exhibit (or provide) at least about 92% elution of bound Ig (particularly IgG) from an affinity purification or separation matrix at pH 4.0 (or higher) after at least 37 hours of incubation in 1 M NaOH include Ig-binding proteins or Ig-binding domains described herein related to any of the monomers of SEQ ID NOS: 1-4, 6, 17, and 19 (including multimers such as pentamers of SEQ ID NOS: 7, 8, 9, 10, 12, 18, and 20).

[0084] In other preferred embodiments of the invention, the ligands of the invention that have demonstrated (or provided) at least about 94% elution of bound Ig (particularly IgG) from an affinity purification or separation matrix at pH 4.0 (or higher) after at least 37 hours of incubation in 1 M NaOH include Ig-binding proteins or Ig-binding domains described herein, particularly related to the monomer of SEQ ID NO: 6 (including multimers such as individual pentamers of SEQ ID NO: 12).

[0085] In a further preferred embodiment of the invention, the ligands of the invention that exhibit (or provide) at least about 97% elution of bound Ig (particularly IgG) from an affinity purification or separation matrix at pH 4.0 (or higher) after at least 37 hours of incubation in 1 M NaOH specifically include the Ig-binding proteins or Ig-binding domains described herein related to the monomers of SEQ ID NOs: 2 and 4 (including multimers such as the respective pentamers of SEQ ID NOs: 8 and 10).

[0086] Furthermore, preferred ligands of the present invention that have demonstrated (or provided) about 99% or 100% elution of bound Ig (particularly IgG) from affinity purification or separation matrices at pH 4.0 (or higher) after at least 37 hours of incubation in 1 M NaOH specifically include the Ig-binding proteins or Ig-binding domains described herein related to the monomers of SEQ ID NOs: 1 and 3 (including multimers such as the respective pentamers of SEQ ID NOs: 7 and 9).

[0087] Furthermore, preferred ligands of the present invention that have demonstrated (or provided) at least about 84% elution of bound Ig (particularly IgG) from an affinity purification or separation matrix at pH 4.3 (or higher) after at least 37 hours of incubation in 1 M NaOH include the Ig-binding proteins or Ig-binding domains described herein, specifically related to the monomer of SEQ ID NO: 17 (including multimers such as individual pentamers of SEQ ID NO: 18).

[0088] Furthermore, preferred ligands of the present invention that have demonstrated (or provided) at least about 84% elution of bound Ig (particularly IgG) from affinity purification or separation matrices at pH 4.5 (or higher) after at least 37 hours of incubation in 1 M NaOH include the Ig-binding proteins or Ig-binding domains described herein, specifically related to the monomer of SEQ ID NO: 17 (including multimers such as individual pentamers of SEQ ID NO: 18).

[0089] Further modifications can be introduced into the protein to modify its specific properties for affinity chromatography. For example, a cysteine ​​can be added to the C-terminus. Alternatively, a cysteine ​​can be introduced at positions corresponding to positions 43 or 46 (e.g., in SEQ ID NOS: 1-6, 13, 15, 17, and 19) to enable efficient coupling to the matrix.

[0090] In some embodiments, the Ig binding protein or Ig binding domain is selected from the following: (1) A protein comprising an amino acid sequence corresponding to an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:19, in which the amino acid residue corresponding to position 5 is Phe (F), the amino acid residue corresponding to position 8 is Ile (I), the amino acid residue corresponding to position 28 is His (H), and the amino acid residue corresponding to position 42 is Lys (K); (2) A protein containing an amino acid sequence that has at least 89.5% sequence identity with the amino acid sequence specified in (1), provided that the amino acids specified in (1) are not modified in (2).

[0091] The term "at least 89.5% or greater sequence identity" as used herein encompasses preferred embodiments in which the sequence identity is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity. Such preferred sequence identities are in accordance with the sequence identities set forth elsewhere herein in relation to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:19.

[0092] Preferred Ig-Binding Domains. In various embodiments, the Ig-binding protein comprises or consists of the amino acid sequence of any of the Ig-binding domains described herein. In various embodiments, the Ig-binding protein comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 13, 15, 17, and 19, or amino acids having at least 89.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to those of those SEQ ID NOs, assuming that the amino acid residue corresponding to position 5 is Phe (F), the amino acid residue corresponding to position 8 is Ile (I), the amino acid residue corresponding to position 28 is His (H), and the amino acid residue corresponding to position 42 is Lys (K). In various embodiments, the Ig binding domain comprises, consists essentially of, or consists of the amino acid sequence of any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:19, or amino acids having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:19, assuming that the amino acid residue corresponding to position 5 is Phe (F), the amino acid residue corresponding to position 8 is Ile (I), the amino acid residue corresponding to position 28 is His (H), and the amino acid residue corresponding to position 42 is Lys (K).

[0093] Preferred Ig-binding proteins. In some embodiments, the Ig-binding protein comprises one or more binding domains, at least one of which domains comprises or consists of the amino acid sequence of any one of SEQ ID NOs:1, 2, 3, 4, 5, 6, 13, 15, 17, and 19, where the amino acid residue corresponding to position 5 is Phe (F), the amino acid residue corresponding to position 8 is Ile (I), the amino acid residue corresponding to position 28 is His (H), and the amino acid residue corresponding to position 42 is Lys (K), or an amino acid sequence having at least 89.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In some embodiments, the Ig binding protein comprises one or more binding domains, at least one of which domains comprises, consists essentially of, or consists of the amino acid sequence of any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:19, or amino acids having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:19, where the amino acid residue corresponding to position 5 is Phe (F), the amino acid residue corresponding to position 8 is Ile (I), the amino acid residue corresponding to position 28 is His (H), and the amino acid residue corresponding to position 42 is Lys (K).

[0094] Affinity to immunoglobulins. All Ig-binding proteins or Ig-binding domains described herein preferably have a dissociation constant K of less than 200 nM, or less than 100 nM, more preferably less than 10 nM. DIn some embodiments, the Ig-binding protein or Ig-binding domain preferably binds to immunoglobulins with a dissociation constant K of less than 200 nM, or less than 100 nM, more preferably 10 nM or less. D and bind to Ig, in particular IgG1, IgG2, IgG4, IgM, and / or IgA, Ig fragments thereof, Fc fragments, Fab fragments, fusion proteins comprising an Ig region or the Fc region of an Ig, and conjugates comprising an Ig region. D Methods for determining the dissociation constant K are known to those skilled in the art and can be selected, for example, from the following methods known in the art: surface plasmon resonance (SPR)-based techniques, equilibrium exclusion analysis (KExA assay), biolayer interferometry (BLI), enzyme-linked immunosorbent assay (ELISA), flow cytometry, isothermal titration catrometry (ITC), analytical ultracentrifugation, radioimmunoassay (RIA or IRMA) and enhanced chemiluminescence (ECL). Some of the methods are further described in the Examples. Typically, the dissociation constant K D is determined at 20°C, 25°C, or 30°C, preferably by SPR at 20°C, 25°C, or 30°C. Unless otherwise specifically indicated, the K D The value is determined by surface plasmon resonance spectroscopy at 22° C.±3° C. In one embodiment, the Ig binding protein has a dissociation constant K for human IgG1 in the range between 0.1 nM and 100 nM, preferably between 0.1 nM and 50 nM. D Preferably, the K ranges from 0.1 nM to 100 nM, preferably from 0.1 nM to 50 nM. D is the K as determined by SPR, more preferably as determined by SPR at 20°C, 25°C, or 30°C. D means.

[0095] High alkaline stability of Ig-binding proteins. The Ig-binding proteins and Ig-binding domains of the present invention surprisingly exhibit a high dynamic binding capacity (DBC) of over 60 mg / ml at a 6-minute residence time, as well as particularly good alkaline stability, as demonstrated by at least about 80% residual IgG binding after prolonged incubation in 1 M NaOH, as shown in the Examples. The alkaline stability of an Ig-binding protein or Ig-binding domain is determined by comparing the loss of Ig-binding activity. In some embodiments, the alkaline solution comprises 0.1 to 1.0 M NaOH or KOH, preferably 0.5 to 1 M NaOH or KOH. Due to the high alkaline stability of the Ig-binding proteins and Ig-binding domains of the present invention, an alkaline solution with a pH greater than 13 can be used to purify affinity matrices bearing immobilized Ig-binding proteins or immobilized Ig-binding domains of the present invention. In some embodiments, the alkaline stability of an Ig-binding protein or Ig-binding domain is determined by comparing the loss of Ig-binding activity after at least 37 hours of incubation in 1 M NaOH (see Examples). In some embodiments, the alkaline stability of an Ig binding protein or Ig binding domain is determined by comparing the loss of Ig binding activity after very long incubation in alkaline solution, for example, after incubation in 1 M NaOH for at least 2 days (at least 48 hours) (see Examples), reflecting the exceptional stability of the Ig binding protein as described herein.

[0096] The Ig-binding proteins and Ig-binding domains of the invention are stable under alkaline conditions, particularly 1 M NaOH, for at least 37 hours. In a preferred embodiment, the Ig-binding proteins or Ig-binding domains of the invention are stable under alkaline conditions, particularly 1 M NaOH, for at least 48 hours, more preferably at least 50 hours.

[0097] The Ig-binding proteins and Ig-binding domains of the invention are alkaline-stable ligands for immunoglobulins. The Ig-binding proteins and Ig-binding domains of the invention retain their binding capacity (or binding affinity) to immunoglobulins after exposure to 1 M NaOH for at least 37 hours. As further described herein, the Ig-binding proteins and Ig-binding domains of the invention retain at least about 80%, or at least about 83%, or at least about 90% of their binding capacity to immunoglobulins after exposure to extreme alkaline conditions as described herein. In even more preferred embodiments, the Ig-binding proteins and Ig-binding domains of the invention retain at least about 80% of their binding capacity to immunoglobulins after exposure to extreme alkaline conditions (1 M NaOH for at least 37 hours). In various embodiments, the Ig-binding proteins and Ig-binding domains of the invention retain their binding capacity to immunoglobulins as described above when immobilized on a solid support, preferably an affinity separation matrix.

[0098] As further described herein, the Ig-binding proteins and Ig-binding domains of the invention are typically stable under alkaline conditions at room temperature. The term room temperature can encompass temperatures between 15°C and 25°C, more specifically temperatures between 20°C and 25°C. In various embodiments, the Ig-binding proteins or Ig-binding domains of the invention are stable under alkaline conditions at 22°C ± 3°C.

[0099] In various embodiments, the alkaline stability of an Ig-binding protein or Ig-binding domain, as described above, refers to the alkaline stability of an Ig-binding protein or Ig-binding domain immobilized on a solid support, preferably on an affinity separation matrix. Thus, in various embodiments, the alkaline stability of an Ig-binding protein or Ig-binding domain is determined by comparing the loss of Ig-binding activity or Ig-binding capacity of the Ig-binding protein or Ig-binding domain when immobilized on a solid support, preferably on an affinity separation matrix. Thus, in other embodiments, the alkaline stability of an Ig-binding protein or Ig-binding domain is determined by comparing the Ig-binding activity of the Ig-binding protein or Ig-binding domain, when immobilized on a solid support, to a reference protein after extended alkaline treatment.

[0100] The binding capacity or binding affinity of the Ig-binding proteins or Ig-binding domains of the invention to immunoglobulins can be determined by methods well known in the art, in particular by measuring the dissociation constant K as described elsewhere herein. D The binding capacity or affinity of an Ig-binding protein or Ig-binding domain of the invention to an immunoglobulin can be assessed by one of skill in the art using methods for determining binding capacity or affinity. In various embodiments, the binding capacity or affinity of an Ig-binding protein or Ig-binding domain of the invention to an immunoglobulin is determined using surface plasmon resonance (SPR) spectroscopy, as described elsewhere herein. In other embodiments, the binding capacity or affinity of an Ig-binding protein or Ig-binding domain of the invention to an immunoglobulin is determined using equilibrium exclusion analysis (KinExA assay) or enzyme-linked immunosorbent assay (ELISA), as described elsewhere herein.

[0101] The binding capacity or binding affinity of an Ig binding protein or Ig binding domain of the invention to immunoglobulins can be assessed for each candidate ligand before and after exposure to alkaline conditions as described herein.

[0102] Multimers. In one embodiment, the Ig binding protein comprises one, two, three, four, five, or six Ig binding domains linked together, i.e., the Ig binding protein can be, for example, a monomer, dimer, trimer, tetramer, pentamer, or hexamer. Multimers may comprise two, three, four, five, or more binding domains. The multimers of the present invention are generally artificially produced fusion proteins using recombinant DNA techniques well known to those skilled in the art.

[0103] In some embodiments, the multimer is a homomultimer, e.g., the amino acid sequences of all Ig binding domains of the Ig binding protein are identical, while in some embodiments, the multimer is a heteromultimer, e.g., at least one Ig binding domain has an amino acid sequence that differs from other Ig binding domains in the Ig binding protein.

[0104] The multimer may comprise two or more Ig binding domains, said Ig binding domains preferably comprising or consisting essentially of amino acid sequences as described above.

[0105] In some preferred embodiments, the multimer is a pentamer. The present invention provides pentamers comprising any of the monomers set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:19. In various embodiments, the Ig-binding protein of the present invention is a pentamer comprising five Ig-binding domains, each of which corresponds to an Ig-binding protein having at least 89.5% amino acid identity to any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:19, in which the amino acid residue corresponding to position 5 is Phe (F), the amino acid residue corresponding to position 8 is Ile (I), the amino acid residue corresponding to position 28 is His (H), and the amino acid residue corresponding to position 42 is Lys (K), and the pentameric Ig-binding protein is stable under alkaline conditions.

[0106] In a preferred embodiment, the Ig-binding protein of the present invention is a pentamer comprising five Ig-binding domains, each of which corresponds to an Ig-binding protein having at least 89.5% amino acid identity to any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:19, in which the amino acid residue corresponding to position 5 is Phe (F), the amino acid residue corresponding to position 8 is Ile (I), the amino acid residue corresponding to position 28 is His (H), and the amino acid residue corresponding to position 42 is Lys (K), and the pentameric Ig-binding protein is stable under alkaline conditions of 1 M NaOH for at least 37 hours. In another preferred embodiment, the Ig-binding protein of the present invention is a pentamer comprising five Ig-binding domains, each of which corresponds to an Ig-binding protein having at least 89.5% amino acid identity to any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:19, where the amino acid residue corresponding to position 5 is Phe (F), the amino acid residue corresponding to position 8 is Ile (I), the amino acid residue corresponding to position 28 is His (H), and the amino acid residue corresponding to position 42 is Lys (K); the pentameric Ig-binding protein is stable under alkaline conditions of 1 M NaOH for at least 37 hours, and the pentameric Ig-binding protein allows elution of the target under mild elution conditions of a pH of at least 4.0. In various embodiments, the pentameric Ig binding protein is stable under alkaline conditions of 1 M NaOH for at least 37 hours, allowing elution of Ig targets (particularly IgG) at elution conditions of at least about pH 4.3, preferably at least about pH 4.5. In further embodiments, the pentameric Ig binding protein is stable under alkaline conditions of 1 M NaOH for at least 37 hours, allowing elution of Ig targets (particularly IgG) at elution conditions of a pH greater than 4.5.

[0107] In some specific embodiments, the Ig binding protein is a pentamer comprising the sequences of 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:14, SEQ ID NO:16, SEQ ID NO:18, and SEQ ID NO:20.

[0108] In other embodiments, the Ig binding protein is a pentamer comprising a sequence having at least 89.5% or at least about 95% identity to any one of 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:14, SEQ ID NO:16, SEQ ID NO:18, and SEQ ID NO:20, assuming that in each monomer of the multimer the amino acid residue corresponding to position 5 is Phe (F), the amino acid residue in each monomer of the multimer corresponding to position 8 is Ile (I), the amino acid residue corresponding to position 28 is His (H), and the amino acid residue corresponding to position 42 is Lys (K).

[0109] In a preferred embodiment, the first monomer of the multimer is deleted at positions 1 and 2, so that the amino acid positions corresponding to positions 9, 11, and 15 are as shown in Table 1.

[0110] TIFF2025539595000001.tif94170

[0111] The present invention encompasses multimeric forms of one or more monomers of any one of SEQ ID NOS: 1-6, 13, 15, 17, and 19 other than pentamers. Such other multimeric forms include, inter alia, trimers, tetramers, and hexamers. In such multimeric forms, the position numbers set forth in Table 1 above apply accordingly.

[0112] In some preferred embodiments, the multimer is a trimer comprising monomers of any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:19, as described elsewhere herein, or an Ig binding protein having at least 89.5% identity to those SEQ ID NOs, respectively.

[0113] In some preferred embodiments, the multimer is a tetramer comprising monomers of any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:19, as described elsewhere herein, or an Ig binding protein having at least 89.5% identity to those SEQ ID NOs, respectively.

[0114] In some preferred embodiments, the multimer is a hexamer comprising monomers of any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:19, as described elsewhere herein, or an Ig binding protein having at least 89.5% identity to each of those SEQ ID NOs.

[0115] Preferably, the multimers of the invention are based on one single type of monomer, and the multimer is reflected in SEQ ID NOs: 7-12, 14, 16, and 18, or an Ig binding protein having at least 89.5% identity to those SEQ ID NOs, as described elsewhere herein.

[0116] In some preferred embodiments, multimers of the invention are based on more than one single type of monomer, such as a multimer reflected in SEQ ID NO:20 or an Ig-binding protein having at least 89.5% identity thereto, as described elsewhere herein. The present invention encompasses pentamers based on SEQ ID NO:19, in which each of the five Ig-binding domains corresponds to an Ig-binding protein having at least 89.5% amino acid identity to SEQ ID NO:19, as described elsewhere herein, except that in one of the two terminal domains, the amino acid corresponding to position 58 of SEQ ID NO:19 is lysine (K), and in all other domains, the amino acid corresponding to position 58 of SEQ ID NO:19 is proline. As will be recognized by those of skill in the art, all of the embodiments relating to the monomer of SEQ ID NO:19 described elsewhere herein apply to pentamers based on SEQ ID NO:19 described herein. In a preferred embodiment, in the other terminal domain (i.e., the other of the two terminal domains), amino acid positions corresponding to positions 1 and 2 of SEQ ID NO:19 are deleted (as reflected in SEQ ID NO:20). More preferably, said one of the two terminal domains carrying a lysine (K) at a position corresponding to position 58 of SEQ ID NO: 19 is the C-terminal domain of the pentamer (as reflected in SEQ ID NO: 20).

[0117] The present invention encompasses pentamers having at least 89.5% amino acid identity to SEQ ID NO:20, wherein in one of the two terminal domains, the amino acid corresponding to position 58 of SEQ ID NO:19 is lysine (K), and in all other domains, the amino acid corresponding to position 58 of SEQ ID NO:19 is proline. As will be recognized by those of skill in the art, all of the embodiments relating to the pentamer of SEQ ID NO:20 described elsewhere herein also apply to the pentamer of SEQ ID NO:20 described herein. In particular, in said pentamer of SEQ ID NO:20, each of the five domains has an (F) at a position corresponding to position 5 of SEQ ID NO:19, an (I) at a position corresponding to position 8 of SEQ ID NO:19, an (H) at a position corresponding to position 28 of SEQ ID NO:19, and a (k) at a position corresponding to position 42 of SEQ ID NO:19. In a preferred embodiment, in each of the five domains, the amino acid corresponding to position 5 is F, the amino acid corresponding to position 8 is I, the amino acid corresponding to position 28 is H, the amino acid corresponding to position 42 is K, the amino acid corresponding to position 4 is glutamine (Q), the amino acid corresponding to position 7 is lysine (K), the amino acids corresponding to positions 9 and 11 are alanine (A), and the amino acid corresponding to position 15 is glutamic acid (E). In a particularly preferred embodiment, in the other terminal domain (i.e., the other of the two terminal domains that does not carry a (K) at position 58 of SEQ ID NO: 19), the amino acid positions corresponding to positions 1 and 2 of SEQ ID NO: 19 are deleted (the amino acid positions correspond to positions 1 and 2 of SEQ ID NO: 20). More preferably, the one of the two terminal domains carrying a (K) at the terminal position (which is the position corresponding to position 58 of SEQ ID NO: 19) is the C-terminal domain of the pentamer (as reflected in SEQ ID NO: 20).

[0118] Furthermore, in a preferred embodiment of the invention, in the multimeric form, the first monomer has the first and second amino acid residues present in the corresponding monomer deleted, as reflected in SEQ ID NOs: 7-12, 14, 16, 18, 20, or an Ig binding protein having at least 89.5% identity to those SEQ ID NOs, respectively, as described elsewhere herein.

[0119] As described elsewhere herein, the present invention encompasses monomers of SEQ ID NOs: 1-6, 13, 15, 17, 19, or Ig binding proteins having at least 89.5% identity to each of those SEQ ID NOs, and incorporating amino acid substitutions at positions 9, 11 and 15 as described relative to SEQ ID NO: 13, and / or amino acid substitutions at positions 4 and 7 as described relative to SEQ ID NO: 15.

[0120] The present invention further encompasses multimers according to any one of SEQ ID NOS: 7-12, 14, 16, 18, and 20, or Ig binding proteins having at least 89.5% identity to those SEQ ID NOS, respectively, which incorporate amino acid substitutions at positions 9, 11, and 15 as set forth relative to SEQ ID NOS: 13, and / or at positions 4 and 7 as set forth relative to SEQ ID NOS: 15. The identification of positions 4, 7, 9, 11, and 15 in the first, second, third, fourth, and fifth monomers set forth in Table 1 above applies, with respect to the corresponding position numbers in such multimers of the invention.

[0121] Linker. In various embodiments, one or more Ig binding domains are directly linked to each other. In other embodiments, one or more Ig binding domains are linked to each other using one or more linkers. In these exemplary embodiments, a peptide linker is preferred. This means that the peptide linker is one or more amino acids, e.g., an amino acid sequence, that connects a first Ig binding domain to a second Ig binding domain. The peptide linker connects the first Ig binding domain to the second Ig binding domain via a peptide bond between the C-terminus and N-terminus of the domains, thereby creating a single linear polypeptide chain. In some embodiments, the Ig binding protein multimer comprises one or more linkers connecting the Ig binding domains, the linkers being the same or different.

[0122] Affinity Separation Matrix In another embodiment, the present invention is directed to an affinity separation matrix comprising the Ig-binding protein or Ig-binding domain of the previous embodiments.

[0123] In a preferred embodiment, the affinity separation matrix is ​​a solid support. The affinity separation matrix comprises at least one Ig-binding protein or Ig-binding domain, as described above.

[0124] The affinity matrix is ​​useful for the separation of immunoglobulins and retains its Ig-binding properties even after the highly alkaline conditions applied during the purification step, which is essential for the long-term repeated use of the matrix.

[0125] Solid support matrices for affinity chromatography are known in the art and include, for example, agarose and stabilized agarose derivatives (e.g., PraestoPure, Praesto Jetted A50, Praesto Jetted A50 HipH, Mabselect, PrismA, Sepharose 6B, CaptivA, rPROTEIN A Sepharose Fast Flow, MabCapturCand, and others), cellulose or cellulose derivatives, controlled pore glass such as ProSep vA Ultra, monoliths such as Convective Interactive Media (CIM) monoliths, 3D printed monolith adsorption (PMA) columns, silica, zirconium oxide (e.g., CM zirconia or CPG), titanium oxide, or synthetic polymers (e.g., UNOsphere Biosciences, such as Poros 50A or Poros MabCapture A). Examples of suitable carriers include, but are not limited to, hydroxylapatite (e.g., SUprA polystyrene), polyvinyl ether, polyvinyl alcohol, monodisperse polyacrylate resins (e.g., UniMab, UniMabPro), polymethacrylates (e.g., Toyopearl), polyhydroxyalkyl acrylates, polyhydroxyalkyl methacrylates, polyacrylamides, polymethacrylamides, and the like), and hydrogels of various compositions. In certain embodiments, the carrier comprises a polyhydroxy polymer, such as a polysaccharide. Examples of polysaccharides suitable as carriers include, but are not limited to, agar, agarose, dextran, starch, cellulose, pullulan, and the like, and stabilized variants thereof.

[0126] The format of the solid support matrix can be of any suitable known type. Such solid support matrices for coupling Ig-binding proteins or Ig-binding domains as described herein may comprise, for example, one of the following: columns, capillaries, particles, membranes, filters, monoliths, fibers, pads, gels, slides, plates, cassettes, or any other format commonly used in chromatography and known to those skilled in the art.

[0127] In one embodiment, the matrix is ​​composed of substantially spherical particles, also known as beads, such as Sepharose or agarose beads, or monodisperse polyacrylate beads. Suitable particle sizes may be in the diameter range of 5 to 500 μm, such as 10 to 100 μm, such as 20 to 80 μm, such as 40 to 70 μm. The matrix in particulate form may be used as a packed bed or in suspended form, including expanded beds.

[0128] In alternative embodiments, the solid support matrix is ​​a membrane, e.g., a hydrogel membrane. In some embodiments, affinity purification involves a membrane as the matrix to which an Ig-binding protein or Ig-binding domain of one embodiment is covalently attached. The solid support can also be in the form of a membrane within a cartridge.

[0129] In some embodiments, affinity purification involves a chromatography column containing a solid support matrix to which an Ig binding protein or Ig binding domain of an embodiment is covalently attached.

[0130] Immobilization to a solid support. In embodiments of the present invention, the Ig-binding protein or Ig-binding domain is conjugated to a solid support. In some embodiments of the present invention, the Ig-binding protein or Ig-binding domain may comprise additional amino acid residues at the N- and / or C-terminus. The Ig-binding protein or Ig-binding domain of the present invention may be attached to a suitable solid support matrix via conventional coupling techniques. Methods for immobilizing protein ligands to solid supports are well known in the art and are readily performed by those skilled in the art using standard techniques and equipment. In some embodiments, coupling may be multipoint coupling, e.g., via multiple lysines, or single coupling, e.g., via cysteines.

[0131] In some embodiments, the alkali-stable Ig-binding protein or Ig-binding domain comprises an attachment site for covalent attachment to a solid phase (matrix). Site-specific attachment sites include natural amino acids such as cysteine ​​or lysine that allow for specific chemical reactions with reactive groups on the solid phase or on a linker between the solid phase and the protein.

[0132] In some embodiments, the attachment site may be directly at the C- or N-terminus of the Ig-binding protein or Ig-binding domain. In some embodiments, a single cysteine ​​is placed at the C-terminus for site-specific immobilization of the Ig-binding protein or Ig-binding domain. The advantage of having a C-terminal cysteine ​​is that coupling of the Ig-binding protein or Ig-binding domain can be achieved by reaction of the cysteine ​​thiol with an electrophilic group on the carrier, resulting in thiol-ether cross-linking. This provides superior mobility of the coupled protein and increases binding capacity.

[0133] In other embodiments, the attachment site may be located within the Ig-binding protein or Ig-binding domain, for example at a position corresponding to position 43 or 46 of any of SEQ ID NOs: 1-6, 13, 15, 17, 19.

[0134] In other embodiments, a linker may be present between the N- or C-terminus and the attachment site. In some embodiments of the invention, the Ig binding protein or Ig binding domain may comprise an N- or C-terminal amino acid sequence of 3 to 20 amino acids, preferably 4 to 10 amino acids, with a terminal cysteine. The amino acids for the terminal attachment site may be selected from the group of proline, glycine, alanine, and serine, with a single cysteine ​​at the C-terminus for coupling.

[0135] In some embodiments of the invention, the Ig binding protein or Ig binding domain may also comprise additional amino acid residues at the N- and / or C-terminus, such as an N-terminal leader sequence and / or a coupling sequence with or without an N- or C-terminal tag.

[0136] Uses of Ig-binding proteins. In one embodiment, the present invention is directed to the use of an Ig-binding protein or Ig-binding domain of an embodiment, or the use of an affinity matrix of an embodiment, for affinity purification of immunoglobulins or variants thereof, i.e., the Ig-binding protein or Ig-binding domain of the present invention is used in affinity chromatography. In some embodiments, the Ig-binding protein or Ig-binding domain of the present invention is immobilized on a solid support, as described in one embodiment of the present invention.

[0137] Method for affinity purification of immunoglobulins. In one embodiment, the present invention is directed to a method for affinity purification of immunoglobulins, the method comprising the steps of: (a) providing a liquid (sample) containing Ig, such as IgG1, IgG2, IgG4, IgM, IgA, Ig fragments, Fc fragments, or Fab fragments (as defined above, including fusion proteins and conjugates); (b) providing an affinity separation matrix comprising an immobilized Ig-binding protein or Ig-binding domain as described above immobilized on said affinity separation matrix; (c) contacting the liquid with the affinity separation matrix under conditions that allow binding of at least one Ig binding protein or Ig binding domain as described above to Ig; (d) eluting the Ig from the matrix, thereby obtaining an eluate containing the Ig; Includes.

[0138] In some embodiments, the affinity purification method may further comprise one or more wash steps carried out between steps (c) and (d) under conditions sufficient to remove from the affinity separation matrix some or all molecules non-specifically bound to the affinity separation matrix, where non-specifically bound means any binding that does not involve an interaction between at least one Ig-binding protein or Ig-binding domain and an Ig.

[0139] Affinity separation matrices suitable for the disclosed uses and methods are those matrices as known to those skilled in the art according to the embodiments described above.

[0140] In some embodiments, elution of immunoglobulins from (a matrix comprising) an Ig-binding protein or Ig-binding domain in step (d) is carried out through a change in pH and / or a change in salt concentration. In general, suitable conditions for performing affinity purification methods are well known to those skilled in the art. In some embodiments, the disclosed uses or methods of affinity purification involving the disclosed Ig-binding proteins or Ig-binding domains may provide elution of at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% of the Ig-containing protein at a pH of 4.0 or higher. Due to the high stability of the Ig-binding proteins and Ig-binding domains of the present invention, solutions at pH 4.0 or higher may be used to elute Ig proteins (see Examples).

[0141] In some embodiments, in step (d) of the affinity purification method, greater than about 95% of proteins containing Ig sequences (e.g., antibodies) are eluted at pH 4.0 (or higher) from a matrix containing immobilized Ig-binding proteins or Ig-binding domains as described above. In some embodiments, a further step (e) for efficient purification of the affinity matrix is ​​added, preferably by using an alkaline solution, e.g., pH 13-14. In certain embodiments, the purification solution contains 0.1-1.0 M NaOH or KOH, preferably 0.5-1 M NaOH or KOH. Due to the high alkaline stability of the Ig-binding proteins or Ig-binding domains of the present invention, such a strong alkaline solution can be used for purification purposes. After the affinity purification matrix has been purified with the alkaline purification solution, in some embodiments, at least about 80% of the Ig-binding proteins or Ig-binding domains retain Ig-binding activity when incubated in 1 M NaOH for at least 37 hours. In some embodiments, the Ig-binding capacity of the Ig-binding protein or Ig-binding domain is at least about 80% or at least about 90% of the Ig-binding capacity before incubation under alkaline conditions, e.g., as determined by the residual Ig-binding capacity after incubation in 1 M NaOH for at least 37 hours.

[0142] The present invention further provides methods for isolating immunoglobulins, comprising: (a) contacting a liquid sample containing immunoglobulins with a separation matrix comprising a plurality of Ig-binding proteins or Ig-binding domains (coupled to a solid support); (b) washing the separation matrix with a wash solution, such as 1 M NaOH; (c) eluting the immunoglobulins from the separation matrix at pH 4.0, pH 4.5, or higher; and (d) obtaining the immunoglobulins. In various preferred embodiments, elution is performed under acidic conditions at about pH 4.0, pH 4.3, or about pH 4.5. In various embodiments, elution may be performed at a pH greater than 4.5.

[0143] Nucleic acid molecule. In one embodiment, the present invention is directed to a nucleic acid molecule, preferably an isolated nucleic acid molecule, encoding an Ig-binding protein or Ig-binding domain as disclosed above. In one embodiment, the present invention is directed to a vector comprising the nucleic acid molecule. A vector refers to any molecule or entity (e.g., a nucleic acid, a plasmid, a bacteriophage, or a virus) that can be used to transfer protein-encoding information into a host. In one embodiment, the vector is an expression vector.

[0144] In one embodiment, the present invention is directed to an expression system comprising a nucleic acid or vector as disclosed above, such as a prokaryotic host cell, e.g., Escherichia coli, or a eukaryotic host, e.g., the yeasts Saccharomyces cerevisiae or Pichia pastoris, or a mammalian cell such as a CHO cell.

[0145] Method for producing an Ig-binding protein. In one embodiment, the present invention is directed to a method for producing an Ig-binding protein or an Ig-binding domain of the present invention, comprising the steps of: (a) culturing a host cell of one embodiment under conditions suitable for expression of said Ig-binding protein or Ig-binding domain to obtain said Ig-binding protein or Ig-binding domain; and (b) optionally isolating said Ig-binding protein or Ig-binding domain. Suitable conditions for culturing prokaryotic or eukaryotic hosts are well known to those skilled in the art.

[0146] The Ig binding molecules of the present invention may be prepared by any of a number of conventional and well-known techniques, such as straightforward organic synthesis strategies, solid-phase assisted synthesis techniques, or by commercially available automated synthesizers, while they may also be prepared by conventional recombinant techniques, alone or in combination with conventional synthetic techniques.

[0147] One embodiment of the present invention is directed to a method for the preparation of an Ig-binding protein or Ig-binding domain according to the invention as detailed above, said method comprising the following steps: (a) preparing a nucleic acid encoding the Ig-binding protein or Ig-binding domain as defined above; (b) introducing said nucleic acid into an expression vector; (c) introducing said expression vector into a host cell; (d) culturing the host cell; (e) subjecting the host cell to culture conditions in which the Ig-binding protein or Ig-binding domain is expressed, thereby (e) producing the Ig-binding protein or Ig-binding domain as described above; optionally (f) isolating the Ig-binding protein or Ig-binding domain produced in step (e); and (g) optionally conjugating the Ig-binding protein or Ig-binding domain to a solid matrix as described above. In a further embodiment of the invention, the production of the Ig-binding protein or Ig-binding domain is carried out by cell-free in vitro transcription / translation. [Example]

[0148] The following examples are provided to further illustrate the present invention, but the present invention is not limited thereto, and the following examples merely demonstrate the feasibility of the present invention based on the preceding description.

[0149] Example 1. Expression Ligands (e.g., 224785 (SEQ ID NO: 12), 224770 (SEQ ID NO: 9), 224771 (SEQ ID NO: 10), 224772 (SEQ ID NO: 8), 224777 (SEQ ID NO: 7), 228302 (SEQ ID NO: 18), and 230620 (SEQ ID NO: 20)) were expressed in E. coli BL21(DE3) using the pNP-016 vector system under the control of a T7 promoter. Proteins were produced in soluble form after induction with lactose in the autoinduction medium. BL21(DE3) competent cells were transformed with the expression plasmids, plated on a selective stearate plate (kanamycin), and incubated overnight at 37°C. A preculture was inoculated from a single colony into 50 ml of 2xYT medium supplemented with 50 μg / ml kanamycin and grown in a shaker flask at 37°C for 7 hours. For the main culture, 350 mL of autoinduction medium (modified H15 medium consisting of 2% glucose, 5% yeast extract, 0.89% glycerol, 0.76% lactose, 250 mM MOPS, 202 mM TRIS, 10 mM MgSO, pH 7.4, antifoam SE15) supplemented with 50 μg / ml kanamycin and trace elements was inoculated to an OD of 0.3 and incubated in a 2.5 L UltraYield™ flask at 37°C in an orbital shaker. Recombinant protein expression was induced by metabolizing glucose and then allowing lactose to enter the cells. Cells were grown overnight for approximately 18 hours to reach a final OD of approximately 40-50. Prior to harvest, the OD was measured, and a sample adjusted to 0.6 / OD was removed, pelleted, and frozen at -20°C. To harvest the biomass, cells were centrifuged at 12,000 × g for 20 min at 22° C. The pellet was weighed (wet weight) and stored at −20° C. before processing.

[0150] Example 2: Expression and Acid-Soluble SDS-PAGE Analysis Samples were resuspended in 90 μl of extraction buffer (PBS supplemented with 0.2 mg / ml lysozyme, 0.5× BugBuster, 6 mM MgSO4, 6 mM MgCl2, and 15 U / ml benzonase) and solubilized by stirring in a thermomixer at 850 rpm for 15 minutes at room temperature, followed by incubation at -80°C for 15 minutes. After thawing, soluble proteins were separated from insoluble proteins by centrifugation (16,000 × g, 2 minutes at room temperature). The supernatant was removed (soluble fraction), and the pellet (insoluble fraction) was resuspended in an equal volume of urea buffer (8 M urea, 0.2 M Tris, 20 mM EDTA, pH 7.0). 35 μl was taken from both the soluble and insoluble fractions, and 10 μl of 5× sample buffer and 5 μl of 0.5 M DTT were added. The samples were boiled at 95°C for 5 minutes. Finally, 5 μl of the samples were applied to a NuPage Novex 4-12% Bis-Tris SDS gel, run according to the manufacturer's recommendations, and stained with Coomassie. Results: Low to high levels of expression were found under optimized conditions within the selected time period. All of the expressed Ig-binding proteins were acid-soluble.

[0151] Example 3: Purification The Ig-binding protein was expressed in the soluble fraction of E. coli. Cells were resuspended in cell disruption buffer and lysed using an ultrasonic cell disruption system (Sonopuls HD 2200, Bandelin). Purification steps were performed using IEC Sepharose SP-HP (Cytiva) with the AKTAvant system (Cytiva) according to the manufacturer's instructions, using citrate buffer pH 3.0 (20 mM citric acid, 1 mM EDTA, pH 3.0). Pure protein fractions were eluted by increasing the sodium chloride concentration to 1 M over a 10-fold linear gradient over the column. Further purification was performed by size exclusion chromatography (Superdex75) using citrate buffer pH 6.0 (20 mM citric acid, 150 mM NaCl, 1 mM EDTA, pH 6.0) according to the manufacturer's instructions. Results: The purity of the variants was 100% after SE-HPLC and >96% after RP HPLC.

[0152] Example 4: Ig-binding proteins bind IgG with high affinity A sensor chip (Bruker) was equilibrated with surface plasmon resonance (SPR) running buffer. Surface-exposed carboxylic acid groups were activated to generate reactive ester groups using a mixture of EDC and NHS. 700–1500 RU of on-ligand was immobilized on one flow cell, and the off-ligand was immobilized on another flow cell. After ligand immobilization, an injection of ethanolamine removed noncovalently bound Ig-binding proteins. Upon ligand binding, protein analytes accumulated on the surface, increasing the refractive index. This change in refractive index was measured in real time and plotted as response or resonance units (RU) versus time. Serial dilutions of analytes were applied to the chip at an appropriate flow rate (μl / min). After each run, the chip surface was regenerated with regeneration buffer and equilibrated with running buffer. A control sample was applied to the matrix. Regeneration and re-equilibration were performed as previously described. Binding studies were performed using a Bruker SPR-32 at 25°C; data evaluation was performed using the Langmuir 1:1 model (RI = 0) via the Bruker evaluation software provided by the manufacturer. The estimated dissociation constants (K D ) against off-targets and the K of Ig-binding proteins of cetuximab (IgG1), natalizumab (IgG4), and panitumab (IgG2) in Table 2. D 228302 (SEQ ID NO: 18) had a K of 3.7 nM. D 228302 binds to bevacizumab IgG1 with a K of 7.6 nM. D It binds to trastuzumab's IgG1.

[0153] TIFF2025539595000002.tif51170

[0154] Example 5: Affinity Chromatography Coupling: Purified ligands (224770, 224771, 224772, 224777, 224785, 228302, 230620) were coupled to agarose-based chromatography beads (Praesto Jetted Expoxy 50, Purolite) according to the manufacturer's instructions (20 mg / ml matrix). Coupling efficiencies were at least 90% for all Ig-binding proteins.

[0155] DBC10%: The coupled resin was packed into a Super Compact 5 / 50 column (Gotec GmbH). Octagam was used as the IgG sample (concentration 2.2 mg / ml in 1x PBS; pH 7.3). The Octagam sample was applied to the matrix containing the immobilized ligand until 10% target breakthrough was achieved with a residence time of 6 minutes (or 5 minutes for 228302 in Table 3A). The unbound sample was washed with 1x PBS containing 1 M NaCl, pH 7.3. The loaded antibody was quantified and calculated as the dynamic binding capacity DBC10%. Results: The binding capacity (DBC10) of all ligands was approximately 60 mg / ml, as shown in Table 3A. Caustic stability reflects the remaining binding capacity (%) compared to the binding capacity at time 0.

[0156] Caustic Stability. Columns were incubated with 1 M NaOH at room temperature (22°C ± -3°C) for 37.5 and 50 hours (Table 3A) and 50 hours (Table 3B), and two sets were measured. The Ig-binding activity of the immobilized ligands was analyzed after incubation with 1 M NaOH. Results: After 37.5 hours in 1 M NaOH, all variants showed at least about 80% of the residual IgG-binding capacity (% DBC10). 224772 had a residual IgG-binding capacity (% DBC10) of 90%. Even after 50 hours in extremely strong alkaline solution, 224770, 224772, and 224777 still showed about 75% of the initial residual binding capacity for Ig (see Table 3A).

[0157] 230620 had a residual IgG (trastuzumab) binding capacity (DBC10 in %) of approximately 91% (after 37.5 hours, 1 M NaOH).

[0158] Multimers (trimer, tetramer, pentamer, hexamer) of SEQ ID NO: 6 exhibit comparable binding activity after 50 hours of incubation with 1 M NaOH (see Table 3B). Similar observations are made with multimers (trimer, pentamer) of SEQ ID NOs: 2, 3, and 17 (data not shown).

[0159] Step pH elution: 15 CV of 0.1 M acetic acid at 1 CV / min, followed by a stepwise pH elution at pH 3.5, pH 3.7, and pH 4.0 with 10 CV of 0.1 M phosphate pH 1.7 (CIP) eluted hIgG bound to the immobilized ligand (load: 2.2 mg / mL Octagam, 6 min residence time). Results: For all variants tested, greater than approximately 95% of the antibody was eluted at pH 4.0. For 224770 and 224777, 100% of the antibody was eluted at pH 4.0 (see Table 3A). 228302 eluted hIgG bound to the immobilized ligand with a stepwise pH elution of 15 CV of 0.1 M acetic acid at 1 CV / min, followed by 10 CV of 0.1 M phosphate pH 1.7 (CIP) at pH 3.7, pH 4.0, and pH 4.5 (load: 2.2 mg / mL Octagam, 5 min residence time). Approximately 92% of the antibody was eluted at pH 4.0. 228302 allowed elution of 84% of the IgG.

[0160] TIFF2025539595000003.tif137170

[0161] array SEQ ID NO: 1 (224777 monomers) IAAQFDKIAQIAFYAILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPK SEQ ID NO: 2 (224772 monomers) IAAQFDKIQQAAFYAILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPK SEQ ID NO: 3 (224770 monomers) IAAQFDKIAQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPK SEQ ID NO: 4 (224771 monomers) IAAQFDKIQQIAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPK SEQ ID NO: 5 (SEQ ID NO: 11 monomer) IAAQFDKIAQAAFYAILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPK SEQ ID NO: 6 (224785 monomers) IAAQFDKIQQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPK SEQ ID NO: 7 (224777 pentamer) AQFDKIAQIAFYAILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIAQIAFYAILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIAQIAFYAILHLPNLTEEQRHAF IQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIAQIAFYAILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIAQIAFYAILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPK SEQ ID NO: 8 (224772 pentamer) AQFDKIQQAAFYAILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIQQAAFYAILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIQQAAFYAILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIQQAAFYAILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIQQAAFYAILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPK Array number 9 (224,770 pentamer)<000062③>AQFDKIAQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIAQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIAQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIAQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIAQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPK Array number 10 (224,⑦71 pentamer) AQFDKIQQIAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIQQIAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIQQIAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIQQIAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIQQIAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPK It should be noted that there may be some inaccuracies in the original text, such as the repeated and seemingly incorrect content. The translation is done as accurately as possible based on the existing text. Also, the tags and seem to have some non - standard or incorrect notations in the original, which are translated as is for the purpose of maintaining consistency with the original text. And for the numbers in the array number descriptions like "224770" and "224771", they are kept in the translated text as they are likely specific identifiers in a technical context. SEQ ID NO: 11 (pentamer) AQFDKIAQAAFYAILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIAQAAFYAILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIAQAAFYAILHLPNLTEEQRHAF IQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIAQAAFYAILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIAQAAFYAILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPK SEQ ID NO: 12 (224785 pentamer) AQFDKIQQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIQQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIQQAAFYEILHLPNLTEEQRHAF IQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIQQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIQQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPK SEQ ID NO: 13 (monomer) IAAQFDKIXQXAFYXILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPK X at position 9 may be A or Q, X at position 11 may be A or I, and X at position 15 may be A or E. SEQ ID NO: 14 (pentamer) AQFDKIXQXAFYXILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIXQXAFYXILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIXQXAFYXILHLPNLTEEQRHAF IQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIXQXAFYXILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIXQXAFYXILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPK X at positions 7, 65, 123, 181, and 239 may be A or Q; X at positions 9, 67, 125, 183, and 241 may be A or I; and X at positions 13, 71, 129, 187, and 245 may be A or E. SEQ ID NO: 15 (monomer) IAAXFDXIXQXAFYXILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPK X at position 4 may be Q or K, X at position 7 may be K or E, X at position 9 may be A or Q, X at position 11 may be A or I, and X at position 15 may be A or E. SEQ ID NO: 16 (pentamer) AXFDXIXQXAFYXILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAXFDXIXQXAFYXILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAXFDXIXQXAFYXILHLPNLTEEQRHAF IQSLRDDPSVSKEILAEAKKLNDAQAPKIAAXFDXIXQXAFYXILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAXFDXIXQXAFYXILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPK X at positions 2, 60, 118, 176, and 234 may be Q or K; X at positions 5, 63, 121, 179, and 237 may be K or E; X at positions 7, 65, 123, 181, and 239 may be A or Q; X at positions 9, 67, 125, 183, and 241 may be A or I; and X at positions 13, 71, 129, 187, and 245 may be A or E. SEQ ID NO: 17 (228302 monomer) IAAKFDEIAQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPK SEQ ID NO: 18 (228302 pentamer) AKFDEIAQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAKFDEIAQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAKFDEIAQAAFYEILHLPNLTEEQRHAF IQSLRDDPSVSKEILAEAKKLNDAQAPKIAAKFDEIAQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAKFDEIAQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPK SEQ ID NO: 19 (230620 monomers) IAAQFDKIAQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPP SEQ ID NO: 20 (230620 pentamer) AQFDKIAQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPPIAAQFDKIAQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPPIAAQFDKIAQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPPIAAQFDKIAQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPPIAAQFDKIAQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPK

Claims

1. An immunoglobulin (Ig) binding protein comprising the amino acid sequence of SEQ ID NO: 13, or an immunoglobulin Ig binding protein comprising an amino acid sequence having at least 89.5% amino acid identity to SEQ ID NO: 13, wherein the amino acid corresponding to position 5 is phenylalanine (F), the amino acid corresponding to position 8 is isoleucine (I), the amino acid corresponding to position 28 is histidine (H), and the amino acid corresponding to position 42 is lysine (K).

2. 2. The Ig-binding protein of claim 1, wherein the amino acid corresponding to position 9 is alanine (A) or glutamine (Q).

3. 3. The Ig-binding protein of claim 1, wherein the amino acid corresponding to position 11 is alanine (A) or isoleucine (I).

4. 4. The Ig-binding protein of claim 1, wherein the amino acid corresponding to position 15 is alanine (A) or glutamic acid (E).

5. The Ig-binding protein of any one of claims 1 to 4, wherein the Ig-binding protein is a multimer and comprises at least three Ig-binding proteins.

6. The Ig binding protein of claim 5 , wherein the multimeric Ig binding protein is a pentamer.

7. 7. The Ig-binding protein of any one of claims 1 to 6, wherein the Ig-binding protein comprises any amino acid sequence selected from the group of SEQ ID NOs: 1 to 14.

8. The protein is IgG １ , IgG ２ , IgG ４ 8. The Ig binding protein of any one of claims 1 to 7, which binds to one or more of the following: IgM, IgA, an Ig fragment, an Fc fragment, a Fab fragment, a fusion protein comprising an Ig region, and a conjugate comprising an Ig region, preferably wherein the Ig binding protein is bound to a protein comprising an Fc region or is bound to an Fc fragment.

9. The Ig-binding protein of any one of claims 1 to 8, wherein the protein is immobilized on a solid support.

10. The Ig-binding protein of any one of claims 1 to 9, wherein the Ig-binding protein is stable under alkaline conditions, preferably in 1 M NaOH, for at least 37 hours.

11. An affinity separation matrix comprising at least one Ig-binding protein according to any one of claims 1 to 10, preferably wherein said at least one Ig-binding protein is coupled to said affinity separation matrix.

12. Use of an Ig-binding protein according to any one of claims 1 to 10 or use of an affinity separation matrix according to claim 11 for affinity purification.

13. 1. A method for purifying a protein comprising the Fc region of an immunoglobulin (Ig), comprising: a) providing a sample, preferably a liquid sample, containing a protein comprising said Fc region of an Ig; b) providing an affinity separation matrix according to claim 11; c) contacting the affinity separation matrix with the sample under conditions that allow binding of the at least one Ig-binding protein of the affinity separation matrix to a protein comprising the Fc region of Ig; and d) recovering, preferably eluting, said protein comprising said Fc region of an Ig from said affinity purification matrix, thereby obtaining said protein comprising said Fc region of an Ig, preferably obtaining an eluate containing said protein comprising said Fc region of an Ig; A method comprising:

14. 14. The method of claim 13, wherein in step (d), greater than about 95% of the proteins comprising the Ig sequences are eluted from the affinity separation matrix comprising the Ig-binding protein of any one of claims 1 to 10 at pH 4.0.

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