Novel ligands for affinity chromatography

Abstract

The present invention relates to novel ligands for affinity chromatography, in particular antibody purification. The novel ligand is an immunoglobulin (Ig) binding protein having excellent properties, and can be used in an efficient purification method of an antibody (immunoglobulin). The invention also relates to affinity matrices comprising ligands according to 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

Field of the Invention

[0001] The present invention relates to new ligands for use in affinity chromatography, particularly for antibody purification. The new ligands are immunoglobulin (Ig) binding proteins with excellent properties and can be used in highly efficient purification methods for antibodies (immunoglobulins). The present invention also relates to affinity matrices comprising the ligands of the present invention. The present invention further relates to the use of these Ig binding proteins or affinity matrices for the affinity purification of immunoglobulins, and to methods for performing affinity purification using the Ig binding proteins of the present invention. Background Art

[0002] Many biotechnological and pharmaceutical applications require the removal of contaminants from samples containing antibodies. An established procedure for capturing and purifying antibodies and molecules containing an Fc domain is affinity chromatography using the bacterial cell surface protein A from Staphylococcus aureus as a selective ligand for immunoglobulins (see, for example, reviewed in Huse et al., J. Biochem. Biophys. Methods 51, 2002: 217 - 231). Wild - type protein A binds to the Fc region of IgG molecules with high affinity and selectivity. Protein A variants with improved properties (such as alkaline stability) can be used for antibody purification, and various chromatographic matrices containing the protein A ligand are commercially available. However, currently available protein A - based chromatographic matrices lose their immunoglobulin - binding ability after exposure to alkaline conditions and require elution at pH values below 4.

[0003] Technical Problem to be Solved by the Present Invention

[0004] Most large - scale production processes for antibodies or Fc - containing (fusion) proteins use protein A for affinity purification. However, due to the limitations in the application of protein A in affinity chromatography, there is a need in the art to provide new Ig binding proteins with improved properties that specifically bind immunoglobulins, thereby facilitating the affinity purification of immunoglobulins. To maximize the value of chromatographic matrices containing Ig binding proteins, repeated use of the affinity ligand matrix is required. Between chromatographic cycles, thorough cleaning procedures are needed to disinfect and remove residual contaminants on the matrix. In this procedure, it is common practice to apply an alkaline solution of high - concentration NaOH (such as 0.5 M NaOH) to the affinity ligand matrix. Wild - type protein A domains cannot withstand such harsh alkaline conditions for a long time and quickly lose their immunoglobulin - binding ability. In addition, for the repeated use of the affinity ligand matrix, a cleaning step is usually required under acidic conditions.

[0005] Therefore, there is a continuing need in the art to obtain new proteins that can bind proteins containing Ig sequences or the immunoglobulin Fc region (such as antibodies) and that can better withstand the harsh conditions applied in immunoglobulin affinity purification.

[0006] The present invention provides an Ig-binding protein that is particularly suitable for the affinity purification of immunoglobulins. Specifically, the Ig-binding protein of the present invention has a variety of advantages. A significant advantage of the Ig-binding protein of the present invention is its improved stability at high pH (alkaline conditions) for a long time (such as 1.5 to 2 days in 1M NaOH), without significantly reducing the Ig-binding ability, and having a high dynamic binding capacity. In addition, the novel protein of the present invention is particularly suitable for the affinity purification of antibodies that require weakly acidic elution conditions at pH 4.0 or above.

[0007] The above summary does not necessarily describe all the problems solved by the present invention. Summary of the Invention

[0008] One aspect of the present invention is to provide an Ig-binding protein suitable for affinity purification.

[0009] [1] This is achieved by an immunoglobulin (Ig)-binding protein comprising the amino acid sequence of SEQ ID NO: 13 or an Ig-binding protein comprising an amino acid sequence having at least 89.5% amino acid identity thereto, 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). 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 glutamate (E). In various embodiments, the amino acid corresponding to position 58 is proline (P).

[0010] [2] The Ig-binding protein according to item [1], wherein the amino acid corresponding to position 9 is alanine (A) or glutamine (Q).

[0011] [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).

[0012] [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 glutamate (E).

[0013] [5] The Ig-binding protein according to any one of items [1] to [4], wherein the Ig-binding protein is a multimer and comprises at least 3 Ig-binding proteins.

[0014] [6] The Ig-binding protein according to item [5], wherein the multimeric Ig-binding protein is a pentamer.

[0015] [7]An 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% identity to any one of SEQ ID NOs: 1-18.

[0016] [8]An 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, Ig fragments, Fc fragments, Fab fragments, fusion proteins comprising Ig regions, and conjugates comprising Ig regions. In various embodiments, the Ig-binding protein binds to a protein comprising an Fc region or binds to an Fc fragment.

[0017] 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 measured by SPR.

[0018] [9]An Ig-binding protein according to any one of items [1] to [8], wherein the protein is immobilized on a solid support.

[0019]

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

[0020]

[11] An affinity separation matrix comprising an Ig-binding protein according to any one of items [1] to

[10] coupled thereto.

[0021] [9]Use of an Ig-binding protein according to any one of items [1] to [7], or the affinity separation matrix of item [8] for affinity purification, particularly for the affinity purification of any protein having an affinity for the Ig-binding protein.

[0022]

[12] Use of an Ig-binding protein according to any one of items [1] to

[10] , or the affinity separation matrix of item

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

[0023]

[13] A method for affinity purifying a protein comprising an immunoglobulin (Ig) Fc region, the method comprising:

[0024] a) providing a sample containing a protein comprising an Fc region of an Ig, preferably a liquid sample;

[0025] b) providing an affinity separation matrix according to item

[11] ;

[0026] c) contacting the affinity separation matrix with a (liquid) sample under conditions that permit binding of at least one Ig-binding protein of the affinity separation matrix to a protein comprising the Fc region of an Ig; and

[0027] d) collecting, preferably eluting, the protein comprising the Fc region of an Ig from the affinity purification matrix, thereby obtaining an (affinity-purified) protein comprising the Fc region of an Ig, preferably obtaining an eluate containing the protein comprising the Fc region of an Ig.

[0028]

[14] The method according to item

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

[0029]

[15] The method according to any one of items

[13] -

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

[0030] The summary of the invention does not necessarily describe all features of the invention. Other embodiments will become apparent by reading the following detailed description. Detailed Description

[0031] Before describing the invention in detail below, it is to be understood that the invention is not limited to the specific methods, protocols and reagents described herein as these may vary. It should also 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 invention, which is 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.

[0032] Preferably, the terminology used herein is consistent with the definitions provided in "A Multilingual Glossary of Biotechnological Terms: (IUPAC Recommendations)", Leuenberger, H.G.W, Nagel, B. and H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

[0033] Throughout the specification and the following claims, unless the context requires otherwise, the word "comprise" and its variations such as "comprises" and "comprising" shall be understood to mean including the stated member, integer or step, or group of members, integers or steps, but not excluding any other member, integer or step, or group of members, integers or steps.

[0034] As used in the description of the present invention and the appended claims, the singular forms "a", "an" and "the" are used interchangeably and are intended to also include the plural forms and belong to each meaning, unless the context clearly dictates otherwise. Further, as used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the listed items, as well as the absence of a combination in the alternative ("or") interpretation.

[0035] The term "about", as used herein, encompasses the explicitly listed amount and its deviation of ±10%. More preferably, the term "about" encompasses a deviation of 5%.

[0036] This specification incorporates by reference multiple documents (e.g., patents, patent applications, scientific publications, manufacturer's specifications, etc.). Nothing in this document shall be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of a prior invention. Some of the documents cited herein are marked as "incorporated by reference". In the event of a conflict between the definitions or teachings of these cited documents and the definitions or teachings set forth in this specification, the text of this specification shall control.

[0037] All sequences mentioned herein are disclosed in the accompanying sequence listing, which sequence listing and all of its contents and disclosures form a part of this specification.

[0038] 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 an immunoglobulin. Additionally, 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 an immunoglobulin. The Ig - binding protein or Ig - binding domain of the present invention is sometimes referred to herein as the ligand of the present invention. As understood herein, "immunoglobulin" or "Ig" can include, but is not limited to, mammalian IgG, such as, 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 containing an Fc region (also referred to as "Fc fragment" or "Fc") and / or immunoglobulin fragments containing a Fab region (also referred to as "Fab fragment" or "Fab"). The Ig - binding protein is capable of binding to the whole immunoglobulin, and to Ig fragments containing an Fc region and / or Ig fragments containing a Fab region. As understood herein, the definition of "immunoglobulin" includes fusion proteins containing immunoglobulins, immunoglobulin fragments containing an Fc region (Fc fragments), immunoglobulin fragments containing a Fab region (Fab fragments), fusion proteins containing immunoglobulin fragments containing an Fc region, fusion proteins containing immunoglobulin fragments containing a Fab region, conjugates containing Ig or Ig fragments (Fc fragments) containing an Fc region, and conjugates containing Ig fragments (Fab fragments) containing a Fab region.

[0039] One of ordinary skill in the art should understand that the terms "immunoglobulin" and "antibody" can be used interchangeably herein. Any definition of the term "immunoglobulin" disclosed herein applies mutatis mutandis to the term "antibody".

[0040] According to the present invention, the term "binding" preferably refers to specific binding. "Specific binding" means that the binding affinity of the Ig - binding protein or Ig - binding domain for the immunoglobulin to which it specifically binds is stronger than its binding affinity for another non - immunoglobulin target.

[0041] 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, the binding activity can be measured before and / or after alkali treatment. The terms "(immunoglobulin) 'binding activity'" and "binding ability" are used interchangeably herein. The binding activity can be measured for an Ig-binding protein or for an Ig-binding protein conjugated to a matrix (i.e., an immobilized Ig-binding protein). In addition, the binding activity can be measured for an Ig-binding domain or for an Ig-binding domain conjugated to a matrix (i.e., an immobilized Ig-binding domain). The term "artificial" refers to an object that is not naturally occurring, i.e., an object produced or modified by a human. For example, a polypeptide or polynucleotide sequence that is generated by a human (e.g., in a laboratory by genetic engineering, by a shuffling method, or by a chemical reaction, etc.) or intentionally modified is artificial.

[0042] The term "dissociation constant" or "K D " defines a specific binding affinity. As used herein, the term "K D " (which is typically measured in "mol / L" (sometimes abbreviated as "M")) is intended to refer to the dissociation equilibrium constant of a specific interaction between a first protein and a second protein. In the context of the present invention, the term K D is specifically used to describe the binding affinity between an Ig-binding protein or Ig-binding domain and an immunoglobulin. If the dissociation constant K D of an Ig-binding protein or Ig-binding domain of the present invention for an immunoglobulin is at least 500 nM or less, or preferably 100 nM or less, more preferably 50 nM or less, and even more preferably 10 nM or less, it is considered to bind to the immunoglobulin.

[0043] 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, the terms "peptide", "protein", "amino acid chain", or any other term used to refer to a chain of two or more amino acids are included in the definition of "polypeptide", and the term "polypeptide" can be used in place of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the product of post-translational modification of a polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, proteolytic cleavage, modification with non-naturally occurring amino acids, and similar modifications well known in the art. Thus, an Ig-binding protein containing two or more protein domains also falls within the definition of the term "protein" or "polypeptide".

[0044] The terms "alkali-stable" or "alkali stability" or "caustic-stable" or "caustic stability" (also abbreviated as "cs" herein) may be used interchangeably herein and refer to the ability of the Ig-binding protein or Ig-binding domain of the present invention to withstand alkaline conditions without significant loss of the ability to bind to immunoglobulins. One skilled in the art can readily test the alkali stability (e.g., as described in the embodiments) by incubating the Ig-binding protein or Ig-binding domain with, for example, a sodium hydroxide solution, and subsequently testing the binding ability or binding activity to immunoglobulins by conventional experiments known to those skilled in the art (e.g., by chromatographic methods). The alkali stability can be determined by conjugating the Ig-binding protein or Ig-binding domain of the present invention to a surface plasmon resonance (SPR) sensor chip and measuring the binding ability or binding activity to immunoglobulins before and after exposure to an alkaline solution. The high-alkali treatment can be carried out, for example, in 1 M NaOH for a relatively long period of time, such as at least 30 h, at least 36 h in various embodiments, or even 48 h. The Ig-binding protein or Ig-binding domain of the present invention and the matrix containing the Ig-binding protein or Ig-binding domain of the present invention exhibit "increased" or "improved" alkali stability, which means that the molecules and matrices incorporating the Ig-binding protein or Ig-binding domain are stable for a relatively long period of time under alkaline conditions relative to a reference. In various embodiments, the reference can be an Ig-binding protein or Ig-binding domain that does not carry specific amino acid residues at positions 5, 8, 28, and 42, and / or does not carry specific amino acid substitutions at positions 9, 11, and 15 described elsewhere herein, and / or does not carry specific amino acid substitutions at positions 4 and / or 7 described elsewhere herein, or a matrix containing the Ig-binding protein or Ig-binding domain. In various embodiments, the reference Ig-binding protein or Ig-binding domain or the matrix containing the Ig-binding protein or Ig-binding domain does not carry a specific amino acid substitution at the position corresponding to position 58 (K to P) of SEQ ID NO: 19.

[0045] As used herein, the term "variant" includes 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 generated by genetic engineering carried out manually or by chemical synthesis or chemical reactions carried out manually.

[0046] As used herein, the term "conjugate" refers to a molecule that comprises at least a first protein or consists essentially of at least a first protein, which is chemically linked to other substances, such as a second protein or a non-protein moiety.

[0047] The term "modification" or "amino acid modification" refers to the exchange, deletion, or insertion of an amino acid at a specific position in a polypeptide sequence. Given the known genetic code and recombinant and synthetic DNA technologies, a skilled scientist can readily construct DNA encoding amino acid variants.

[0048] The term "substitution" or "amino acid substitution" refers to the exchange of an amino acid at a specific position in a polypeptide sequence. The term "deletion" or "amino acid deletion" refers to the removal of an amino acid at a specific position in a polypeptide sequence.

[0049] The term "insertion" or "amino acid insertion" refers to the addition of an amino acid in a polypeptide sequence.

[0050] Throughout the specification, amino acid residue positions are numbered as corresponding to those in, for example, 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 of SEQ ID NO:1. In embodiments of the present invention relating to SEQ ID NO: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). Further, in embodiments of the present invention relating to SEQ ID NO: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). Further, in embodiments of the present invention relating to SEQ ID NO: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). Regarding the corresponding positions in the multimers, particularly pentamers, of the present invention, refer to Table 1 below. The term "amino acid sequence identity" refers to a quantitative comparison of the identity (or difference) of the amino acid sequences of two or more proteins. The "percent amino acid sequence identity" or "percent identical" or "identity percent" associated with a reference polypeptide sequence is defined as the percentage of amino acid residues in the sequence that are identical to 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, when optimally aligned (e.g., by the programs GAP or BESTFIT using default gap weights), have 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.

[0051] To determine sequence identity, the sequence of the query protein is aligned with and compared to the sequence of the reference protein. Sequence alignment methods and sequence comparison algorithms are well known in the art. For example, to determine the degree of amino acid sequence identity of any polypeptide relative to a reference amino acid sequence, it is preferred to employ the SIM local similarity program. For multiple alignment analysis, it is preferred to use ClustalW known to those skilled in the art.

[0052] The degree of sequence identity is typically calculated based on the total length of the unmodified sequences. 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 the percentage of amino acid residues that two or more sequences or subsequences have in common when compared and aligned for maximum correspondence using one of the following sequence comparison algorithms or by visual inspection, which in some embodiments is 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 at least 100%. For clarity, for example, sequences having at least 89.5% identity include all sequences having an identity higher than 89.5%, such as 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.

[0053] In some embodiments, the percent identity exists within a region of at least 52 residues, in some embodiments within a region of at least 53 residues, in some embodiments within a region of at least 54 residues, in some embodiments within a region of at least 55 residues, in some embodiments within a region of at least 56 residues, in some embodiments within a region of at least 57 residues, and in some embodiments within a region of at least 58 residues. In certain embodiments, the percent identity exists within a region of (at least) 56 residues, such as with respect to the Ig-binding proteins described herein, which have a deletion at the amino acid positions corresponding to positions 1 and 2 of any of SEQ ID NOs: 1-6, 13, 15, 17, and 19.

[0054] The term "fusion" refers to polypeptide components or units that are directly linked by a peptide bond or linked by a peptide linker. In different embodiments, the term "fusion" may refer to polypeptide components or units that are linked by a non-peptide linker (e.g., by chemical conjugation).

[0055] The term "fusion protein" refers to a protein comprising at least a first protein and at least a second protein joined by a genetic linkage. A fusion protein is produced by joining two or more genes that originally encoded separate proteins. Thus, a fusion protein can comprise a multimer of the same or different proteins expressed as a single linear polypeptide. In various embodiments, a fusion protein is produced by joining two or more polypeptides by a non-peptide linker (e.g., by chemical conjugation). In various embodiments, a dimer of an Ig-binding protein or Ig-binding domain of the present invention can be considered a "fusion protein".

[0056] As used herein, the term "linker" in its broadest sense refers to a molecule that covalently links at least two other molecules. In exemplary embodiments of the present invention, a "linker" should be understood as the moiety that links an Ig-binding protein or Ig-binding domain to at least one additional Ig-binding protein or Ig-binding domain, i.e., the moiety that joins two protein domains together to produce a dimer or multimer. In preferred embodiments, a "linker" is a peptide linker, i.e., the moiety that joins two binding proteins or binding domains is a single amino acid or a peptide comprising two or more amino acids. In various embodiments, a dimer or multimer of the present invention can comprise a linker that joins two or more Ig-binding proteins or Ig-binding domains together.

[0057] 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., an immunoglobulin) in a sample from other molecules (e.g., contaminants or other immunoglobulins). The liquid mobile phase contains a mixture of molecules and transports them through or across the stationary phase (e.g., a solid matrix). Due to the differential interactions between the different molecules in the mobile phase and the stationary phase, the molecules in the mobile phase can be separated.

[0058] The term "affinity chromatography" refers to a specific mode of chromatography in which a ligand coupled to the stationary phase interacts with a molecule (i.e., an immunoglobulin) in the mobile phase (the sample), i.e., the ligand has a specific binding affinity or binding capacity for the molecule to be purified. As understood in the context of the present invention, affinity chromatography involves adding a (liquid) sample containing an immunoglobulin to a stationary phase comprising a chromatographic ligand (e.g., an Ig-binding protein or Ig-binding domain of the present invention).

[0059] The terms "solid support" or "solid matrix" are used interchangeably herein and are used for the stationary phase in various embodiments.

[0060] The terms "affinity matrix", "affinity separation matrix", or "affinity chromatography matrix" are used interchangeably herein and refer to a matrix to which an affinity ligand (e.g., an Ig-binding protein or Ig-binding domain of the present invention) is attached, such as a chromatography matrix. 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). Those of ordinary skill in the art will understand that the terms "affinity matrix", "affinity separation matrix", or "affinity chromatography matrix" describe the separation of a molecule of interest (particularly an immunoglobulin) by using an Ig-binding protein or Ig-binding domain of the present invention. Thus, the terms "affinity matrix", "affinity separation matrix", "affinity chromatography matrix", or "separation matrix" are used interchangeably herein.

[0061] As used herein, the term "affinity purification" refers to a method of purifying an immunoglobulin of interest as defined above from a liquid (sample) by binding the immunoglobulin of interest as defined above to an Ig-binding protein or Ig-binding domain immobilized on a matrix. Thereby, all other components in the mixture other than the immunoglobulin of interest are removed. In various embodiments, the other components of the mixture may include, for example, other immunoglobulins that are not of interest. In a further step, the immunoglobulin of interest is eluted in a purified form. The terms "affinity purification", "affinity chromatography purification", "affinity separation", or "affinity chromatography separation" are used interchangeably herein.

[0062] Embodiments of the present invention

[0063] The present invention will now be further described. Different embodiments of the present invention will be defined in more detail hereinafter. Unless otherwise explicitly stated, each embodiment defined below can be combined with any other embodiment. In particular, any feature indicated as preferred or advantageous can be combined with any one or more other features indicated as preferred or advantageous.

[0064] The present invention provides an immunoglobulin (Ig)-binding protein comprising the amino acid sequence of SEQ ID NO: 13, or an Ig-binding protein comprising an amino acid sequence having at least 89.5% amino acid identity thereto, 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).

[0065] In various embodiments, the amino acid corresponding to position 9 of SEQ ID NO:13 is alanine (A) or glutamine (Q).

[0066] In various embodiments, the amino acid corresponding to position 11 of SEQ ID NO:13 is alanine (A) or isoleucine (I).

[0067] In various embodiments, the amino acid corresponding to position 15 of SEQ ID NO:13 is alanine (A) or glutamic acid (E).

[0068] In various embodiments, the amino acid corresponding to position 4 of SEQ ID NO:13 is lysine (K) or glutamine (Q).

[0069] In various embodiments, the amino acid corresponding to position 7 of SEQ ID NO:13 is lysine (K) or glutamic acid (E).

[0070] In various embodiments, the amino acid corresponding to position 58 of SEQ ID NO:13 is lysine (K) or proline (P).

[0071] In some embodiments, the Ig-binding protein or Ig-binding domain of the present invention is selected from the following (1) to (2):

[0072] (1) A protein comprising the amino acid sequence corresponding to SEQ ID NO:13;

[0073] (2) A protein comprising an amino acid sequence having at least 89.5% or higher 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.

[0074] In some embodiments, the Ig-binding protein or Ig-binding domain of the present invention is selected from the following (1) to (6):

[0075] (1) A protein comprising the amino acid sequence 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, wherein the amino acid residue corresponding to position 5 is Phe (F), and the amino acid residue corresponding to position 8 is Ile (I), and the amino acid residue corresponding to position 28 is His (H), and the amino acid residue corresponding to position 42 is Lys (K);

[0076] (2) A protein comprising an amino acid sequence having at least 89.5% or higher 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 NO:1, 2, 3, 4, 5, 6, 17, or 19 specified in (1) are not mutated;

[0077] (3) A protein comprising an amino acid sequence having at least 89.5% or higher sequence identity with the amino acid sequence specified in (1), wherein the amino acid at position 4 corresponding to any one of SEQ ID NO:1, 2, 3, 4, 5, 6, or 17 is lysine (K), and the amino acid at position 7 corresponding to any one of SEQ ID NO:1, 2, 3, 4, 5, 6, or 17 is glutamic acid (E);

[0078] (4) A protein comprising an amino acid sequence having at least 89.5% or higher sequence identity with the amino acid sequence specified in (1), wherein the amino acid at position 4 corresponding to any one of SEQ ID NO:1, 2, 3, 4, 5, 6, 17, or 19 is glutamine (Q), and the amino acid at position 7 corresponding to any one of SEQ ID NO:1, 2, 3, 4, 5, 6, or 17 is lysine (K);

[0079] (5) A protein comprising an amino acid sequence having at least 89.5% or higher sequence identity with the amino acid sequence specified in (1), wherein the amino acid at position 4 corresponding to any one of SEQ ID NO:1, 2, 3, 4, 5, 6, or 17 is glutamine (Q), and the amino acid at position 7 corresponding to any one of SEQ ID NO:1, 2, 3, 4, 5, 6, or 17 is glutamic acid (E);

[0080] (6) A protein comprising an amino acid sequence having at least 89.5% or higher sequence identity with the amino acid sequence specified in (1), wherein the amino acid at position 4 corresponding to any one of SEQ ID NO:1, 2, 3, 4, 5, 6, or 17 is lysine (K), and the amino acid at position 7 corresponding to any one of SEQ ID NO:1, 2, 3, 4, 5, 6, or 17 is lysine (K).

[0081] In some embodiments, the Ig-binding protein or Ig-binding domain of the present invention is selected from the following (1) to (4):

[0082] (1) A protein comprising the amino acid sequence of SEQ ID NO:13;

[0083] (2) A protein comprising the amino acid sequence specified in (1), wherein the amino acid residue corresponding to position 9 of SEQ ID NO: 13 is Ala (A) or Gln (Q), and / or wherein the amino acid residue corresponding to position 11 of SEQ ID NO: 13 is Ile (I) or Ala (A), and / or wherein the amino acid residue corresponding to position 15 of SEQ ID NO: 13 is Glu (E) or Ala (A);

[0084] (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 wherein the amino acid residue corresponding to position 7 of SEQ ID NO: 13 is lysine (K) or glutamate (E), and / or wherein the amino acid residue corresponding to position 9 of SEQ ID NO: 13 is Ala (A) or Gln (Q), and / or wherein the amino acid residue corresponding to position 11 of SEQ ID NO: 13 is Ile (I) or Ala (A), and / or wherein the amino acid residue corresponding to position 15 of SEQ ID NO: 13 is Glu (E) or Ala (A); (4) A protein comprising an amino acid sequence having at least 89.5% or higher 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.

[0085] The present invention further provides an immunoglobulin (Ig) binding protein comprising the amino acid sequence of SEQ ID NO:15, or an Ig binding protein comprising an amino acid sequence having at least 89.5% amino acid identity thereto, wherein the amino acid at position 5 corresponding to SEQ ID NO:15 is phenylalanine (F), the amino acid at position 8 corresponding to SEQ ID NO:15 is isoleucine (I), the amino acid at position 28 corresponding to SEQ ID NO:15 is histidine (H), and the amino acid at position 42 corresponding to SEQ ID NO:15 is lysine (K). In various preferred embodiments, the amino acid at position 4 corresponding to SEQ ID NO:15 is lysine (K), and the amino acid at position 7 corresponding to SEQ ID NO:15 is glutamic acid (E). In various other embodiments, the amino acid at position 4 corresponding to SEQ ID NO:15 is glutamine (Q), and the amino acid at position 7 corresponding to SEQ ID NO:15 is lysine (K). In various further embodiments, the amino acid at position 4 corresponding to SEQ ID NO:15 is glutamine (Q), and the amino acid at position 7 corresponding to SEQ ID NO:15 is glutamic acid (E). Still further, in each embodiment, the amino acid at position 4 corresponding to SEQ ID NO:15 is lysine (K), and the amino acid at position 7 corresponding to SEQ ID NO:15 is lysine (K). In various further embodiments, the amino acid at position 58 corresponding to SEQ ID NO:15 is proline (P). In various further embodiments, the amino acid at position 4 corresponding to SEQ ID NO:15 is glutamine (Q), the amino acid at position 7 corresponding to SEQ ID NO:15 is lysine (K), the amino acids at positions 9 and 11 corresponding to SEQ ID NO:15 are alanine (A), the amino acid at position 15 corresponding to SEQ ID NO:15 is glutamic acid (E), and the amino acid at position 58 corresponding to SEQ ID NO:15 is proline (P).

[0086] In some embodiments, the Ig binding protein or Ig binding domain of the present invention is selected from the following (1) to (4):

[0087] (1) A protein comprising the amino acid sequence corresponding to SEQ ID NO:15;

[0088] (2) A protein comprising an amino acid sequence having at least 89.5% or higher sequence identity to the amino acid sequence specified in (1), provided that the amino acids at positions 5, 8, 28, and 42 of SEQ ID NO:15 specified in (1) are not mutated.

[0089] (3) A protein comprising an amino acid sequence having at least 89.5% or higher sequence identity with the amino acid sequence specified in (1), provided that the amino acids corresponding to the 5th, 8th, 28th, and 42nd amino acids of SEQ ID NO:15 specified in (1) are not mutated, and 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).

[0090] (4) A protein comprising an amino acid sequence having at least 89.5% or higher sequence identity with the amino acid sequence specified in (1), provided that the amino acids corresponding to the 5th, 8th, 28th, and 42nd amino acids of SEQ ID NO:15 specified in (1) are not mutated, and 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), and wherein 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, the amino acid corresponding to the amino acid at position 4 of SEQ ID NO:15 is lysine (K), and the amino acid corresponding to the amino acid at position 7 of SEQ ID NO:15 is glutamic acid (E); in various other embodiments, the amino acid corresponding to the amino acid at position 4 of SEQ ID NO:15 is glutamine (Q), and the amino acid corresponding to the amino acid at position 7 of SEQ ID NO:15 is lysine (K); in various further embodiments, the amino acid corresponding to the amino acid at position 4 of SEQ ID NO:15 is glutamine (Q), and the amino acid corresponding to the amino acid at position 7 of SEQ ID NO:15 is glutamic acid (E); further still, in various embodiments, the amino acid corresponding to the amino acid at position 4 of SEQ ID NO:15 is lysine (K), and the amino acid corresponding to the amino acid at position 7 of SEQ ID NO:15 is lysine (K).

[0091] In some embodiments, the Ig-binding protein or Ig-binding domain of the present invention is selected from the following (1) to (6):

[0092] (1) A protein comprising the amino acid sequence corresponding to SEQ ID NO:1;

[0093] (2) A protein comprising the amino acid sequence specified in (1), which further has one or more amino acid residue substitutions at positions other than positions 5, 8, 28, and 42 of SEQ ID NO:1 specified in (1), preferably having one or more amino acid residue substitutions at any one of positions 4, 7, 9, 11, and 15 of SEQ ID NO:1 specified in (1);

[0094] (3) A protein comprising an amino acid sequence having at least 89.5% or higher 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.

[0095] (4) A protein comprising an amino acid sequence having at least 89.5% or higher 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;

[0096] (5) A protein comprising an amino acid sequence having at least 89.5% or higher 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;

[0097] (6) A protein comprising an amino acid sequence having at least 89.5% or higher 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, and the amino acids at positions 4 and 7 of SEQ ID NO:1 specified in (1) are not mutated.

[0098] In some embodiments, the Ig-binding protein or Ig-binding domain of the present invention is selected from the following (1) to (6):

[0099] (1) A protein comprising the amino acid sequence corresponding to SEQ ID NO:2;

[0100] (2) A protein comprising the amino acid sequence specified in (1), which further has one or more amino acid residue substitutions at one or more positions other than positions 5, 8, 28, and 42 of SEQ ID NO:2 specified in (1), preferably having one or more amino acid residue substitutions at any one of positions 4, 7, 9, 11, and 15 of SEQ ID NO:2 specified in (1);

[0101] (3) A protein comprising an amino acid sequence having at least 89.5% or higher 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;

[0102] (4) A protein comprising an amino acid sequence having at least 89.5% or higher 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;

[0103] (5) A protein comprising an amino acid sequence having at least 89.5% or higher 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;

[0104] (6) A protein comprising an amino acid sequence having at least 89.5% or higher 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.

[0105] In some embodiments, the Ig-binding protein or Ig-binding domain of the present invention is selected from the following (1) to (6):

[0106] (1) A protein comprising the amino acid sequence corresponding to SEQ ID NO:3;

[0107] (2) A protein comprising the amino acid sequence specified in (1), which further has one or more amino acid residue substitutions at one or more positions other than positions 5, 8, 28, and 42 of SEQ ID NO:3 specified in (1), preferably having one or more amino acid residue substitutions at any one of positions 4, 7, 9, 11, and 15 of SEQ ID NO:3 specified in (1);

[0108] (3) A protein comprising an amino acid sequence having at least 89.5% or higher 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;

[0109] (4) A protein comprising an amino acid sequence having at least 89.5% or higher 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;

[0110] (5) A protein comprising an amino acid sequence having at least 89.5% or higher 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;

[0111] (6) A protein comprising an amino acid sequence having at least 89.5% or higher 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.

[0112] In various embodiments involving SEQ ID NO:3 described above in (1) and (6) and elsewhere herein, the amino acid corresponding to position 58 of SEQ ID NO:15 is proline (P).

[0113] In some embodiments, the Ig-binding protein or Ig-binding domain of the present invention is selected from the following (1) to (6):

[0114] (1) A protein comprising the amino acid sequence corresponding to SEQ ID NO:4;

[0115] (2) A protein comprising the amino acid sequence specified in (1), which further has one or more amino acid residue substitutions at one or more positions other than positions 5, 8, 28, and 42 of SEQ ID NO: 4 specified in (1), preferably having one or more amino acid residue substitutions at any one of positions 4, 7, 9, 11, and 15 of SEQ ID NO: 4 specified in (1);

[0116] (3) A protein comprising an amino acid sequence having at least 89.5% or higher 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;

[0117] (4) A protein comprising an amino acid sequence having at least 89.5% or higher 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;

[0118] (5) A protein comprising an amino acid sequence having at least 89.5% or higher 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;

[0119] (6) A protein comprising an amino acid sequence having at least 89.5% or higher 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.

[0120] In some embodiments, the Ig-binding protein or Ig-binding domain of the present invention is selected from the following (1) to (6):

[0121] (1) A protein comprising the amino acid sequence corresponding to SEQ ID NO: 5;

[0122] (2) A protein comprising the amino acid sequence specified in (1), which further has one or more amino acid residue substitutions at one or more positions other than positions 5, 8, 28, and 42 of SEQ ID NO:5 specified in (1), preferably having one or more amino acid residue substitutions at any one of positions 4, 7, 9, 11, and 15 of SEQ ID NO:5 specified in (1);

[0123] (3) A protein comprising an amino acid sequence having at least 89.5% or higher 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.

[0124] (4) A protein comprising an amino acid sequence having at least 89.5% or higher 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;

[0125] (5) A protein comprising an amino acid sequence having at least 89.5% or higher 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;

[0126] (6) A protein comprising an amino acid sequence having at least 89.5% or higher 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.

[0127] In some embodiments, the Ig-binding protein or Ig-binding domain of the present invention is selected from the following (1) to (6):

[0128] (1) A protein comprising the amino acid sequence corresponding to SEQ ID NO:6;

[0129] (2) A protein comprising the amino acid sequence specified in (1), which further has one or more amino acid residue substitutions at one or more positions other than positions 5, 8, 28, and 42 of SEQ ID NO: 6 specified in (1), preferably having one or more amino acid residue substitutions at any one of positions 4, 7, 9, 11, and 15 of SEQ ID NO: 6 specified in (1);

[0130] (3) A protein comprising an amino acid sequence having at least 89.5% or higher 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;

[0131] (4) A protein comprising an amino acid sequence having at least 89.5% or higher 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;

[0132] (5) A protein comprising an amino acid sequence having at least 89.5% or higher 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;

[0133] (6) A protein comprising an amino acid sequence having at least 89.5% or higher 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.

[0134] In some embodiments, the Ig-binding protein or Ig-binding domain of the present invention is selected from the following (1) to (6):

[0135] (1) A protein comprising the amino acid sequence corresponding to SEQ ID NO: 17;

[0136] (2) A protein comprising the amino acid sequence specified in (1), which further has one or more amino acid residue substitutions at one or more positions other than positions 5, 8, 28, and 42 of SEQ ID NO: 17, preferably having one or more amino acid residue substitutions at any one of positions 4, 7, 9, 11, and 15 of SEQ ID NO: 17;

[0137] (3) A protein comprising an amino acid sequence having at least 89.5% or higher 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;

[0138] (4) A protein comprising an amino acid sequence having at least 89.5% or higher 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;

[0139] (5) A protein comprising an amino acid sequence having at least 89.5% or higher 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;

[0140] (6) A protein comprising an amino acid sequence having at least 89.5% or higher 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.

[0141] The present invention relates to an immunoglobulin (Ig) - binding protein comprising the amino acid sequence of SEQ ID NO: 19, or an Ig - binding protein comprising an amino acid sequence having at least 89.5% amino acid identity thereto, 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). In a preferred embodiment relating to 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 glutamate (E), and the amino acid corresponding to position 58 is proline (P).

[0142] In some embodiments, the Ig - binding protein or Ig - binding domain of the present invention is selected from the following (1) to (7):

[0143] (1) A protein comprising the amino acid sequence corresponding to SEQ ID NO: 19;

[0144] (2) A protein comprising the amino acid sequence specified in (1), which further has one or more amino acid residue substitutions at one or more positions other than positions 5, 8, 28, and 42 of SEQ ID NO: 19, preferably having one or more amino acid residue substitutions at any one of positions 4, 7, 9, 11, and 15 of SEQ ID NO: 19;

[0145] (3) A protein comprising an amino acid sequence having at least 89.5% or higher 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;

[0146] (4) A protein comprising an amino acid sequence having at least 89.5% or higher 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;

[0147] (5) A protein comprising an amino acid sequence having at least 89.5% or higher 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;

[0148] (6) A protein comprising an amino acid sequence having at least 89.5% or higher 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;

[0149] (7) A protein comprising an amino acid sequence having at least 89.5% or higher 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.

[0150] A surprising advantage of the Ig-binding proteins and Ig-binding domains of the present invention is that they are stable under extreme conditions (e.g., high pH (pH 13 and higher)) without losing their Ig-binding properties. The Ig-binding proteins and Ig-binding domains described herein exhibit high alkaline stability for a long time (at least 37 hours) without impairing their Ig-binding properties (see Examples). In addition, they are stable at low pH without significantly losing their Ig-binding properties. The extremely high alkaline stability property is particularly important for chromatographic methods because chromatographic methods employ harsh cleaning procedures using a NaOH alkaline solution with a concentration up to 1 M to remove contaminants on the matrix so that the matrix can be used multiple times. In addition to the extremely high strong base stability, the Ig-binding proteins also exhibit high coupling efficiency, as shown in the Examples.

[0151] In addition, an important step in affinity chromatography is to elute the protein of interest that is bound to the Ig-binding protein or Ig-binding domain of the present invention, particularly the immunoglobulin of interest. This step is typically carried out at low pH. The affinity ligands of the present invention do not lose their binding properties to Ig after such treatment, while the protein of interest can also be eluted at low pH. In certain cases, the conditions for eluting antibodies (immunoglobulins) from the ligand at a pH higher than 3.7 (e.g., pH 4.0 or pH 4.5 and above) are important. The ligands of the present invention have a remaining IgG-binding capacity of about 80% after at least 37 hours in 1M NaOH, and elute at least about 92%, at least about 95%, preferably at least 97%, more preferably 100% of the bound IgG from the matrix at a mild pH of 4.0 or higher.

[0152] In a preferred embodiment of the present invention, the ligand of the present invention shows (or provides) that after incubation in 1M NaOH for at least 37 hours, at least about 92% of the bound Ig (particularly IgG) is eluted from the affinity purification or separation matrix at pH 4.0 (or higher), including Ig-binding proteins or Ig-binding domains related to any one of the monomers (including multimers, such as the corresponding pentamers of SEQ ID NO:7, 8, 9, 10, 12, 18, and 20) of SEQ ID NO:1-4, 6, 17, 19 described herein.

[0153] In other preferred embodiments of the present invention, the ligand of the present invention shows (or provides) that after incubation in 1M NaOH for at least 37 hours, at least about 94% of the bound Ig (particularly IgG) is eluted from the affinity purification or separation matrix at pH 4.0 (or higher), specifically including Ig-binding proteins or Ig-binding domains related to the monomer (including multimers, such as the corresponding pentamer of SEQ ID NO:12) of SEQ ID NO:6 described herein.

[0154] In a further preferred embodiment of the present invention, the ligand of the present invention shows (or provides) that after incubation in 1M NaOH for at least 37 hours, at least about 97% of the bound Ig (particularly IgG) is eluted from the affinity purification or separation matrix at pH 4.0 (or higher), specifically including Ig-binding proteins or Ig-binding domains related to the monomers of SEQ ID NO:2 and 4 (including multimers, such as the corresponding pentamers of SEQ ID NO:8 and 10) described herein.

[0155] Further, the preferred ligand of the present invention shows (or provides) that after incubation in 1M NaOH for at least 37 hours, about 99% or even 100% of the bound Ig (especially IgG) is eluted from an affinity purification or separation matrix at pH 4.0 (or higher), specifically including the Ig-binding proteins or Ig-binding domains related to the monomers of SEQ ID NO:1 and 3 (including multimers, such as the corresponding pentamers of SEQ ID NO:7 and 9) described herein.

[0156] Further, the preferred ligand of the present invention shows (or provides) that after incubation in 1M NaOH for at least 37 hours, at least about 84% of the bound Ig (especially IgG) is eluted from an affinity purification or separation matrix at pH 4.3 (or higher), specifically including the Ig-binding proteins or Ig-binding domains related to the monomers of SEQ ID NO:17 (including multimers, such as the corresponding pentamers of SEQ ID NO:12) described herein.

[0157] Further, the preferred ligand of the present invention shows (or provides) that after incubation in 1M NaOH for at least 37 hours, at least about 84% of the bound Ig (especially IgG) is eluted from an affinity purification or separation matrix at pH 4.5 (or higher), specifically including the Ig-binding proteins or Ig-binding domains related to the monomers of SEQ ID NO:17 (including multimers, such as the corresponding pentamers of SEQ ID NO:18) described herein.

[0158] The protein can be further modified to modify certain properties of the affinity chromatography. For example, cysteine can be added at the C-terminus. Alternatively, cysteine can be introduced at the position corresponding to position 43 or position 46 (such as any one of SEQ ID NO:1-6, 13, 15, 17, and 19) to enable efficient coupling to the matrix.

[0159] In some embodiments, the Ig-binding protein or Ig-binding domain is selected from the following (1) to (2):

[0160] (1) A protein comprising an amino acid sequence corresponding to the 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, wherein the amino acid residue corresponding to the 5th position is Phe (F), the amino acid residue corresponding to the 8th position is Ile (I), the amino acid residue corresponding to the 28th position is His (H), and the amino acid residue corresponding to the 42nd position is Lys (K);

[0161] (2) A protein comprising an amino acid sequence having at least 89.5% or higher sequence identity with the amino acid sequence specified in (1), provided that the amino acids specified in (1) are not modified in (2).

[0162] As described herein, the term "at least 89.5% or higher sequence identity" includes preferred embodiments where 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 consistent with the sequence identities described 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.

[0163] Preferred Ig binding domainsIn 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 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 an amino acid 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, provided 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 or consists essentially of or consists of: the amino acid sequence of 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, or an amino acid having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% 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, provided 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).

[0164] Preferred Ig binding proteinsIn some embodiments, the Ig-binding protein comprises one or more binding domains, wherein at least one domain comprises or consists of: the amino acid sequence of 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, or an amino acid 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, wherein the amino acid residue corresponding to position 5 is Phe (F), and the amino acid residue corresponding to position 8 is Ile (I), and the amino acid residue corresponding to position 28 is His (H), and the amino acid residue corresponding to position 42 is Lys (K). In some embodiments, the Ig-binding protein comprises one or more domains, wherein at least one domain comprises the following or consists essentially of the following or consists of: the amino acid sequence of 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, or an amino acid having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% 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, wherein the amino acid residue corresponding to position 5 is Phe (F), and the amino acid residue corresponding to position 8 is Ile (I), and the amino acid residue corresponding to position 28 is His (H), and the amino acid residue corresponding to position 42 is Lys (K).

[0165] Affinity for immunoglobulins All Ig-binding proteins or Ig-binding domains described herein bind to immunoglobulins, with a dissociation constant K DPreferably less than 200 nM, or less than 100 nM, even more preferably 10 nM or less. In some embodiments, the Ig-binding protein or Ig-binding domain binds to Ig, particularly to IgG1, IgG2, IgG4, IgM, and / or IgA, Ig fragments thereof, Fc fragments, Fab fragments, fusion proteins comprising Ig regions or the Fc region of Ig, and conjugates comprising Ig regions, and its dissociation constant K D Preferably less than 200 nM or less than 100 nM, even more preferably 10 nM or less. Methods for determining the binding affinity or binding capacity of an Ig-binding protein or domain (i.e., determining the dissociation constant K D ) are known to those of ordinary skill in the art and can be selected, for example, from the following methods known in the art: surface plasmon resonance (SPR)-based techniques, kinetic exclusion analysis (KinExA analysis), biolayer interferometry (BLI), enzyme-linked immunosorbent assay (ELISA), flow cytometry, isothermal titration calorimetry (ITC), analytical ultracentrifugation, radioimmunoassay (RIA or IRMA), and enhanced chemiluminescence (ECL). Some methods are further described in the examples. Generally, 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 specified, the K D values described herein are determined by surface plasmon resonance spectroscopy at 22 °C ± 3 °C. In one embodiment, the dissociation constant K D of the Ig-binding protein with human IgG1 is in the range of 0.1 nM to 100 nM, preferably 0.1 nM to 50 nM. Preferably, the K D in the range of 0.1 nM to 100 nM, preferably 0.1 nM to 50 nM refers to the K D determined by SPR, more preferably the K D determined by SPR at 20 °C, 25 °C, or 30 °C.

[0166] High alkaline stability of Ig binding proteinsThe Ig-binding proteins and Ig-binding domains of the present invention exhibit surprisingly good basic stability, as evidenced by retaining at least about 80% of IgG binding after prolonged incubation in 1 M NaOH, as shown in the Examples, and in addition having a high dynamic binding capacity (DBC) of more than 60 mg / ml at a residence time of 6 min. The basic stability of an Ig-binding protein or Ig-binding domain is determined by comparing the loss of Ig-binding activity. In some embodiments, the basic liquid comprises 0.1 - 1.0 M NaOH or KOH, preferably 0.5 - 1 M NaOH or KOH. Due to the high basic stability of the Ig-binding proteins and Ig-binding domains of the present invention, basic liquids with a pH above 13 can be used to wash affinity matrices having immobilized Ig-binding proteins or immobilized Ig-binding domains of the present invention. In some embodiments, the basic stability of an Ig-binding protein or Ig-binding domain is determined by comparing the loss of Ig-binding activity after incubation in 1 M NaOH for at least 37 h (see Examples). In some embodiments, the basic stability of an Ig-binding protein or Ig-binding domain is determined by comparing the loss of Ig-binding activity after prolonged incubation in a basic solution, such as incubation in 1 M NaOH for at least 2 days (at least 48 h) (see Examples), which reflects the extremely high stability of the Ig-binding proteins described herein.

[0167] The Ig-binding proteins and Ig-binding domains of the present invention are stable under basic conditions, particularly stable in 1 M NaOH basic conditions for at least 37 h. In a preferred embodiment, the Ig-binding protein or Ig-binding domain of the present invention is stable under basic conditions, particularly stable in 1 M NaOH basic conditions for at least 48 h, more preferably at least 50 h.

[0168] The Ig-binding proteins and Ig-binding domains of the present invention are basic-stable ligands of immunoglobulins. The Ig-binding proteins and Ig-binding domains of the present invention retain immunoglobulin-binding ability (or binding affinity) after exposure to 1 M NaOH for at least 37 h. As further described herein, the Ig-binding proteins and Ig-binding domains of the present invention retain at least about 80%, at least about 83%, or at least about 90% of immunoglobulin-binding ability after exposure to the extreme basic conditions described herein. In a further preferred embodiment, the Ig-binding proteins and Ig-binding domains of the present invention retain at least about 80% of immunoglobulin-binding ability after exposure to extreme basic conditions (1 M NaOH for at least 37 h). In various embodiments, when immobilized on a solid support, preferably on the solid support of an affinity separation matrix, the Ig-binding proteins and Ig-binding domains of the present invention retain the immunoglobulin-binding ability as described above.

[0169] As further described herein, the Ig-binding proteins and Ig-binding domains of the invention are generally stable under alkaline conditions at room temperature. The term room temperature can include temperatures from 15°C to 25°C, more specifically, temperatures from 20°C to 25°C. In various embodiments, the Ig-binding protein or Ig-binding domain of the invention is stable under alkaline conditions at 22°C ± 3°C.

[0170] In various embodiments, the alkaline stability of the Ig-binding protein or Ig-binding domain as described above refers to the alkaline stability of the Ig-binding protein or Ig-binding domain immobilized to a solid support, preferably a solid support of an affinity separation matrix. Thus, in various embodiments, the alkaline stability of the Ig-binding protein or Ig-binding domain is determined by comparing the loss of Ig-binding activity or Ig-binding capacity when the Ig-binding protein or Ig-binding domain is immobilized to a solid support, preferably a solid support of an affinity separation matrix. Thus, in other embodiments, the alkaline stability of the Ig-binding protein or Ig-binding domain is determined by comparing the Ig-binding activity of the Ig-binding protein or Ig-binding domain after prolonged alkaline treatment with that of a reference protein when the Ig-binding protein or Ig-binding domain is immobilized to a solid support.

[0171] Those skilled in the art can use methods well known in the art, particularly the method for determining the dissociation constant K D described elsewhere herein, to evaluate the binding ability or binding affinity of the Ig-binding protein or Ig-binding domain of the invention for immunoglobulins. In various embodiments, the binding ability or binding affinity of the Ig-binding protein or Ig-binding domain of the invention for immunoglobulins is determined using surface plasmon resonance (SPR) spectroscopy as described elsewhere herein. In other embodiments, kinetic exclusion analysis (KinExA assay) or enzyme-linked immunosorbent assay (ELISA) as described elsewhere herein is used to determine the binding ability or binding affinity of the Ig-binding protein or Ig-binding domain of the invention for immunoglobulins.

[0172] The binding ability or binding affinity of the Ig-binding protein or Ig-binding domain of the invention for immunoglobulins can be evaluated for each candidate ligand before and after exposure to the alkaline conditions described herein.

[0173] Polymers In one embodiment, the Ig-binding protein comprises 1, 2, 3, 4, 5, or 6 interconnected Ig-binding domains, i.e., the Ig-binding protein can be, for example, a monomer, dimer, trimer, tetramer, pentamer, or hexamer. The multimer can comprise two, three, four, five, or more binding domains. The multimers of the invention are artificially produced fusion proteins, typically produced by recombinant DNA techniques well known to those skilled in the art.

[0174] In some embodiments, the multimer is a homomultimer, e.g., the amino acid sequences of all Ig-binding domains of an Ig-binding protein are identical. In some embodiments, the multimer is a heteromultimer, e.g., at least one Ig-binding domain has an amino acid sequence different from other Ig-binding domains within the Ig-binding protein.

[0175] The multimer may comprise two or more Ig-binding domains, wherein the Ig-binding domain preferably comprises the amino acid sequence as described above or consists essentially of the amino acid sequence as described above.

[0176] In some preferred embodiments, the multimer is a pentamer. The present invention provides pentamers comprising any one of the monomers 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 various embodiments, the Ig-binding protein of the present invention is a pentamer comprising five Ig-binding domains, wherein each of the five Ig-binding domains 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, wherein the amino acid residue corresponding to position 5 is Phe (F), and the amino acid residue corresponding to position 8 is Ile (I), and the amino acid residue corresponding to position 28 is His (H), and the amino acid residue corresponding to position 42 is Lys (K), and wherein the Ig-binding protein of the pentamer is stable under alkaline conditions.

[0177] In a preferred embodiment, the Ig-binding protein of the present invention is a pentamer comprising five Ig-binding domains, wherein each of the five Ig-binding domains 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, 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), and wherein the pentameric Ig-binding protein is stable for at least 37 h under alkaline conditions of 1 M NaOH. In other preferred embodiments, the Ig-binding protein of the present invention is a pentamer comprising five Ig-binding domains, wherein each of the five Ig-binding domains 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, 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), and wherein the pentameric Ig-binding protein is stable for at least 37 h under alkaline conditions of 1 M NaOH, and wherein the pentameric Ig-binding protein allows elution of the target under mild elution conditions at a pH of at least 4.0. In various embodiments, the pentameric Ig-binding protein is stable for at least 37 h under alkaline conditions of 1 M NaOH and allows elution of the Ig target (especially IgG) under elution conditions of at least about pH 4.3, preferably at least about pH 4.5. In a further embodiment, the pentameric Ig-binding protein is stable for at least 37 h under alkaline conditions of 1 M NaOH and allows elution of the Ig target (especially IgG) under elution conditions at a pH above 4.5.

[0178] 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.

[0179] In another embodiment, the Ig-binding protein is a pentamer comprising a sequence having at least 89.5% or at least 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, provided that in each monomer of the multimer, the amino acid residue corresponding to position 5 is Phe (F), and the amino acid residue corresponding to position 8 in each monomer of the multimer is Ile (I), and the amino acid residue corresponding to position 28 is His (H), and the amino acid residue corresponding to position 42 is Lys (K).

[0180] In a preferred embodiment, the first monomer of the multimer is deleted at positions 1 and 2, such that the amino acid positions corresponding to positions 9, 11, and 15 are as shown in Table 1.

[0181] Table 1. Positions in the pentamer as shown in SEQ ID NO:7-12, 14, 16, and 18 (Monomer 1: 56 amino acids, Monomers 2, 3, 4, 5: 58 amino acids)

[0182] Monomers <![CDATA[Positions 5, 8, 28, 42 1 )corresponding positions]]> <![CDATA[Positions 9, 11, 15 2 )corresponding positions]]> <![CDATA[Positions 4 and 7 3 )corresponding positions]]> First monomer 3、6、26、40 7、9、13 2、5 Second monomer 61、64、84、98 65、67、71 60、63 Third monomer 119、122、142、156 123、125、129 118、121 Fourth monomer 177、180、200、214 181、183、187 176、179 Fifth monomer 235、238、258、272 239、241、245 234、237

[0183] 1) These positions describe the amino acid residues at positions 5, 8, 28, and 42 described elsewhere herein in relation to SEQ ID NO:1-6, 13, 15, 17, and 19.

[0184] 2) These positions describe the amino acid residues at positions 9, 11, and 15 described elsewhere herein in relation to, for example, SEQ ID NO:1-6, 13, 15, 17, and 19.

[0185] 3) These positions describe the amino acid residues at positions 4 and 7 described elsewhere herein in relation to, for example, SEQ ID NO:1-6, 13, 15, 17, and 19.

[0186] The present invention encompasses multimeric forms (except pentamers) of one or more monomers of any of SEQ ID NO: 1-6, 13, 15, 17, and 19. Such other multimeric forms specifically include trimers, tetramers, and hexamers. In such multimeric forms, the corresponding position numbers shown in Table 1 above are applicable.

[0187] In some preferred embodiments, the multimer is a trimer that comprises a monomer 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 an Ig-binding protein having at least 89.5% identity thereto, respectively, as described elsewhere herein.

[0188] In some preferred embodiments, the multimer is a tetramer that comprises a monomer 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 an Ig-binding protein having at least 89.5% identity thereto, respectively, as described elsewhere herein.

[0189] In some preferred embodiments, the multimer is a hexamer that comprises a monomer 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 an Ig-binding protein having at least 89.5% identity thereto, respectively, as described elsewhere herein.

[0190] Preferably, the multimer of the present invention is based on a single type of monomer, which is reflected in SEQ ID NO: 7-12, 14, 16, and 18 or an Ig-binding protein having at least 89.5% identity thereto, as described elsewhere herein.

[0191] In some embodiments, the multimers of the invention are based on more than one single type of monomer, such as the multimer shown in the Ig-binding protein of SEQ ID NO: 20 or having at least 89.5% identity thereto, as described elsewhere herein. The invention encompasses pentamers based on SEQ ID NO: 19, wherein 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 at the position corresponding to position 58 of SEQ ID NO: 19 is lysine (K), while in all other domains the amino acid at the position corresponding to position 58 of SEQ ID NO: 19 is proline (P). Those skilled in the art will understand that all embodiments described elsewhere herein relating to the monomer of SEQ ID NO: 19 are applicable to the 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), the amino acid positions corresponding to positions 1 and 2 of SEQ ID NO: 19 are deleted (as shown in SEQ ID NO: 20). More preferably, the one of the two terminal domains that carries lysine (K) at the position corresponding to position 58 of SEQ ID NO: 19 is the C-terminal domain of the pentamer (as shown in SEQ ID NO: 20).

[0192] The present invention encompasses pentamers having at least 89.5% amino acid identity with 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), while in all other domains, the amino acid corresponding to position 58 of SEQ ID NO:19 is proline (P). Those skilled in the art should understand that otherwise, all embodiments of the pentamers of SEQ ID NO:20 described elsewhere herein are applicable to the pentamers of SEQ ID NO:20 described herein. Specifically, in the pentamers of SEQ ID NO:20, each of the five domains has (F) at the position corresponding to position 5 of SEQ ID NO:19, (I) at the position corresponding to position 8 of SEQ ID NO:19, (H) at the position corresponding to position 28 of SEQ ID NO:19, and (k) at the 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 glutamate (E). In a particularly preferred embodiment, in the other terminal domain (i.e., the other of the two terminal domains, which does not carry (K) at the position corresponding to position 58 of SEQ ID NO:19), the amino acid positions corresponding to positions 1 and 2 of SEQ ID NO:19 are deleted (these amino acid positions correspond to positions 1 and 2 of SEQ ID NO:20). More preferably, the one of the two terminal domains that carries (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 shown in SEQ ID NO:20).

[0193] In addition, in a preferred embodiment of the present invention, in the multimeric form, the first monomer deletes the first and second amino acid residues present in the corresponding monomer, as shown in SEQ ID NO:7 - 12, 14, 16, 18, 20 or Ig-binding proteins having at least 89.5% identity therewith, respectively, as described elsewhere herein.

[0194] As described elsewhere herein, the invention encompasses monomers of Ig-binding proteins of SEQ ID NO: 1-6, 13, 15, 17, 19 or having at least 89.5% identity thereto, the latter incorporating amino acid substitutions at positions 9, 11 and 15 relative to SEQ ID NO: 13, and / or the latter incorporating amino acid substitutions at positions 4 and 7 relative to SEQ ID NO: 15.

[0195] The invention further encompasses multimers of Ig-binding proteins according to any one of SEQ ID NO: 7-12, 14, 16, 18 and 20 or having at least 89.5% identity thereto, the latter incorporating amino acid substitutions at positions 9, 11 and 15 relative to SEQ ID NO: 13, and / or the latter incorporating amino acid substitutions at positions 4 and 7 relative to SEQ ID NO: 15. For the corresponding position numbering in such multimers of the invention, the identification of positions 4, 7, 9, 11 and 15 in monomers 1, 2, 3, 4 and 5 as described in Table 1 above applies.

[0196] Linkers In various embodiments, one or more Ig-binding domains are directly connected to each other. In other embodiments, one or more Ig-binding domains are connected to each other through one or more linkers. A peptide linker is preferred in these exemplary embodiments. This means that the peptide linker is one or more amino acids, for example, an amino acid sequence that connects a first Ig-binding domain to a second Ig-binding domain. The peptide linker is connected to the first and second Ig-binding domains through peptide bonds between the C-terminus and N-terminus of the domains, thereby producing a single linear polypeptide chain. In some embodiments, the multimer of the Ig-binding protein comprises one or more linkers that connect the Ig-binding domains, where the linker is the same or different.

[0197] Affinity separation matrices In another embodiment, the invention relates to an affinity separation matrix comprising the Ig-binding protein or Ig-binding domain of the previous embodiments.

[0198] 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.

[0199] The affinity matrix can be used to separate immunoglobulins and should retain its Ig-binding properties even after exposure to highly alkaline conditions during a cleaning procedure. Such matrix cleaning is crucial for the long-term repeated use of the matrix.

[0200] Solid support matrices for affinity chromatography are known in the art and include, for example but not limited to, agarose and stabilized agarose derivatives (such as PraestoPure, Praesto Jetted A50, Praesto Jetted A50HipH, Mabselect, PrismA, Sepharose 6B, CaptivA, rPROTEIN ASepharose Fast Flow, MabCapturC, etc.), cellulose or cellulose derivatives, controlled pore glass, etc. ProSepvA Ultra, monoliths (such as Convective Interaction Media (CIM) monoliths, 3D printed monolithic adsorption (PMA) columns), silica, zirconia (such as CM zirconia or CPG), titanium oxide or synthetic polymers (such as hydroxyapatite, such as UNOsphere SUPrA polystyrene, such as Poros50A or Poros MabCapture A, polyethylene ether, polyvinyl alcohol, monodisperse polyacrylate resins (such as UniMab, UniMabPro), polymethacrylate, such as Toyopearl, polyhydroxyalkyl acrylate, polyhydroxyalkyl methacrylate, polyacrylamide, polymethacrylamide, etc.) and hydrogels of various components. In certain embodiments, the support comprises a polyhydroxy polymer, such as a polysaccharide. Examples of polysaccharides suitable for use as a support include, but are not limited to, agar, agarose, dextran, starch, cellulose, pullulan, etc., and stable variants of these substances.

[0201] The form of the solid support matrix can be any suitable and well-known type. Such solid support matrices for coupling the Ig-binding proteins or Ig-binding domains described herein can include, for example, one of the following: columns, capillaries, particles, membranes, filters, monoliths, fibers, pads, gels, slides, plates, cartridges, or any other form commonly used in chromatography and known to those skilled in the art.

[0202] In one embodiment, the matrix consists of substantially spherical particles (also known as beads), such as sepharose beads or agarose beads or monodisperse polyacrylate beads. Suitable particle sizes can be in the diameter range of 5 - 500 μm, such as 10 - 100 μm, such as 20 - 80 μm, such as 40 - 70 μm. The matrix in particle form can be used as a packed bed or in a suspended form (including an expanded bed).

[0203] In an alternative embodiment, the solid support matrix is a membrane, such as 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 bound. The solid support can also be in the form of a membrane in a column cartridge.

[0204] 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 one embodiment is covalently bound.

[0205] Immobilized on a solid support In embodiments of the present invention, an 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 contain additional amino acid residues at the N-terminus and / or C-terminus. The Ig-binding protein or Ig-binding domain of the present invention can be attached to a suitable solid support matrix by conventional coupling techniques. Methods for immobilizing protein ligands to solid supports are well known in the art and can be readily performed by those skilled in the art using standard techniques and equipment. In some embodiments, the coupling can be a multi-point coupling (e.g., via several lysines), or a single-point coupling (e.g., via cysteine).

[0206] In some embodiments, the basic-stable Ig-binding protein or Ig-binding domain contains an attachment site for covalent attachment to a solid phase (matrix). Site-specific attachment sites include natural amino acids, such as cysteine or lysine, which are capable of specific chemical reactions with reactive groups of the solid phase or a linker between the solid phase and the protein.

[0207] In some embodiments, the attachment site can be directly at the C-terminus or N-terminus of the Ig-binding protein or Ig-binding domain. In some embodiments, a single cysteine is located 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 the 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 support to form a thioether bridge coupling. This provides excellent mobility for the coupled protein, thereby enhancing the binding ability.

[0208] In other embodiments, the attachment site can be located within the Ig-binding protein or Ig-binding domain, e.g., at a position corresponding to position 43 or position 46 of any one of, for example, SEQ ID NO: 1-6, 13, 15, 17, 19.

[0209] In other embodiments, there may be a linker between the N-terminus or C-terminus and the attachment site. In some embodiments of the present invention, the Ig-binding protein or Ig-binding domain may contain an N-terminal or C-terminal amino acid sequence (with a terminal cysteine) of 3-20 amino acids, preferably 4-10 amino acids. The amino acids of the terminal attachment site can be selected from the group consisting of proline, glycine, alanine, and serine, where a single cysteine at the C-terminus is used for coupling.

[0210] In some embodiments of the present invention, the Ig-binding protein or Ig-binding domain may also contain additional amino acid residues at the N-terminus and / or C-terminus, such as, for example, a leader sequence at the N-terminus and / or a coupling sequence with or without a tag at the N-terminus or C-terminus.

[0211] Use of Ig binding proteins In one embodiment, the present invention relates to the use of an Ig-binding protein or Ig-binding domain of one embodiment or an affinity matrix of one embodiment for the affinity purification of immunoglobulins or variants thereof, i.e., the Ig-binding protein or Ig-binding domain of the present invention for 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.

[0212] Method for affinity purification of immunoglobulins In one embodiment, the present invention relates to a method for the affinity purification of immunoglobulins, the method comprising the following steps:

[0213] (a) providing a liquid (sample) containing Ig, such as IgG1, IgG2, IgG4, IgM, IgA, Ig fragments, Fc fragments or Fab fragments (including fusion proteins and conjugates, as defined above);

[0214] (b) providing an affinity separation matrix comprising an immobilized Ig-binding protein or Ig-binding domain as described above immobilized to the affinity separation matrix;

[0215] (c) contacting the liquid with the affinity separation matrix under conditions that allow at least one Ig-binding protein or Ig-binding domain as described above to bind to Ig; and

[0216] (d) eluting the Ig from the matrix to obtain an eluate containing the Ig.

[0217] In some embodiments, the affinity purification method may further include one or more washing steps carried out between steps (c) and (d), under conditions sufficient to remove some or all of the molecules that bind non-specifically to the affinity separation matrix. Non-specific binding refers to any binding that does not involve the interaction between at least one Ig-binding protein or Ig-binding domain and Ig.

[0218] The affinity separation matrices suitable for the disclosed uses and methods are those matrices according to the above embodiments and known to those skilled in the art.

[0219] In some embodiments, the elution of immunoglobulins from the Ig-binding protein or Ig-binding domain or from a matrix comprising an Ig-binding protein or Ig-binding domain in step (d) is achieved by changing the pH and / or changing the salt concentration. Generally, suitable conditions for performing the affinity purification method are well known to those skilled in the art. In some embodiments, the disclosed use or method of affinity purification comprising the disclosed Ig-binding protein or Ig-binding domain can 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 equal to or greater than pH 4.0. Due to the high stability of the Ig-binding proteins and Ig-binding domains of the present invention, solutions with a pH greater than or equal to 4.0 can be used to elute Ig proteins (see Examples).

[0220] In some embodiments, in step (d) of the affinity purification method, more than about 95% of the proteins (such as antibodies) comprising Ig sequences are eluted from the matrix comprising the immobilized Ig-binding protein or Ig-binding domain as described above at pH 4.0 (or higher). In some embodiments, a further step (e) is added for effective cleaning of the affinity matrix, preferably by using an alkaline liquid, such as a pH of 13 - 14. In certain embodiments, the cleaning liquid comprises 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 protein or Ig-binding domain of the present invention, such strongly alkaline solutions can be used for cleaning purposes. In some embodiments, after cleaning the affinity purification matrix with the alkaline cleaning liquid, if incubated in 1 M NaOH for at least 37 h, at least about 80% of the Ig-binding protein or Ig-binding domain has Ig-binding activity. In some embodiments, the Ig-binding ability of the Ig-binding protein or Ig-binding domain is at least about 80% or at least about 90% of the Ig-binding ability before incubation under alkaline conditions, e.g., as determined by the remaining Ig-binding ability after incubation in 1 M NaOH for at least 37 h.

[0221] The present invention also provides a method for separating immunoglobulins, comprising the following steps: (a) contacting a liquid sample comprising 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 washing liquid such as 1 M NaOH; (c) eluting the immunoglobulins from the separation matrix with a solution at pH 4.0 or pH 4.5 or higher; and (d) obtaining the immunoglobulins. In various preferred embodiments, the elution is carried out under acidic conditions at about pH 4.0, pH 4.3 or about pH 4.5. In various embodiments, the elution can be carried out at a pH higher than pH 4.5.

[0222] Nucleic acid moleculesIn one embodiment, the present invention relates to a nucleic acid molecule, preferably an isolated nucleic acid molecule, which encodes an Ig-binding protein or Ig-binding domain as disclosed above. In one embodiment, the present invention relates to a vector comprising the nucleic acid molecule. A vector refers to any molecule or entity (such as a nucleic acid, plasmid, phage or virus) that can be used to transfer protein-coding information into a host cell. In one embodiment, the vector is an expression vector.

[0223] In one embodiment, the present invention relates to an expression system comprising the above nucleic acid or vector, such as a prokaryotic host cell, such as Escherichia coli (E. coli), or a eukaryotic host, such as Saccharomyces cerevisiae or Pichia pastoris, or a mammalian cell, such as a CHO cell.

[0224] Method for producing Ig binding proteins In one embodiment, the present invention relates to a method for producing the Ig-binding protein or Ig-binding domain of the present invention, comprising the steps of: (a) culturing a host cell of one embodiment under conditions suitable for expressing the binding protein or Ig-binding domain to obtain the Ig-binding protein or Ig-binding domain; and (b) optionally isolating the Ig-binding protein or Ig-binding domain. Suitable conditions for culturing prokaryotic or eukaryotic hosts are well known to those skilled in the art.

[0225] The Ig-binding molecules of the present invention can be prepared by any of a variety of conventional and well-known techniques, such as simple organic synthesis strategies, solid-phase assisted synthesis techniques or by commercially available automated synthesizers. On the other hand, they can also be prepared individually by conventional recombinant techniques or in combination with conventional synthesis techniques.

[0226] One embodiment of the present invention relates to a method for preparing an Ig-binding protein or Ig-binding domain according to the present invention as detailed above, the method comprising the steps of: (a) preparing a nucleic acid encoding an Ig-binding protein or Ig-binding domain as defined above; (b) introducing the nucleic acid into an expression vector; (c) introducing the expression vector into a host cell; (d) culturing the host cell; (e) subjecting the host cell to culture conditions for expressing the Ig-binding protein or Ig-binding domain, whereby (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 another embodiment of the present invention, the production of the Ig-binding protein or Ig-binding domain is carried out by cell-free in vitro transcription / translation.

[0227] Examples

[0228] The following examples are provided to further illustrate the present invention. However, the present invention is not limited thereto, and the following examples merely demonstrate the practicality of the present invention based on the above description.

[0229] Example 1. Expression

[0230] Using the pNP-016 vector system, ligands (such as 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), 230620 (SEQ ID NO:20)) were expressed in Escherichia coli BL21(DE3) under the regulation of the T7 promoter. Soluble proteins were produced after induction by lactose included in the medium (auto-induction medium). BL21(DE3) competent cells were transformed with the expression plasmid, spread on a selective agar plate (containing kanamycin), and incubated overnight at 37 °C. The pre-culture was inoculating a single colony into 50 ml of 2xYT medium (supplemented with 50 μg / ml kanamycin) and culturing at 37 °C in a shake flask for 7 h. For the main culture, 350 mL of auto-induction 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 MgSO4, pH 7.4, antifoaming agent SE1) (supplemented with 50 μg / ml kanamycin and trace elements) was inoculated to an OD600 of 0.3 and incubated in a 2.5 L Ultra YieldTM flask in an orbital shaker at 37 °C. Recombinant protein expression was induced by metabolizing glucose and then allowing lactose to enter the cells. The cells grew overnight for about 18 hours to reach a final OD600 of about 40 - 50. Before harvesting, the OD600 was measured, and samples adjusted to 0.6 / OD600 were taken out, precipitated, and frozen at -20 °C. To collect the biomass, the cells were centrifuged at 12,000 xg for 20 min at 22 °C. The precipitate (wet weight) was weighed and stored at -20 °C for further processing.

[0231] Example 2: Expression and acid-soluble SDS-PAGE analysis

[0232] The sample was resuspended in 90 μl of extraction buffer (PBS supplemented with 0.2 mg / ml lysozyme, 0.5x BugBuster, 6 mM MgSO4, 6 mM MgCl2, 15 U / mL Benzonase) and dissolved by stirring at 850 rpm at room temperature for 15 minutes in a thermomixer, followed by incubation at -80 °C for 15 min. After thawing, the soluble proteins were separated from the insoluble proteins by centrifugation (16,000 x g, 2 minutes, room temperature). The supernatant (soluble fraction) was removed, 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 were taken from both the soluble and insoluble fractions, and 10 μl of 5x sample buffer and 5 μl of 0.5 M DTT were added. The samples were boiled at 95 °C for 5 min. Finally, 5 μl of these samples were applied to a NuPage Novex 4-12% Bis-Tris SDS gel, electrophoresed according to the manufacturer's recommendations, and stained with Coomassie Brilliant Blue. Results: Medium to high levels of expression were found under optimized conditions over the selected time period. All expressed Ig-binding proteins showed acidic solubility.

[0233] Example 3: Purification

[0234] The Ig-binding protein was expressed in the soluble fraction of E. coli. The cells were resuspended in cell lysis buffer and lysed by an ultrasonic cell disruption system (Sonopuls HD2200, Bandelin). The purification step was performed using IEC Sepharose SP-HP (Cytiva) using system (Cytiva) according to the manufacturer's instructions using a citric acid buffer at pH 3.0 (20 mM citric acid, 1 mM EDTA, pH 3.0). The pure protein fraction was eluted by increasing the NaCl concentration to 1 M in a linear gradient over 10 column volumes. Further purification was performed by size exclusion chromatography (Superdex 75) using a citric acid 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 variant was 100% after SE-HPLC and >96% after RP HPLC.

[0235] Example 4. The Ig-binding protein binds IgG with high affinity

[0236] The sensor chip (Bruker) was equilibrated with surface plasmon resonance (SPR) running buffer. The surface-exposed carboxyl groups were activated with a mixture of EDC and NHS to generate reactive ester groups. Substances in the on-ligand state with 700 - 1500 RU of bound ligand were immobilized on one flow cell, and substances in the off-ligand state were immobilized on another flow cell. After ligand immobilization, ethanolamine was injected to remove non-covalently bound Ig-binding proteins. After ligand binding, the protein analyte accumulated on the surface, thereby increasing the refractive index. The change in refractive index was measured in real time and plotted as a graph of response or response units (RU) versus time. The analyte was serially diluted at an appropriate flow rate (μl / min) and applied to the chip. After each run, the chip surface was regenerated with regeneration buffer and equilibrated with running buffer. Control samples were applied to the matrix. Regeneration and re-equilibration were performed as mentioned previously. Binding studies were carried out at 25 °C using a Bruker SPR-32; data evaluation was performed using the Langmuir 1:1 model (RI = 0) with Bruker evaluation software provided by the manufacturer. The dissociation constant (K D ) evaluated was normalized against the off-target effects and the K D values of the Ig-binding proteins for cetuximab (IgG1), natalizumab (IgG4), and panitumumab (IgG2) in Table 2. 228302 (SEQ ID NO:18) binds to the IgG1 of bevacizumab with a K D of 3.7 nM. 228302 binds to the IgG1 of trastuzumab with a K D of 7.6 nM.

[0237] Table 2. K D values of Ig-binding proteins for IgG

[0238]

[0239] Example 5. Affinity Chromatography

[0240] 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). The coupling efficiency of all Ig-binding proteins was at least 90%.

[0241] DBC 10%Load the coupling resin into a super-compact 5 / 50 column ( GmbH). Octagam is used as the IgG sample (concentration 2.2 mg / ml; 1xPBS, pH 7.3). The Octagam sample is applied to the matrix containing the immobilized ligand until 10% target breakthrough is reached within a residence time of 6 min (or 5 min for 228302 in Table 3A). Unbound samples are washed with 1xPBS containing 1 M NaCl, pH 7.3. The loaded antibody is quantified and calculated as the dynamic binding capacity DBC10%. Results: The binding capacity (DBC10) of all ligands is approximately 60 mg / ml, as shown in Table 3A. Strong base stability reflects the remaining binding capacity (in %) compared to the binding capacity at 0 h.

[0242] Strong alkaline stability The chromatography column is incubated with 1 M NaOH at room temperature (22 °C + / - 3 °C) for 37.5 h and 50 h (Table 3A), and 50 h (Table 3B); 2 sets are measured. The Ig-binding activity of the immobilized ligand is analyzed after incubation with 1 M NaOH. Results: After 37.5 h in 1 M NaOH, all variants show at least approximately 80% remaining IgG-binding capacity (DBC10, in %). 224772 has 90% remaining IgG-binding capacity (DBC10, in %). Even after 50 h in a very strong alkaline solution, 224770, 224772, and 224777 still show approximately 75% of the initial remaining binding capacity to Ig (see Table 3A).

[0243] 230620 has approximately 91% remaining IgG (trastuzumab) binding capacity (DBC10, in %) (after 37.5 h, 1 M NaOH).

[0244] The multimer (trimer, tetramer, pentamer, hexamer) of SEQ ID NO:6 shows comparable binding activity after incubation in 1 M NaOH for 50 h (see Table 3B). Similar observations are also made for the multimers (trimers, pentamers) of SEQ ID NO:2, 3, and 17 (data not shown).

[0245] Stepwise pH elutionStepwise pH elution was performed at pH 3.5, pH 3.7, and pH 4.0. First, 0.1 M acetic acid was used to elute at 1 CV / min for 15 CV, and then 10 CV of 0.1 M phosphoric acid (pH 1.7) (CIP) was used to elute the hIgG bound to the immobilized ligand (loading: 2.2 mg / mL Octagam, 6 min residence time). Results: For all tested variants, more than about 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). For 228302, stepwise pH elution was performed at pH 3.7, pH 4.0, and pH 4.5. First, 0.1 M acetic acid was used to elute at 1 CV / min for 15 CV, and then 10 CV of 0.1 M phosphoric acid (pH 1.7) (CIP) was used to elute the hIgG bound to the immobilized ligand (loading: 2.2 mg / mL Octagam, 5 min residence time). Approximately 92% of the antibody was eluted at pH 4.0. At pH 4.5, 84% of the IgG of 228302 was eluted.

[0246] Table 3. Affinity Chromatography Characteristics of Ligands

[0247] Table 3A. DBC10, Residual IgG Binding Capacity, Elution

[0248]

[0249] *DBC10% (mg / ml) was measured after 5 min residence time

[0250] Table 3B. Residual Binding Activity of the Multimer of SEQ ID NO:6 after Incubation in 1 M NaOH for 50 h

[0251]

[0252] Sequence

[0253] SEQ ID NO:1 (224777 monomer)

[0254] IAAQFDKIAQIAFYAILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEA KKLNDAQAPK

[0255] SEQ ID NO:2 (224772 monomer)

[0256] IAAQFDKIQQAAFYAILHLPNLTEEQRHAFIQSLRDDPSVSKEILAE AKKLNDAQAPK

[0257] SEQ ID NO:3 (224770 monomer)

[0258] IAAQFDKIAQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAE AKKLNDAQAPK

[0259] SEQ ID NO:4 (224771 monomer)

[0260] IAAQFDKIQQIAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEA KKLNDAQAPK

[0261] SEQ ID NO:5 (SEQ ID NO:11 monomer)

[0262] IAAQFDKIAQAAFYAILHLPNLTEEQRHAFIQSLRDDPSVSKEILAE AKKLNDAQAPK

[0263] SEQ ID NO:6 (224785 monomer)

[0264] IAAQFDKIQQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAE AKKLNDAQAPK

[0265] SEQ ID NO:7 (224777 pentamer)

[0266] AQFDKIAQIAFYAILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIAQIAFYAILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIAQIAFYAILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIAQIAFYAILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIAQIAFYAILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPK

[0267] SEQ ID NO:8 (224772 pentamer)

[0268] AQFDKIQQAAFYAILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIQQAAFYAILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIQQAAFYAILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIQQAAFYAILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIQQAAFYAILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPK

[0269] SEQ ID NO:9 (224770 pentamer)

[0270] AQFDKIAQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIAQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIAQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIAQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIAQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPK

[0271] SEQ ID NO:10 (224771 pentamer)

[0272] AQFDKIQQIAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIQQIAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIQQIAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIQQIAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIQQIAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPK

[0273] SEQ ID NO:11 (pentamer)

[0274] AQFDKIAQAAFYAILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIAQAAFYAILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIAQAAFYAILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIAQAAFYAILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIAQAAFYAILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPK

[0275] SEQ ID NO:12 (224785 pentamer)

[0276] AQFDKIQQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIQQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIQQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIQQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIQQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPK

[0277] SEQ ID NO:13 (monomer)

[0278] IAAQFDKIXQXAFYXILHLPNLTEEQRHAFIQSLRDDPSVSKEILAE AKKLNDAQAPK

[0279] 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.

[0280] SEQ ID NO:14 (pentamer)

[0281] AQFDKIXQXAFYXILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIXQXAFYXILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIXQXAFYXILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIXQXAFYXILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAQFDKIXQXAFYXILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPK

[0282] 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.

[0283] SEQ ID NO:15 (monomer)

[0284] IAAXFDXIXQXAFYXILHLPNLTEEQRHAFIQSLRDDPSVSKEILAE AKKLNDAQAPK

[0285] The X at position 4 may be Q or K, the X at position 7 may be K or E, the X at position 9 may be A or Q, the X at position 11 may be A or I, and the X at position 15 may be A or E.

[0286] SEQ ID NO:16 (pentamer)

[0287] AXFDXIXQXAFYXILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAXFDXIXQXAFYXILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAXFDXIXQXAFYXILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAXFDXIXQXAFYXILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAXFDXIXQXAFYXILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPK

[0288] The X at positions 2, 60, 118, 176, and 234 may be Q or K, the X at positions 5, 63, 121, 179, and 237 may be K or E, the X at positions 7, 65, 123, 181, and 239 may be A or Q, the X at positions 9, 67, 125, 183, and 241 may be A or I, and the X at positions 13, 71, 129, 187, and 245 may be A or E.

[0289] SEQ ID NO:17 (228302 monomer)

[0290] IAAKFDEIAQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPK

[0291] SEQ ID NO:18 (228302 pentamer)

[0292] AKFDEIAQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAKFDEIAQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAKFDEIAQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAKFDEIAQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPKIAAKFDEIAQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPK

[0293] SEQ ID NO:19 (230620 monomer)

[0294] IAAQFDKIAQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPP

[0295] SEQ ID NO:20 (230620 pentamer)

[0296] AQFDKIAQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPPIAAQFDKIAQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPPIAAQFDKIAQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPPIAAQFDKIAQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPPIAAQFDKIAQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPK。

Claims

1. An immunoglobulin (Ig)-binding protein comprising the amino acid sequence of SEQ ID NO:13, or an Ig-binding protein comprising an amino acid sequence having at least 89.5% amino acid identity with 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. The Ig-binding protein according to claim 1, wherein the amino acid corresponding to position 9 is alanine (A) or glutamine (Q).

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

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

5. The Ig-binding protein according to any one of claims 1 to 4, wherein the Ig-binding protein is polymeric and comprises at least 3 Ig-binding proteins.

6. The Ig-binding protein according to claim 5, wherein the polymeric Ig-binding protein is a pentamer.

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

8. The Ig-binding protein according to any one of claims 1 to 7, wherein the protein binds to one or more of IgG1, IgG2, IgG4, IgM, IgA, Ig fragments, Fc fragments, Fab fragments, fusion proteins comprising Ig regions, and conjugates comprising Ig regions, preferably, the Ig-binding protein binds to a protein comprising an Fc region or to an Fc fragment.

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

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

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

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

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

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

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