New ligands for the purification of antibodies via affinity chromatography

Abstract

The present invention relates to novel ligands for affinity chromatography for the purification of antibodies. The novel ligands are immunoglobulin (Ig) binding proteins with superior properties for highly efficient purification methods for antibodies (immunoglobulins) in terms of extreme alkaline stability of the ligands combined with mild pH elution conditions for the antibodies. The invention further relates to affinity matrices comprising the novel ligands of the invention. The invention also relates to a use of these ligands or affinity matrices comprising these ligands for affinity purification of immunoglobulins and to methods of affinity purification.

Description

[0001] NEW LIGANDS FOR THE PURIFICATION OF ANTIBODIES VIA AFFINITY CHROMATOGRAPHY

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to novel ligands for affinity chromatography for the purification of antibodies. The novel ligands are immunoglobulin (Ig) binding proteins with superior properties for highly efficient purification methods for antibodies (immunoglobulins) in terms of extreme alkaline stability of the ligands combined with mild pH elution conditions for the antibodies. The invention further relates to affinity matrices comprising the novel ligands of the invention. The invention also relates to a use of these ligands or affinity matrices comprising these ligands for affinity purification of immunoglobulins and to methods of affinity purification.

[0004] BACKGROUND OF THE INVENTION

[0005] Many biotechnological and pharmaceutical applications require the removal of contaminants from a sample containing antibodies. An established procedure for capturing and purifying antibodies and molecules that contain an Fc-domain is affinity chromatography using the bacterial cell surface Protein A from Staphylococcus aureus as selective ligand for immunoglobulins (see, for example, review by 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. Variants of Protein A with improved properties such as alkaline stability are available for purifying antibodies and various chromatographic matrices comprising Protein A ligands are commercially available. However, currently available Protein A based chromatography matrices show a loss of binding capacity for immunoglobulins following exposure to alkaline conditions and require elution conditions at pH below 4.

[0006] Most large scale production processes for antibodies or Fc-containing (fusion) proteins use Protein A for affinity purification. However, due to limitations of Protein A applications in affinity chromatography there is a need in the art to provide novel Ig binding proteins with improved properties that specifically bind to immunoglobulins in order to facilitate affinity purification of immunoglobulins. To maximally exploit the value of the chromatographic matrices comprising Ig binding proteins it is desirable to use the affinity ligand matrices multiple times. Between chromatography cycles a thorough cleaning procedure is required for sanitization and removal of residual contaminants on the matrix. In this procedure, it is general practice to apply alkaline solutions with high concentrations of NaOH such as 0.5 M NaOH to the affinity ligand matrices. Wild-type Protein A domains cannot withstand such alkaline conditions for an extended time and quickly lose binding capacity for immunoglobulin. For some purification methods, it might be required to use even harsher alkaline conditions such as 1 M NaOH to clean the matrices, but without disturbing the ligand and the Ig binding capacity activity of the ligand. Accordingly, there is an ongoing need in this field to obtain novel proteins capable of binding proteins comprising an Ig sequence or the Fc region of an Immunoglobulin, for example antibodies, and to better withstand the extreme harsh conditions applied in affinity purification of immunoglobulins combined with properties that allow elution of the Ig sequences from the ligand or matrix at mild pH, starting at pH 4.0.

[0007] The present invention provides ligands that are particularly well-suited for affinity purification of immunoglobulins. In particular, the ligands of the invention have several advantages. One significant advantage of the Ig binding proteins of the invention is their extreme stability at high pH (alkaline conditions) for a prolonged time period (such as 1.5 to 3 days at 1 M NaOH) without significantly reducing the Ig binding capacities in combination with high dynamic binding capacities. Further, the novel proteins of the invention are particularly useful for affinity purification of antibodies where mild acidic elution conditions at about pH 4.0 or above are required.

[0008] The above overview does not necessarily describe all problems solved by the present invention.

[0009] SUMMARY OF THE INVENTION

[0010] An item of the present invention is to provide novel ligands suitable for affinity purification of immunoglobulins or molecules, including fragments if immunoglobulins, comprising the Fc region of antibodies. In particular, the present invention provides Immunoglobulin (Ig) binding proteins, which are characterized by an exceptional alkaline stability and which allow elution of immunoglobulins from affinity matrices at mild pH.

[0011] Items and embodiments of the present invention include:

[0012] [1] An Immunoglobulin (Ig) binding protein comprising the amino acid sequence of SEQ ID NO:

[0013] 1 , or an Ig binding protein comprising an amino acid sequence with at least 96 % amino acid identity thereto, wherein the Ig binding protein has a remaining Ig binding activity, in particular IgG binding activity, of at least 90 % after incubation at 1 M NaOH for at least 37 h.

[0014] [2] The Ig binding protein of item 1, wherein the Ig binding protein has a remaining Ig binding activity, in particular IgG binding activity, of at least 85 % after incubation at 1 M NaOH for at least 50 h.

[0015] [3] The Ig binding protein of item 1 , having at least 96 % amino acid identity thereto, wherein the amino acid at the positions corresponding to positions 56, 114, 172, and 230 of SEQ ID NO: 1 is proline (P).

[0016] [4] The Ig binding protein of any one of items 1 to 3, wherein the amino acid at the positions corresponding to positions 3, 61 , 119, 177, and 235 of SEQ ID NO: 1 is phenylalanine (F), the amino acid corresponding to positions 6, 64, 122, 180, and 238 of SEQ ID NO: 1 is isoleucine (I), the amino acid corresponding to position 26, 84, 142, 200, and 258 of SEQ ID NO: 1 is histidine (H), and the amino acid corresponding to position 40, 98, 156, 214, and 272 of SEQ ID NO: 1 is lysine (K).

[0017] [5] The Ig binding protein of any one of items 1 to 4, wherein the amino acid at the position corresponding to position 288 of SEQ ID NO: 1 is lysine (K).

[0018] [6] The Ig binding protein according to any one of items 1 to 5, which is immobilized to a solid support.

[0019] [7] The Ig binding protein of any one of items 1 to 6, wherein the Ig is any one of IgGi, lgG2, lgG4, IgM, IgA, an Ig fragment, a fusion protein comprising an Ig or a fragment thereof, or a conjugate comprising an Ig.

[0020] [8] An affinity separation matrix comprising the Ig binding protein of any one of items 1 to 7 coupled thereto.

[0021] [9] The affinity separation matrix of item 8, which allows 97 % recovery of Ig bound to the Ig binding protein by elution at pH 4.0 from the affinity separation matrix.

[0022]

[0010] Use of the Ig binding protein of any one of items 1 to 7, or the affinity separation matrix of item 8 or 9, for affinity purification of an Ig protein.

[0023]

[0011] A method for affinity purifying an immunoglobulin (Ig), the method comprising: a) providing a sample, preferably a liquid sample, that contains an Ig; b) providing an affinity separation matrix according to item 8 or 9; c) contacting said affinity separation matrix with the (liquid) sample under conditions that permit binding of the at least one Ig binding protein of the affinity separation matrix to an Ig; and d) collecting, preferably eluting, said Ig from said affinity purification matrix, thereby obtaining the (affinity purified) Ig, preferably obtaining an eluate containing said Ig, wherein in step (d) more than about 97 % of the Ig is eluted at pH 4.0 from the affinity separation matrix.

[0024]

[0012] The method of item 11 , comprising the additional step (e) of washing the affinity purification matrix with an alkaline cleaning liquid, optionally or preferably wherein the Ig binding protein retains at least about 90 % Ig binding activity after incubation for at least 37 h at 1 M NaOH, and / or at least about 85 % Ig binding activity after incubation.

[0025] This summary of the invention does not necessarily describe all features of the present invention. Other embodiments will become apparent from a review of the ensuing detailed description.

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

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

[0028] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated member, integer or step or group of members, integers or steps but not the exclusion of any other member, integer or step or group of members, integers or steps.

[0029] As used in the description of the invention and the appended claims, the singular forms “a”, “an” and “the” are used interchangeably and intended to include the plural forms as well and fall within each meaning, unless the context clearly indicates otherwise. Also, 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 lack of combinations when interpreted in the alternative (“or”).

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

[0031] Several documents (for example: patents, patent applications, scientific publications, manufacturer's specifications etc.) are cited throughout the text of this specification. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. Some of the documents cited herein are characterized as being “incorporated by reference". In the event of a conflict between the definitions or teachings of such incorporated references and definitions or teachings recited in the present specification, the text of the present specification takes precedence.

[0032] All sequences referred to herein are disclosed in the attached sequence listing that, with its whole content and disclosure, is a part of this specification.

[0033] In the context of the present invention, the term “Ig binding protein” or “immunoglobulin-binding protein” or “ligand” is used to describe proteins that are capable to specifically bind to an immunoglobulin. The Ig binding proteins of the present invention are sometimes referred to herein as ligands of the invention. The “immunoglobulin” or “Ig” as understood herein can include, but is not necessarily limited to, mammalian IgG, such as for example human IgGi, human lgG2, human lgG4, mouse IgG, rat IgG, goat IgG, bovine IgG, guinea pig IgG, rabbit IgG; human IgM, human IgA; and an immunoglobulin or an immunoglobulin fragment comprising an Fc region (also referred to as “Fc fragment” or “Fc”). The Ig binding proteins are capable of binding to entire immunoglobulins, and to Ig fragments comprising an Fc region. The definition “immunoglobulin” as understood herein includes fusion proteins comprising an immunoglobulin, fragment of an immunoglobulin comprising an Fc region (Fc fragment), fusion proteins comprising (a fragment of) an immunoglobulin comprising an Fc region, and conjugates comprising an Ig or an Ig fragment comprising an Fc region (Fc fragment).

[0034] As will be appreciated by a person of ordinary skill in the art, the terms “immunoglobulin” and “antibody” may be used interchangeably herein. Any definitions disclosed herein concerning the term “immunoglobulin” apply to the term “antibody” accordingly.

[0035] The term “binding” according to the invention preferably relates to a specific binding. “Specific binding” means that an Ig binding protein binds stronger to an immunoglobulin for which it is specific compared to the binding to another non-immunoglobulin target.

[0036] The term "binding activity" refers to the ability of an Ig binding protein of the invention to bind to immunoglobulin. For example, the binding activity can be determined before and / or after alkaline treatment. The terms (immunoglobulin) “binding activity” and “binding capacity” may be used interchangeably herein. The binding activity can be determined for an Ig binding protein or for an Ig binding protein coupled to a matrix, / .e., for an immobilized Ig binding protein. Also, the binding activity can be determined for Ig coupled to a matrix, i.e., for an immobilized Ig binding protein. The term “artificial” refers to an object that is not naturally occurring, i.e. the term refers to an object that has been produced or modified by man. For example, a polypeptide or polynucleotide sequence that has been generated by man (e.g. for example in a laboratory by genetic engineering, by shuffling methods, or by chemical reactions, etc.) or intentionally modified is artificial.

[0037] The term “dissociation constant” or “KD” defines the specific binding affinity. As used herein, the term “KD” (usually measured in “mol / L”, sometimes abbreviated as “M”) is intended to refer to the dissociation equilibrium constant of the particular interaction between a first protein and a second protein. In the context of the present invention, the term KD is particularly used to describe the binding affinity between an Ig binding protein and an immunoglobulin. An Ig binding protein of the invention is considered to bind to an immunoglobulin, if it has a dissociation constant KD to immunoglobulin of 50 nM or less, preferably of 20 nM or less, preferably of 15 nM or less, more preferably of 10 nM or less, or even 5 nM or less.

[0038] The terms "protein" and "polypeptide" refer to any linear molecular chain of two or more amino acids linked by peptide bonds and does not refer to a specific length of the product. Thus, “peptides”, "protein", "amino acid chain," or any other term used to refer to a chain of two or more amino acids, are included within the definition of "polypeptide," and the term "polypeptide" may be used instead of, or interchangeably with any of these terms.

[0039] The terms “alkaline stable” or “alkaline 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 of the invention to withstand alkaline conditions without significantly losing the ability to bind to immunoglobulins. The skilled person in this field can easily test alkaline stability by incubating an Ig binding protein with, for example, sodium hydroxide solutions, e.g., as described in the Examples, and subsequent testing of the binding capacity or binding activity to immunoglobulin by routine experiments known to someone skilled in the art, for example, by chromatographic approaches. The alkaline stability may be determined by coupling the ligand to a surface plasmon resonance (SPR) sensor chip, and assaying the binding capacity or binding activity for immunoglobulin before and after exposure to an alkaline solution. The high alkaline treatment can be performed, for instance, in 1 M NaOH for an extended period of time, e.g., in various embodiments at least 37 h (1.5 days), or at least 48 h (2 days), or even at least 50 h.

[0040] Ligands of the invention as well as matrices comprising ligands of the invention exhibit an "increased" or "improved" alkaline stability, meaning that the molecules and matrices incorporating said Ig binding proteins s are stable under alkaline conditions for an extended period of time relative to a reference.

[0041] The term “modification” or "amino acid modification" refers to an exchange, a deletion, or an insertion of an amino acid at a particular position in a polypeptide sequence by another amino acid. Given the known genetic code, and recombinant and synthetic DNA techniques, the skilled scientist can readily construct DNAs encoding the amino acid variants.

[0042] The term “substitution” or "amino acid substitution" refers to an exchange of an amino acid at a particular position in a polypeptide sequence by another amino acid. The term “deletion” or "amino acid deletion" refers to the removal of an amino acid at a particular position in a polypeptide sequence.

[0043] The term “insertions” or "amino acid insertion” refers to the addition of amino acids to the polypeptide sequence.

[0044] Throughout this description, the amino acid residue position numbers are designated as corresponding to those for example in SEQ ID NO: 1. Accordingly, for example, an amino acid corresponding to position 56 means an amino acid residue at the position corresponding to position 56 of SEQ ID NO: 1. The term “amino acid sequence identity” refers to a quantitative comparison of the identity (or differences) of the amino acid sequences of two or more proteins. "Percent (%) amino acid sequence identity" or “percent identical” or "“percent identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. In various embodiments, the term “sequence identity” means that two (nucleotide or) amino acid sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 96 % sequence identity or more.

[0045] To determine the sequence identity, the sequence of a query protein is aligned and compared to the sequence of a reference protein. Methods for sequence alignment and sequence comparison algorithms are well known in the art. For example, for determining the extent of an amino acid sequence identity of an arbitrary polypeptide relative to the reference amino acid sequence, the SIM Local similarity program is preferably employed. For multiple alignment analysis, ClustalW as known to someone skilled in the art is preferably used.

[0046] The extent of sequence identity is generally calculated with respect to the total length of the unmodified sequence. As used herein, the phrases “percent identical” or "percent (%) amino acid sequence identity" or “percent identity”, in the context of two polypeptide sequences, refer to two or more sequences or subsequences that have in some embodiments 96 %at least 96 %, at least 97 %, at least 98 %, and 100 % amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. For clarity reasons, for example a sequence with at least 96 % identity includes all sequences with identities higher than 96 % identity, e.g. embodiments with at least 96 %, at least 97 %, at least 98 %, at least 99 %, or 100 % amino acid identity.

[0047] The term “chromatography” refers to separation technologies which employ a mobile phase and a stationary phase to separate one type of molecules (e.g., immunoglobulins) from other molecules (e.g. contaminants or other immunoglobulins) in the sample. The liquid mobile phase contains a mixture of molecules and transports these across or through a stationary phase (such as a solid matrix). Due to the differential interaction of the different molecules in the mobile phase with the stationary phase, molecules in the mobile phase can be separated. The term “affinity chromatography” refers to a specific mode of chromatography in which a ligand coupled to a stationary phase interacts with a molecule (i.e. 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 invention, affinity chromatography involves the addition of a (liquid) sample containing an immunoglobulin to a stationary phase which comprises a chromatography ligand, such as an Ig binding protein of the invention.

[0048] The terms “solid support” or “solid matrix” are used interchangeably herein, and in various embodiments are used for the stationary phase. The terms "affinity matrix" or "affinity separation matrix" or "affinity chromatography matrix", as used interchangeably herein, refer to a matrix, e.g. a chromatographic matrix, onto which an affinity ligand e.g., an Ig binding protein of the invention is attached. The ligand (e.g., Ig binding protein) is capable of specific binding to a molecule of interest (e.g., an immunoglobulin) which is to be purified or removed from a mixture (in a liquid sample). As will be appreciated by a person of ordinary skill in the art, the terms "affinity matrix" or "affinity separation matrix" or "affinity chromatography matrix" describe the separation of a molecule of interest (in particular an immunoglobulin) by using an Ig binding protein of the invention. Accordingly, the terms "affinity matrix" or "affinity separation matrix" or "affinity chromatography matrix" or "separation matrix" may be used interchangeably herein.

[0049] The term “affinity purification” as used herein refers to a method of purifying immunoglobulins of interest as defined above from a liquid (sample) by binding immunoglobulins of interest as defined above to an Ig binding protein that is immobilized to a matrix. Thereby, all other components of the mixture except immunoglobulins of interest are removed. In various embodiments, said other components of the mixture may include, e.g., other immunoglobulins that are not of interest. In a further step, immunoglobulins of interest are eluted in purified form. The terms "affinity purification" or "affinity chromatography purification" or "affinity separation" or "affinity chromatography separation" may be used interchangeably herein.

[0050] EMBODIMENTS OF THE INVENTION

[0051] The present invention will now be further described. In the following passages different embodiments of the invention are defined in more detail. Each embodiment defined below may be combined with any other embodiments unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0052] The present invention provides novel immunoglobulin (Ig) binding proteins (ligands) comprising the amino acid sequence of SEQ ID NO: 1 , or an comprising an amino acid sequence with at least 96 % amino acid identity to SEQ ID NO: 1. The novel immunoglobulin (Ig) binding proteins (ligands) 96 %have been demonstrated to exhibit a remaining Ig binding activity, in particular IgG binding activity, of at least 90 % after incubation at 1 M NaOH for (at least) 37 h. In preferred embodiments, the novel Ig binding proteins have a remaining Ig binding activity of (at least) 85 % after incubation at 1 M NaOH for at least 50 h.

[0053] In some embodiments, the Ig binding protein of the invention is selected from the following (1) to (4):

[0054] (1) a protein comprising an amino acid sequence corresponding to SEQ ID NO: 1 , and wherein the Ig binding protein has a remaining Ig binding activity, in particular IgG binding activity of (at least) 90 % after incubation at 1 M NaOH for (at least) 37 h; (2) a protein comprising an amino acid sequence having a sequence identity of at least 96 % or more with the amino acid of SEQ ID NO: 1 , wherein the Ig binding protein has a remaining Ig binding activity, in particular IgG binding activity, of (at least) 90 % after incubation at 1 M NaOH for (at least) 37 h;

[0055] (3) a protein comprising an amino acid sequence corresponding to SEQ ID NO: 1 , and wherein the Ig binding protein has a remaining Ig binding activity, in particular IgG binding activity, of (at least) 85 % after incubation at 1 M NaOH for (at least) 50 h;

[0056] (4) a protein comprising an amino acid sequence having a sequence identity of at least 96 % or more with the amino acid of SEQ ID NO: 1 , wherein the Ig binding protein has a remaining Ig binding activity, in particular IgG binding activity, of at least 85 % after incubation at 1 M NaOH for (at least) 37 h.

[0057] The present invention encompasses an Immunoglobulin (Ig) binding protein comprising the amino acid sequence of SEQ ID NO: 1 , or an Ig binding protein comprising an amino acid sequence with at least 96 % amino acid identity thereto, wherein the amino acid at the positions corresponding to positions 56, 114, 172, 230 of SEQ ID NO: 1 is a proline (P) and wherein the Ig binding protein has a remaining Ig binding activity, in particular IgG binding activity, of at least 90 % after incubation at 1 M NaOH for at least 37 h.

[0058] The present invention encompasses an Immunoglobulin (Ig) binding protein of SEQ ID NO: 1 , or an Ig binding protein comprising an amino acid sequence with at least 96 % amino acid identity to SEQ ID NO: 1 wherein

[0059] (a) the amino acid corresponding to positions 3, 61 , 119, 177, and 235 of SEQ ID NO: 1 is phenylalanine (F),

[0060] (b) the amino acid corresponding to positions 6, 64, 122, 180, and 238 of SEQ ID NO: 1 is isoleucine (I),

[0061] (c) the amino acid corresponding to positions 26, 84, 142, 200, and 258 of SEQ ID NO: 1 is histidine (H),

[0062] (d) the amino acid corresponding to positions 40, 98, 156, 214, and 272 of SEQ ID NO: 1 is lysine (K), and / or

[0063] (e) the amino acid corresponding positions 56, 114, 172, 230 of SEQ ID NO: 1 is proline (P), and wherein the Ig binding protein has a remaining Ig binding activity, in particular IgG binding activity, of at least 90 % after incubation at 1 M NaOH for at least 37 h.

[0064] The present invention encompasses an Immunoglobulin (Ig) binding protein of SEQ ID NO: 1 , or an Ig binding protein comprising an amino acid sequence with at least 96 % amino acid identity to SEQ ID NO: 1 wherein (a) the amino acid corresponding to positions 3, 61 , 119, 177, and 235 of SEQ ID NO: 1 is phenylalanine (F),

[0065] (b) the amino acid corresponding to positions 6, 64, 122, 180, and 238 of SEQ ID NO: 1 is isoleucine (I),

[0066] (c) the amino acid corresponding to position 26, 84, 142, 200, and 258 of SEQ ID NO: 1 is histidine (H),

[0067] (d) the amino acid corresponding to position 40, 98, 156, 214, and 272 of SEQ ID NO: 1 is lysine (K), and / or

[0068] (e) the amino acid corresponding positions 56, 114, 172, 230 of SEQ ID NO: 1 is proline (P), and wherein the Ig binding protein has a remaining Ig binding activity, in particular IgG binding activity, of at least 85 % after incubation at 1 M NaOH for at least 50 h.

[0069] In some embodiments, the Ig binding protein of the invention is selected from the following (1) to (5):

[0070] (1) a protein comprising an amino acid sequence corresponding to SEQ ID NO: 1 ;

[0071] (2) a protein consisting of the amino acid sequence of SEQ ID NO: 1

[0072] (3) a protein comprising an amino acid sequence having a sequence identity of at least 96 % or more with the amino acid sequence specified in the (1) or (2), provided that the amino acid at the positions corresponding to positions 3, 6, 26, 40, 56, 64, 84, 98, 114, 122, 142, 156, 172, 180, 200, 214, 230, 238, 258, 272 of SEQ ID NO: 1 specified in the (1) are not mutated;

[0073] (4) a protein comprising an amino acid sequence having a sequence identity of at least 96 % or more with the amino acid sequence specified in the (1) or (2), provided that the amino acid at the positions corresponding to positions 3, 6, 26, 40, 56, 64, 84, 98, 114, 119, 122, 142, 156, 172, 177, 180, 200, 214, 230, 235, 238, 258, 272 of SEQ ID NO: 1 specified in the (1) are not mutated;

[0074] (5) a protein comprising an amino acid sequence having a sequence identity of at least 96 % or more with the amino acid sequence specified in the (1) or (2), provided that the amino acid at the positions corresponding to positions 3, 6, 26, 40, 56, 64, 84, 98, 114, 119, 122, 142, 156, 172, 177, 180, 200, 214, 230, 235, 238, 258, 272, 288 of SEQ ID NO: 1 specified in the (1) are not mutated.

[0075] The surprising advantage of the ligands of the invention is the stability under extreme conditions such as high pH (pH 13 and higher) without losing Ig binding properties. More specifically, the surprising advantage of the ligands of the invention is the stability under extreme conditions such as high pH (pH 13 and higher) while showing extraordinary high Ig binding properties. The ligands as described herein demonstrate extreme high alkaline stability for a prolonged period of time (at least 37 hours, preferably at least 50 h or 50h) without impairing the Ig-binding properties (see Examples). Further, they are stable at low pH without significantly losing Ig binding properties. The extreme high alkali stability feature is particularly important for chromatography approaches with harsh cleaning procedures using alkaline solutions with NaOH concentrations as high as 1 M to remove contaminants on the matrix so that the matrix can be used several times. In addition to extreme high caustic stability, ligands show high coupling efficiencies, as shown in the Examples.

[0076] As described herein, the remaining binding capacity (in %) is an expression of the caustic stability of the novel Ig binding ligands of the present invention. The terms “caustic stability” and “remaining binding capacity” may be used interchangeably herein. The caustic stability reflects remaining binding capacity (in %) after incubation at 1 M NaOH for (at least) 37 h or 50 h as described elsewhere herein, as compared to the binding capacity at 0 h under the same conditions.

[0077] In various embodiments, a novel Ig binding protein of the present invention has a remaining IgGi binding capacity of at least 90 % after incubation at 1 M NaOH for at least 37 h, as described elsewhere herein.

[0078] In various embodiments, a novel Ig binding protein of the present invention has a remaining IgGi binding capacity of at least 85 % after incubation at 1 M NaOH for at least 50 h, as described elsewhere herein.

[0079] As described herein, the remaining Ig binding capacity is preferably a remaining dynamic binding capacity (DBG), more specifically a remaining DBC10, and in particular a remaining DBC10 at 5 or 6 min contact time. The remaining binding capacity is typically determined when the Ig binding ligand is coupled to a solid support such as a chromatography matrix.

[0080] In particularly preferred embodiments, a novel Ig binding protein of the present invention has a remaining (dynamic) binding capacity (DBC10) for Trastuzumab of more than 90 % after incubation at 1 M NaOH for 37.5 h, and in particular a remaining DBC10 at 5 or 6 min contact time. The remaining binding capacity is typically determined when the Ig binding ligand is coupled to a solid support such as a chromatography matrix.

[0081] In particularly preferred embodiments, a novel Ig binding protein of the present invention has a remaining (dynamic) binding capacity (DBC10) for Trastuzumab of more than 80 %, in particular of about 85% after incubation at 1 M NaOH for 50 h, and in particular a remaining DBC10 at 5 or 6 min contact time. The remaining binding capacity is typically determined when the Ig binding ligand is coupled to a solid support such as a chromatography matrix.

[0082] In various embodiments, a novel Ig binding protein of the present invention has a remaining Ig binding capacity after incubation at 1 M NaOH for (at least) 37 h, which is higher as compared to the Ig binding protein comprising the control sequence A described in Example 5, and / or as compared to the Ig binding protein comprising the control sequence B described in Example 5. In various embodiments, a novel Ig binding protein of the present invention has a remaining Ig binding capacity after incubation at 1 M NaOH for (at least) 50 h, which is higher as compared to the Ig binding protein comprising the control sequence A described in Example 5, and / or as compared to the Ig binding protein comprising the control sequence B described in Example 5.

[0083] The Ig binding protein comprising the control sequence A comprises the amino acid sequence SEQ ID NO: 1 , except for having histidine (H) at positions 3, 61 , 119, 177, and 235, glutamine (Q) at positions 7, 65, 123, 181 , and 239, lysine (K) at position 40, 98, 156, 214, and 272, and lysine (K) at position 56, 114, 172, 230.

[0084] The Ig binding protein comprising the control sequence A comprises the amino acid sequence SEQ ID NO: 1, except for having lysine (K) at positions 2, 60, 118, 176, and 234, histidine (H) at positions 3, 61 , 119, 177, and 235, aspartic acid (D) at positions 6, 64, 122, 180, and 238, glutamine (Q) at positions 7, 65, 123, 181 , and 239, aspartic acid (D) at positions 23, 81 , 139, 197, and 255, asparagine (N) at position 26, 84, 142, 200, and 258 is asparagine (N), and lysine (K) at position 56, 114, 172, 230. Further, an important step in affinity chromatography is the elution of the protein of interest, particular an immunoglobulin of interest, that is bound to the Ig binding protein of the invention. This step is usually done at low pH. The affinity ligands of the invention do not lose binding properties to Ig after this treatment, while elution of the protein of interest is possible at low pH. In some circumstances, it is important to have conditions for the elution of antibodies (immunoglobulins) from the ligand at pH 4.0, pH 4.1 , pH 4.2, pH 4.3, pH 4.4, pH 4.5, or higher. The ligands of the invention have a remaining IgG binding capacity of about 80 % after at least 37 hours at 1 M NaOH and elution of at least about 95 %, preferably at least 97 % of the bound IgG from the matrix at mild pH of pH 4.0, or higher.

[0085] Still further, preferred ligands of the invention showing (or providing for) (after incubation of at least 37 hours or at least 50 hours at 1 M NaOH) an elution of about 97 %, of bound antibody (in particular IgG) from an affinity purification or separation matrix at pH 4.0 (or higher) specifically include ligands described herein in relation to SEQ ID NO: 1. In preferred embodiments, a novel Ig binding protein of the present invention allows an elution of bound Ig, in particular bound IgG, more specifically bound IgGi, at pH 4.0 of more than 97 %, preferably more than 95 %, even more 97 %, after incubation at 1 M NaOH for (at least) 37 h, and in particular a remaining DBC10 at 5 or 6 min contact time. The remaining binding capacity is typically determined when the Ig binding ligand is coupled to a solid support such as an chromatography matrix. In other preferred embodiments, a novel Ig binding protein of the present invention allows an elution of bound Ig, in particular bound IgG, more specifically bound IgGi, at pH 4.5 of more than 70 %, preferably more than 75 %, even more 79 %, after incubation at 1 M NaOH for (at least) 37 h. in. Ligands as described herein bind to Immunoglobulin with a dissociation constant KD preferably below 20 nM or less. In some embodiments, the Ig binding protein binds to Ig, in particular IgGi, lgG2, lgG4, IgM, and / or IgA, Ig fragments (thereof), Fc fragments, fusion proteins comprising an Ig region or an Fc region of an Ig, and conjugates comprising an Ig region with a dissociation constant KD preferably below 20 nM or less.

[0086] Methods for determining binding affinities or binding capacities of Ig binding proteins, i.e. for determining the dissociation constant KD, are known to a person of ordinary skill in the art and can be selected for instance from the following methods known in the art: Surface Plasmon Resonance (SPR) based technology, kinetic exclusion analysis (KinExA assay), Bio-layer interferometry (BLI), enzyme-linked immunosorbent assay (ELISA), flow cytometry, isothermal titration calorimetry (ITC), analytical ultracentrifugation, radioimmunoassay (RIA or IRMA) and enhanced chemiluminescence (ECL). Some of the methods are described further in the Examples. Typically, the dissociation constant KD is determined at 20 °C, 25 °C, or 30 °C, preferably at 20 °C, 25 °C, or 30 °C by SPR. If not specifically indicated otherwise, the KD values recited herein are determined at 22 °C + / - 3 °C by surface plasmon resonance spectroscopy. In one embodiment, the Ig binding protein has a dissociation constant KD to human IgGi in the range between 0.1 nM and 10 nM. Preferably, the KD in the range between 0.1 nM and 5 nM for human IgGi, means a KD as determined by SPR, more preferably as determined by SPR at 20 °C, 25 °C, or 30 °C.

[0087] In various embodiments, the Ig binding protein of the present invention has a binding affinity (KD) to an IgG, in particular human IgG, of less than 20 nM, in particular less than 15 nM as determined by SPR, more specifically less than 20 nM as determined by SPR at 25°C.

[0088] In preferred embodiments, the Ig binding protein of the present invention has a binding affinity (KD) to an IgGi, in particular human IgGi, of less than 10 nM, in particular less than 10 nM as determined by SPR, more specifically less than 10 nM as determined by SPR at 25°C. The Ig binding protein of SEQ ID NO: 1 has been shown to have a binding affinity (KD) to Cetuximab (IgGi) of less than 10 nM, in particular less than 5 nM as determined by SPR, more specifically less than 5 nM as determined by SPR at 25°C (Example 4). ins. The ligands of the invention provide surprisingly particularly good alkaline stability, shown as remaining IgG binding of at least about 85 % after long term incubation at 1 M NaOH, as shown in the Examples, in addition to high dynamic binding capacities (DBC) of more than 60 mg / ml at 6 min residence time. The alkaline stability of the ligand is determined by comparing the loss in Ig binding activity. In some embodiments, the alkaline 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 proteins of the invention, an alkaline liquid with pH higher than 13 can be used for cleaning affinity matrices with immobilized Ig binding proteins of the invention. In some embodiments, the alkaline stability of the Ig binding protein is determined by comparing the loss in Ig binding activity after at least 37 h incubation in 1 M NaOH (see Examples). In some embodiments, the alkaline stability of the Ig binding protein is determined by comparing the loss in Ig binding activity after very long incubation in alkaline solution, e.g. for at least 2 days (at least 48 h) incubation in 1 M NaOH (see Examples), reflecting an extraordinary stability of the Ig binding proteins as described herein.

[0089] The Ig binding proteins of the invention are stable under alkaline conditions, in particular under alkaline conditions of 1 M NaOH for at least 37 h. In preferred embodiments, the Ig binding protein of the invention is stable under alkaline conditions, in particular under alkaline conditions of 1 M NaOH for at least 48 h, more preferably for at least 50 h.

[0090] The Ig binding proteins of the invention are alkaline-stable ligands for immunoglobulins. The Ig binding proteins of the invention retain binding capacity (or binding affinity) for immunoglobulin after exposure to 1 M NaOH for at least 37 h. As further described herein, the Ig binding proteins of the invention retain at least about 85 % or at least about 90 % binding capacity for immunoglobulin after exposure to extreme alkaline conditions as described herein. In further preferred embodiments, the Ig binding proteins of the invention retain at least about 90 % binding capacity for immunoglobulin after exposure to extreme alkaline conditions (1 M NaOH for at least 37 h). In various embodiments, the Ig binding proteins of the invention retain binding capacity for immunoglobulin as described above when immobilized to a solid support, preferably to a solid support of an affinity separation matrix.

[0091] As further described herein, the Ig binding proteins of the invention are typically stable under alkaline conditions at room temperature. The term room temperature may include temperatures between 15°C and 25°C, more specifically temperatures between 20°C and 25°C. In various embodiments, the Ig binding protein of the invention is stable under alkaline conditions at 22°C ± 3°C.

[0092] In various embodiments, the alkaline stability of the Ig binding protein as described above means alkaline stability of the Ig binding protein immobilized to a solid support, preferably to a solid support of an affinity separation matrix. Hence, in various embodiments, the alkaline stability of the Ig binding protein is determined by comparing the loss in Ig binding activity or Ig binding capacity of the Ig binding protein when immobilized to a solid support, preferably to a solid support of an affinity separation matrix. Hence, in other embodiments, the alkaline stability of the Ig binding protein is determined by comparing the Ig binding activity of the Ig binding protein to a reference protein after alkaline treatment for a prolonged time when immobilized to a solid support.

[0093] The binding capacity or binding affinity for immunoglobulin of the Ig binding protein of the present invention can be evaluated by a skilled person using methods well known in the art, in particular methods for determining the dissociation constant KD as described elsewhere herein. In various embodiments, the binding capacity or binding affinity for immunoglobulin of the Ig binding protein of the present invention is determined using Surface Plasmon Resonance (SPR) spectroscopy, as also described elsewhere herein. In other embodiments, the binding capacity or binding affinity for immunoglobulin of the Ig binding protein of the present invention is determined using kinetic exclusion analysis (KinExA assay), or enzyme-linked immunosorbent assay (ELISA), as described elsewhere herein.

[0094] The binding capacity or binding affinity for immunoglobulin of the Ig binding protein of the present invention can be assessed for each candidate ligand before and after exposure to alkaline conditions as described herein. matrix. In another embodiment the present invention is directed to an affinity separation matrix, comprising an Ig binding protein of the previous embodiments.

[0095] In preferred embodiments, the affinity separation matrix is a solid support. The affinity separation matrix comprises at least one Ig binding protein as described above.

[0096] An affinity matrix is useful for separation of immunoglobulins and should retain the Ig binding property even after highly alkaline conditions as applied during cleaning processes. Such cleaning of matrices is essential for long-term repeated use of matrices.

[0097] Solid support matrices for affinity chromatography are known in the art and include for example but are not limited to, agarose and stabilized derivatives of agarose (e.g. PraestoPure, Praesto Jetted A50, Praesto Jetted A50 HipH, Mabselect, PrismA, Sepharose 6B, CaptivA, rPROTEIN A Sepharose Fast Flow, MabCapture other), cellulose or derivatives of cellulose, controlled pore glass such as. ProSep vA Ultra, monolith such as convective interactive media (CIM) monolith, 3D printed monolith adsorption (PMA) column), silica, zirconium oxide (e.g. CM Zirconia or CPG), titanium oxide, or synthetic polymers (e.g. hydroxapaptite such as U NOsphere SUPrA polystyrene such as Poros 50A or Poros MabCapture A, polyvinylether, polyvinyl alcohol, monodisperse polyacrylate resin (e.g. UniMab’ UniMabPro), polymethacrylate such as Toyopearl, polyhydroxyalkyl acrylates, polyhydroxyalkyl methacrylates, polyacrylamides, polymethacrylamides etc) and hydrogels of various compositions. In certain embodiments the support comprises a polyhydroxy polymer, such as a polysaccharide. Examples of polysaccharides suitable for supports include but are not limited to agar, agarose, dextran, starch, cellulose, pullulan, etc, and stabilized variants of these.

[0098] The formats for solid support matrices can be of any suitable well-known kind. Such solid support matrix for coupling the Ig binding protein as described herein might comprise for example, one of the following: columns, capillaries, particles, membranes, filters, monoliths, fibers, pads, gels, slides, plates, cassettes, or any other format commonly used in chromatography and known to someone skilled in the art.

[0099] In one embodiment, the matrix is comprised of substantially spherical particles, also known as beads, for example Sepharose or Agarose beads or monodisperse polyacrylate beads. Suitable particle sizes may be in the diameter range of 5-500 pm, such as 10-100 pm, such as

[0100] 20-80 pm, such as 40-70 pm. Matrices in particle form can be used as a packed bed or in a suspended form including expanded beds.

[0101] In an alternative embodiment, the solid support matrix is a membrane, for example a hydrogel membrane. In some embodiments, the affinity purification involves a membrane as matrix to which the Ig binding protein of the one embodiment is covalently bound. The solid support can also be in the form of a membrane in a cartridge.

[0102] In some embodiments, the affinity purification involves a chromatography column containing a solid support matrix to which the Ig binding protein of the one embodiment is covalently bound.

[0103] Immobilization to a solid . In embodiments of the invention, the Ig binding protein is conjugated to a solid support. In some embodiments of the invention, the Ig binding protein may comprise additional amino acid residues at the N- and / or C-terminal end. The Ig binding protein of the invention may be attached to a suitable solid support matrix via conventional coupling techniques. Methods for immobilization of protein ligands to solid supports are well- known in this field and easily performed by the skilled person in this field using standard techniques and equipment. In some embodiments, the coupling may be a multipoint coupling, for example via several lysines, or a single point coupling, for example via cysteine.

[0104] In some embodiments, the alkaline stable Ig binding protein comprises an attachment site for covalent attachment to a solid phase (matrix). Site-specific attachment sites comprise natural amino acids, such as cysteine or lysine, which enable specific chemical reactions with a reactive group of the solid phase or a linker between the solid phase and the protein.

[0105] In some embodiments, the attachment site may be directly at the C- or N-terminal end of the Ig binding protein. In some embodiments, a single cysteine is located at the C-terminal end for site-specific immobilization of the Ig binding protein. An advantage of having a C-terminal cysteine is that coupling of the Ig binding protein can be achieved through reaction of the cysteine thiol with an electrophilic group on a support resulting in a thioether bridge coupling. This provides excellent mobility of the coupled protein which provides increased binding capacity.

[0106] In other embodiments, there may be a linker between the N- or C-terminus and the attachment site. In some embodiments of the invention, the Ig binding protein may comprise a N- or C- terminal amino acid sequence of 3 - 20 amino acids, preferably of 4 - 10 amino acids, with a terminal cysteine. Amino acids for a terminal attachment site may be selected from the group of proline, glycine, alanine, and serine, with a single cysteine at the C-terminal end for coupling. In some embodiments of the invention, the Ig binding protein may also comprise additional amino acid residues at the N- and / or C-terminal end, such as for example a leader sequence at the N-terminal end and / or a coupling sequence with or without a tag at the N- or C-terminal end. Use of the In a one embodiment the present invention is directed to the use of the Ig binding protein of the one embodiment or an affinity matrix of the one embodiment for affinity purification of immunoglobulins or variants thereof, i.e. the Ig binding protein of the invention is used for affinity chromatography. In some embodiments, the Ig binding protein of the invention is immobilized onto a solid support as described in the one embodiment of the invention.

[0107] Method of affinity purification of immunoglobulins, In one embodiment the present invention is directed to a method of affinity purification of immunoglobulins, the method comprising the following steps:

[0108] (a) providing a liquid (sample) that contains an Ig such as IgGi, lgG2, lgG4, IgM, IgA, Ig fragments, Fc fragments, or Fab fragments (including fusion proteins and conjugates, as defined above);

[0109] (b) providing an affinity separation matrix comprising an immobilized Ig binding protein as described above immobilized to said affinity separation matrix;

[0110] (c) contacting said liquid with said affinity separation matrix, under conditions that permit binding of the at least one Ig binding protein as described above to an Ig; and

[0111] (d) eluting said Ig from said matrix, thereby obtaining an eluate containing said Ig.

[0112] In some embodiments, the method of affinity purification may further comprise one or more washing steps carried out between steps (c) and (d) under conditions sufficient to remove from the affinity separation matrix some or all molecules that are non-specifically bound thereto. Non-specifically bound means any binding that does not involve an interaction between the at least one Ig binding protein and an Ig.

[0113] Affinity separation matrices suitable for the disclosed uses and methods are those matrices according to the embodiments described above and as known to someone skilled in the art.

[0114] In some embodiments, the elution of the immunoglobulin from (the matrix comprising) the Ig binding protein in step (d) is effected through a change in pH and / or a change in salt concentration. In general, suitable conditions for performing the method of affinity purification are well known to someone skilled in the art. In some embodiments, the disclosed uses or methods of affinity purification comprising the disclosed Ig binding proteins may provide elution of at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, at least about 99 %, or 100 % of Ig (molecules) at a pH of equal or greater than pH 4.0. Due to the high stability of the Ig binding proteins of the invention, solutions with greater than or equal to pH 4.0 can be used for the elution of Ig proteins (see Examples).

[0115] In some embodiments, in step (d) of the method of affinity purification more than about 95 % of the protein comprising the Ig sequence (e.g. antibody) is eluted at pH 4.0 (or higher) from the matrix comprising the immobilized Ig binding protein as described above. In some embodiments, a further step (e) for efficient cleaning of the affinity matrix is added, preferably by using an alkaline liquid, for example, with pH of 13 - 14. In certain embodiments, the cleaning liquid comprises 0.5 - 1 M NaOH or KOH. Due to the high alkaline stability of the Ig binding proteins s of the invention, such strong alkaline solution can be used for cleaning purposes. After cleaning the affinity purification matrix with an alkaline cleaning liquid, in some embodiments, at least about 80 % of the Ig binding protein have Ig binding activity if incubated for at least 37 h at 1 M NaOH. In some embodiments, the Ig-binding capacity of the Ig binding protein is at least about 85 % or at least about 90 % of the Ig binding capacity before the incubation under alkaline conditions, for example, as determined by the remaining Ig-binding capacity after at least 37 h incubation in 1 M NaOH.

[0116] The present invention further provides a method of isolating an immunoglobulin, comprising the steps (a) contacting a liquid sample comprising an immunoglobulin with a separation matrix comprising a plurality of Ig binding proteins (coupled to a solid support); (b) washing the separation matrix with a washing liquid such as 1 M NaOH; (c) eluting the immunoglobulin from the separation matrix with about pH 4.2; and (d) obtaining the immunoglobulin. In various preferred embodiments, the elution is performed under mild acidic conditions of about pH 4.0, pH 4.1 , pH 4.2, pH 4.3, pH 4.4 or pH 4.5.

[0117] Nucleic acid molecule. In one embodiment, the present invention is directed to a nucleic acid molecule, preferably an isolated nucleic acid molecule, encoding an Ig binding protein as disclosed above. In one embodiment, the present invention is directed to a vector comprising the nucleic acid molecule. A vector means any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage or virus) that can be used to transfer protein coding information into a host cell. In one embodiment, the vector is an expression vector.

[0118] In one embodiment, the present invention is directed to an expression system which comprises a nucleic acid or a vector as disclosed above, for example a prokaryotic host cell, for example E. coli, or a eukaryotic host, for example yeast Saccharomyces cerevisiae or Pichia pastoris or mammalian cells such as CHO cells.

[0119] Method for the production of a In one embodiment the present invention is directed to a method for the production of a Ig binding protein of the invention, comprising the step(s): (a) culturing the host cell of the one embodiment under suitable conditions for the expression of the binding protein in order to obtain said Ig binding protein; and (b) optionally isolating said Ig binding protein. Suitable conditions for culturing a prokaryotic or eukaryotic host are well-known to the person skilled in the art.

[0120] Ig binding molecules of the invention may be prepared by any of the many conventional and well-known techniques such as plain organic synthetic strategies, solid phase-assisted synthesis techniques or by commercially available automated synthesizers. On the other hand, they may also be prepared by conventional recombinant techniques alone or in combination with conventional synthetic techniques. One embodiment of the present invention is directed to a method for the preparation of a Ig binding protein according to the invention as detailed above, said method comprising the following steps: (a) preparing a nucleic acid encoding an Ig binding protein as defined above; (b) introducing said nucleic acid into an expression vector; (c) introducing said expression vector into a host cell; (d) cultivating the host cell; (e) subjecting the host cell to culturing conditions under which an Ig binding protein is expressed, thereby (e) producing an Ig binding protein as described above; optionally (f) isolating the Ig binding protein produced in step (e); and (g) optionally conjugating the Ig binding protein to solid matrices as described above. In a further embodiment of the present invention the production of the Ig binding protein is performed by cell-free in vitro transcription I translation.

[0121] EXAMPLES

[0122] The following Examples are provided for further illustration of the invention. The invention, however, is not limited thereto, and the following Examples merely show the practicability of the invention on the basis of the above description.

[0123] Example 1. Expression

[0124] SEQ ID NO: 1 was expressed in Escherichia coli BL21 (DE3) fermentation process using a pNP-001 vector system under regulation of a T7 promoter. The seed culture was grown in preculture medium (34.5 g / L yeast extract, 0.61 g / L MgSO4, 14.2 K2HPO4, 0.5 g / L NH4CI, 50 pg / mL kanamycin). The fermentation process was performed in a 50 L stainless steel bioreactor as a fed-batch process. The culture medium (17.25 g / L yeast extract, 0.61 g / L MgSO4, 14.2 K2HPO4, 0.5 g / L NH4CI) was inoculated with the seed culture and the culture grown until the substrate was depleted (37 °C, pH 7.1 , 30 % pO2 saturation, aeration 2.5 VVM, pressure 0.5 bar). Exponential feeding was performed with glucose as the main substrate (200 g / L glucose, 276 g / L yeast extract, 1.1 g / L MgSC>4). Protein expression was induced by isopropyl p-D-1 -thiogalactopyranoside (IPTG, end concentration of 1 mM) at 37 °C for 4 h at a constant feeding rate. To collect biomass cells were centrifuged at 12,000 x g for 25 min at 10 °C. Bacterial pellets were stored at -20 °C before processing. Expression was analyzed via SDS-PAGE.

[0125] Example 2: SDS-PAGE analysis of expression and acidic solubility

[0126] Samples were resuspended in 90 pl extraction buffer (PBS supplemented with 0.2 mg / ml Lysozyme, 0.5x BugBuster, 6 mM MgSO4, 6 mM MgCh, 15 U / rnL Benzonase) and solubilized by agitation in a thermomixer at 850 rpm, rt for 15 min with a subsequent incubation at -80 °C for 15 min. After thawing, soluble proteins were separated from insoluble proteins by centrifugation (16000 x g, 2 min, rt). Supernatant was withdrawn (soluble fraction) and the pellet (insoluble fraction) was resuspended in equivalent amount of urea buffer (8 M urea, 0.2 M Tris, 20 mM EDTA, pH 7.0). 35 pl were taken both from the soluble and insoluble fraction, and 10 pl 5x sample buffer as well as 5 pl 0.5 M DTT were added. Samples were boiled at 95 °C for 5 min. Finally, 5 pl of those samples were applied to NuPage Novex 4-12 % Bis-Tris SDS gels which were run in accordance to the manufacturer’s recommendations and stained with Coomassie. Mid- to high level expression was found under optimized conditions within the chosen period of time. SEQ ID NO: 1 showed acidic solubility.

[0127] Example 3: Purification

[0128] Small scale purification was performed via IgG Sepharose (Cytiva, according to manufacturers instructions) using an AKTA avant system (Cytiva) followed by size exclusion chromatography (Cytiva: Superdex75) in 1xPBS, 5 mM TCEP, pH=7.3. For large scale production of ligand, cells were solubilized in 50 mM BisT ris, 1 mM EDTA, 150 mM NaCI pH 6.5. After cell disruption an acetic acid concentration of 100 mM was adjusted in the lysate. Afterwards pH was adjusted to 2.0 followed by a centrifugation step (30 min, 30000xg). pH of supernatant was adjusted to 3.5 using 1 M Tris pH 10. Supernatant was loaded on IEC Sepharose SP-HP (Cytiva) or SP65 (Purolite) using an AKTAvant system (Cytiva) according to the manufacturer’s instructions using acetic acid buffer at pH 3.5 (50 mM Na-Acetat, 1 mM EDTA, pH 3.5). Protein fractions were eluted by increasing sodium chloride concentration to 1 M with a linear gradient. Further purification was performed by size exclusion chromatography (Superdex 75, Cytiva) for SEQ ID NO: 1 according to manufactures instructions using citric acid buffer pH 6.5 (20 mM Citric acid, 150 mM NaCI, 1 mM EDTA, pH 6.5). The purity of SEQ ID NO: 1 was 100 % after SE- HPLC and >96 % after RP HPLC.

[0129] Example 4. SEQ ID NO: 1 binds to IgG with high affinity

[0130] A sensor chip (Bruker) was equilibrated with surface plasmon resonance (SPR) running buffer. Surface-exposed carboxylic groups were activated by passing a mixture of EDC and NHS to yield reactive ester groups. 400-700 Rll on-ligand were immobilized on a flow cell, off- ligand was immobilized on another flow cell. Injection of ethanolamine after ligand immobilization deactivates remaining active groups. Upon ligand binding, protein analyte was accumulated on the surface increasing the refractive index. This change in the refractive index was measured in real time and plotted as response or resonance units (RU) versus time. The analytes (Ig) were applied to the chip in serial dilutions with a suitable flow rate (pl / min). After each run, the chip surface was regenerated with regeneration buffer and equilibrated with running buffer. As a control-samples were applied to activated / deactivated chip spots. Regeneration and re-equilibration were performed as previously mentioned. Binding studies were carried out by the use of the Bruker SPR-32 at 25 °C; data evaluation was operated via the Bruker evaluation software, provided by the manufacturer, by the use of the Langmuir 1 : 1 model (RI=0).

[0131] Evaluated dissociation constants (KD) were standardized against off-target and KD values of SEQ ID NO: 1 for Cetuximab (IgGi), Natalizumab (lgG4), and Panitumumab (lgG2). SEQ ID NO: 1 has an affinity of less than 20 nM to each of the monoclonal antibodies. The KD of SEQ ID NO: 1 for hlgGi was 2.2 nM, for hlgG2 17 nM, and for hlgG4 4.4 nM.

[0132] Example 5. Affinity purification of antibodies

[0133] COUPLING: Purified SEQ ID NO: 1 was coupled to agarose-based chromatography beads (Praesto Jetted Expoxy 50, Purolite) according to the manufacturer’s instructions (20 mg / ml matrix). The coupling efficiency was 18.5 mg / ml.

[0134] DBC10%: The IgG sample (2.2 g / L) was applied to the matrix comprising immobilized SEQ ID NO: 1 until 10 % target breakthrough at 6 min residence time (Trastuzumab as IgG sample) or at 5 min residence time (Octagam as IgG sample). Unbound sample was washed with 1xPBS with 1 M NaCI, pH 7.3. Loaded IgG was quantified and calculated as dynamic binding capacity DBC10 %. Caustic stability reflects the remaining binding capacity (in %) compared to the binding capacity at 0 h.

[0135] The matrix comprising immobilized SEQ ID NO: 1 had a remaining IgGi (Trastuzumab) binding capacity (DBC10 in %) of about 91 % (after 37.5 h, 1 M NaOH). The matrix comprising immobilized SEQ ID NO: 1 had a remaining IgGi (Trastuzumab) binding capacity (DBC10 in %) of about 85 % (after 50 h, 1 M NaOH).

[0136] Control sequence A (228314) is SEQ ID NO: 1 with histidine (H) at positions 3, 61 , 119, 177, and 235, glutamine (Q) at positions 7, 65, 123, 181 , and 239, lysine (K) at position 40, 98, 156, 214, and 272, and lysine (K) at position 56, 114, 172, 230. Control sequence B (230351) is SEQ ID NO: 1 with lysine (K) at positions 2, 60, 118, 176, and 234, histidine (H) at positions 3, 61 , 119, 177, and 235, aspartic acid (D) at positions 6, 64, 122, 180, and 238, glutamine (Q) at positions 7, 65, 123, 181 , and 239, aspartic acid (D) at positions 23, 81 , 139, 197, and 255, asparagine (N) at position 26, 84, 142, 200, and 258 is asparagine (N), and lysine (K) at position 56, 114, 172, 230.

[0137] Table 1 : Stability of SEQ ID NO: 1 after long term incubation in extreme alkaline solution Step pH elution: Step pH elution at pH 4.5, pH 4.0, and pH 3.7 with 0.1 M acetic acid for 15 CV at 1 CV / min followed by 10 CV 0.1 M phosphoric acid pH 1.7 (CIP) to elute hlgG (Load: 2.2 mg / mL Octagam, 5 min residence time) that was bound to the immobilized SEQ ID NO: 1. From the resin comprising SEQ ID NO: 1 , more than 97 % (as compared to 92 % to control sequence A and 94 % to control sequence B) of the antibody was eluted at pH at pH 4.0.

[0138] Furthermore, 79 % of the antibody was eluted at pH 4.5, as compared to 73 % and 56 % to control sequence A and control sequence B, respectively.

[0139] SEQUENCES SEQ ID NO: 1

[0140] AQFDKIAQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPPIAAQFDKI AQAAFYEILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPPIAAQFDKIAQAAFY EILHLPNLTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPPIAAQFDKIAQAAFYEILHLPN LTEEQRHAFIQSLRDDPSVSKEILAEAKKLNDAQAPPIAAQFDKIAQAAFYEILHLPNLTEEQR HAFIQSLRDDPSVSKEILAEAKKLNDAQAPK

Claims

CLAIMS1. Immunoglobulin (Ig) binding protein comprising the amino acid sequence of SEQ ID NO: 1 , or an Ig binding protein comprising an amino acid sequence with at least 96 % amino acid identity thereto, wherein the Ig binding protein has a remaining Ig binding activity, in particular IgG binding activity, of at least 90 % after incubation at 1 M NaOH for at least 37 h.

2. The Ig binding protein of claim 1 , wherein the Ig binding protein has a remaining Ig binding activity, in particular IgG binding activity, of at least 85 % after incubation at 1 M NaOH for at least 50 h.

3. The Ig binding protein of claim 1 comprising an amino acid sequence with at least 96 % amino acid identity to SEQ ID NO: 1 wherein the amino acid at the positions corresponding to positions 56, 114, 172, and 230 of SEQ ID NO: 1 is proline (P).

4. The Ig binding protein of any one of claims 1 to 3, wherein the amino acid at the positions corresponding to positions 3, 61 , 119, 177, and 235 of SEQ ID NO: 1 is phenylalanine (F), the amino acid corresponding to positions 6, 64, 122, 180, and 238 of SEQ ID NO: 1 is isoleucine (I), the amino acid corresponding to position 26, 84, 142, 200, and 258 of SEQ ID NO: 1 is histidine (H), and the amino acid corresponding to position 40, 98, 156, 214, and 272 of SEQ ID NO: 1 is lysine (K).

5. The Ig binding protein of any one of claims 1 to 4, wherein the amino acid at the position corresponding to position 288 of SEQ ID NO: 1 is lysine (K).

6. The Ig binding protein according to any one of claims 1 to 5, which is immobilized to a solid support.

7. The Ig binding protein of any one of claims 1 to 6, wherein the Ig is any one of IgGi, lgG2, lgG4, IgM , IgA, an Ig fragment, a fusion protein comprising an Ig or a fragment thereof, or a conjugate comprising an Ig.

8. An affinity separation matrix comprising the Ig binding protein of any one of claims 1 to 7 coupled thereto.

9. The affinity separation matrix of claim 8, which allows 97 % recovery of Ig bound to the Ig binding protein by elution at pH 4.0 from the affinity separation matrix.

10. Use of the Ig binding protein of any one of claims 1 to 7, or the affinity separation matrix of claim 8 or 9, for affinity purification of an Ig protein.

11. A method for affinity purifying an immunoglobulin (Ig), the method comprising: a) providing a sample, preferably a liquid sample, that contains an Ig; b) providing an affinity separation matrix according to claim 8 or 9; c) contacting said affinity separation matrix with the (liquid) sample under conditions that permit binding of the at least one Ig binding protein of the affinity separation matrix to an Ig; and d) collecting, preferably eluting, said Ig from said affinity purification matrix, thereby obtaining the (affinity purified) Ig, preferably obtaining an eluate containing said Ig, wherein in step (d) more than about 97 % of the Ig is eluted at pH 4.0 from the affinity separation matrix.

12. The method of claim 11 , comprising the additional step (e) of washing the affinity purification matrix with an alkaline cleaning liquid, optionally or preferably wherein the Ig binding protein retains at least about 90 % Ig binding activity after incubation for at least 37 h at 1 M NaOH, and / or at least about 85 % Ig binding activity after incubation for at least 50 h at 1 M NaOH.

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