Mutated immunoglobulin-binding polypeptides

Abstract

An Fc-binding polypeptide with improved alkaline stability comprising a mutant of the Fc-binding domain of Staphylococcus protein A (SpA), said mutant being defined by 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: 7, SEQ ID NO: 22, SEQ ID NO 51 or EQ ID NO 52, wherein at least the asparagine or serine residue at a position corresponding to position 11 in SEQ ID NO: 4-7 has been mutated to an amino acid selected from the group consisting of glutamic acid, lysine, tyrosine, threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine, valine, alanine, histidine and arginine.

Description

Technical Field

[0001] This invention relates to the field of affinity chromatography, and more particularly to a mutated immunoglobulin-binding domain of protein A, which can be used for affinity chromatography of immunoglobulins. The invention also relates to polymers of the mutated domain and to separation matrices containing the mutated domain or polymers. Background of the Invention

[0003] Immunoglobulins represent the most widely produced or developed biopharmaceutical products worldwide. The high commercial demand and inherent value of this specific therapeutic market have led pharmaceutical companies to focus on maximizing the productivity of their respective mAb production processes while controlling associated costs.

[0004] Affinity chromatography is used in most cases as a key step in the purification of immunoglobulin molecules, such as monoclonal or polyclonal antibodies. A class of affinity reagents of particular interest are proteins capable of specifically binding to invariant portions of immunoglobulin molecules, with such interactions independent of the antibody's antigen-binding specificity. Such reagents can be widely used to recover immunoglobulins from diverse samples (e.g., but not limited to, serum or plasma preparations or raw materials derived from cell cultures) via affinity chromatography. An example of such a protein is staphylococcal protein A, which contains Fc and Fab domains capable of binding to IgG immunoglobulins from different species. These domains are commonly referred to as E-, D-, A-, B-, and C-domains.

[0005] Staphylococcal protein A (SpA)-based reagents have found widespread use in the biotechnology field due to their high affinity and selectivity, such as affinity chromatography for antibody capture and purification, and for detection or quantification. Currently, SpA-based affinity media are likely the most widely used affinity media for separating monoclonal antibodies and their fragments from diverse samples, including industrial cell culture supernatants. Therefore, various matrices containing protein A-ligands are commercially available, for example, in the form of natural protein A (e.g., protein A SEPHAROSE™, GE Healthcare, Uppsala, Sweden), and also containing recombinant protein A (e.g., rProtein A SEPHAROSE™, GE Healthcare). More specifically, genetic manipulation performed in commercial recombinant protein A products aims to facilitate its attachment to the support and enhance ligand productivity.

[0006] These applications, like other affinity chromatography applications, require comprehensive consideration of the precise removal of contaminants. Such contaminants can be, for example, uneluted molecules adsorbed onto the stationary phase or matrix during the chromatographic process, such as undesirable biomolecules or microorganisms, including, for example, proteins, carbohydrates, lipids, bacteria, and viruses. Removal of such contaminants from the matrix typically occurs after the first elution of the desired product to regenerate the matrix before subsequent use. Such removal usually involves a procedure called in-situ cleaning (CIP), in which reagents capable of eluting contaminants from the stationary phase are used. One class of such reagents frequently used is an alkaline solution of the stationary phase. The most widely used cleaning and disinfecting agent is currently NaOH, and its concentration can range from 0.1 to, for example, 1 M, depending on the degree and nature of the contamination. This strategy involves exposing the matrix to a solution with a pH value above 13. For many affinity chromatography matrices containing protein affinity ligands, such an alkaline environment is a very harsh condition and subsequently leads to a decline in capability due to the instability of the ligand pairs at the high pH involved.

[0007] Therefore, extensive research has focused on the development of engineered protein ligands that exhibit improved tolerance to alkaline pH conditions. For example, Gülich et al. (Susanne Gülich, Martin Linhult, Per-Åke Nygren, Mathias Uhlén, Sophia Hober, Journal of Biotechnology 80 (2000), 169-178) proposed an engineered protein to improve the stability of the streptococcal albumin-binding domain (ABD) in alkaline environments. Gülich et al. created an ABD mutant in which all four asparagine residues were replaced by leucine (1 residue), aspartic acid (2 residues), and lysine (1 residue). Furthermore, Gülich et al. reported that their mutant exhibited target protein binding behavior similar to that of the native protein, and that affinity columns containing engineered ligands showed higher binding capacity after repeated exposure to alkaline conditions than columns prepared using non-engineered ligands from the parent protein. Therefore, it can be concluded that all four asparagine residues can be substituted without any significant effect on structure and function.

[0008] Recent work shows that protein A (SpA) can also be modified to achieve similar properties. US Patent Application Publication US 2005 / 0143566, which is incorporated herein by reference in its entirety, discloses that when at least one asparagine residue is mutated to an amino acid that is not glutamine or aspartic acid, the mutation confers increased chemical stability at pH values ​​of up to about 13-14 compared to the parental SpA, such as the B-domain of SpA, or protein Z (a synthetic construct derived from the B-domain of SpA) (US 5,143,844, incorporated herein by reference in its entirety). The authors show that when these mutated proteins are used as affinity ligands, the separation medium is as expected to be better resistant to cleaning procedures using alkaline reagents. To increase alkaline stability, additional mutations of the protein A domain have been disclosed in WO 2008 / 039141, JP2006304633A, EP 1992692A1, EP 2202310A2, WO 2010 / 110288, WO 2012 / 086660, WO 2012 / 083425, WO 2012 / 087230, and WO 2014 / 146350, all of which are incorporated herein by reference in their entirety. However, the currently available mutants remain sensitive to alkaline pH, and the NaOH concentration during cleaning is typically limited to 0.1 M, meaning complete cleaning is difficult to achieve. Higher NaOH concentrations (which would improve cleaning) result in unacceptable loss of ability.

[0009] Therefore, there remains a need in the art to obtain separation matrices containing protein ligands that have further improved stability for alkaline cleaning procedures. Invention Overview

[0011] One aspect of the invention is to provide a polypeptide with improved base stability. This is achieved using a polypeptide as defined in claim 1.

[0012] One advantage is that its alkaline stability is improved compared to the parent peptide, while maintaining highly selective binding to immunoglobulins and other Fc-containing proteins.

[0013] A second aspect of the invention is to provide a polymer with improved alkali stability, comprising a plurality of polypeptides. This is achieved using a polymer as defined in the claims.

[0014] A third aspect of the invention is to provide a nucleic acid or vector encoding a polypeptide or polymer having improved base stability. This is achieved using a nucleic acid or vector as defined in the claims.

[0015] A fourth aspect of the invention is to provide an expression system capable of expressing polypeptides or polymers with improved base stability. This is achieved using an expression system as defined in the claims.

[0016] A fifth aspect of the invention is to provide a separation matrix capable of selectively binding immunoglobulins and other Fc-containing proteins and exhibiting improved alkali stability. This is achieved using a separation matrix as defined in the claims.

[0017] A sixth aspect of the invention is to provide an efficient and economical method for isolating immunoglobulins or other Fc-containing proteins. This is achieved using the method as defined in the claims.

[0018] More suitable embodiments of the invention are described in the dependent claims.

[0019] definition

[0020] The terms “antibody” and “immunoglobulin” are used interchangeably in this document and are understood to also include fragments of antibodies, fusion proteins containing antibodies or antibody fragments, and conjugates containing antibodies or antibody fragments.

[0021] The terms “Fc-binding polypeptide” and “Fc-binding protein” refer to polypeptides or proteins that are capable of binding to the crystallizable portion (Fc) of an antibody and include, for example, protein A and protein G, or any fragment or fusion protein thereof that maintains the binding property.

[0022] The term "connector" in this article refers to the element that links two polypeptide units, monomers, or domains in a polymer to each other.

[0023] The term "spacer region" in this article refers to the element that connects a polypeptide or polypeptide multimer to a support. Brief description of the attached diagram

[0025] Figure 1 The comparison shows the Fc-binding domains as defined by SEQ ID NO:1-7 and 51-52.

[0026] Figure 2 The results from Example 2 show the alkaline stability of the parental and mutated tetramer Zvar (SEQ ID NO 7) polypeptide variant coupled to the SPR biosensor chip.

[0027] Figure 3 The results from Example 4 show the alkaline stability (0.5 M NaOH) of the parent and mutant tetramer Zvar (SEQ ID NO 7) polypeptide variant coupled to agarose beads.

[0028] Figure 4The results from Example 4 show the alkaline stability (1.0 M NaOH) of the parent and mutant tetramer Zvar (SEQ ID NO 7) polypeptide variant coupled to agarose beads.

[0029] Detailed description of the implementation plan

[0030] On one hand, this invention discloses an Fc-binding polypeptide comprising a mutant of the Fc-binding domain of staphylococcal protein A (SpA), or essentially composed of a mutant of the Fc-binding domain of staphylococcal protein A (SpA), said mutant being defined by, or having at least 90%, at least 95%, or at least 98% identity with, the following sequences: Figure 1 The following are described in SEQ ID NO: 1 (E-domain), SEQ ID NO: 2 (D-domain), SEQ ID NO: 3 (A-domain), SEQ ID NO: 22 (variant A-domain), SEQ ID NO: 4 (B-domain), SEQ ID NO: 5 (C-domain), SEQ ID NO: 6 (protein Z), SEQ ID NO: 7 (Zvar), SEQ ID NO 51 (Zvar without linker region amino acids 1-6) or SEQ ID NO 52 (C-domain without linker region amino acids 1-6), wherein at least the asparagine (or serine, in the case of SEQ ID NO 2) residue at position 11* corresponding to SEQ ID NO: 4-7 has been mutated to an amino acid selected from glutamic acid, lysine, tyrosine, threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine, valine, alanine, histidine and arginine. Protein Z (SEQ ID NO:6) is a mutant B-domain as disclosed in US5143844, while SEQ ID NO:7 represents another mutant variant of protein Z, referred to herein as Zvar, having mutations N3A, N6D, and N23T. SEQ ID NO:22 is a natural variant of the A-domain of protein A from Staphylococcus aureus strain N315 (having the A46S mutation, using...) Figure 1 (Position terminology). The N11 mutation in these domains confers improved base stability compared to the parental domain / peptide without impairing immunoglobulin-binding properties. Therefore, the peptide may also be described as an Fc- or immunoglobulin-binding peptide, or alternatively as an Fc- or immunoglobulin-binding peptide unit.

[0031] *Use throughout this instruction manual Figure 1The amino acid residue position numbering convention is used, and the position number is specified as corresponding to the number in SEQ ID NO 4-7.

[0032] In alternative languages, this invention discloses an Fc-binding polypeptide comprising a sequence as defined by SEQ ID NO 53, or having at least 90%, at least 95%, or at least 98% identity with SEQ ID NO 53.

[0033] SEQ ID NO 53

[0034] KEX1Q X2AFYEILX3LP NLTEEQRX4X5F IX6X7LKDX8PSX9 SX 10 X 11 X 12 LAEAKX 13 X 14 NDAQAPK

[0035] Among them, each is independent of the others:

[0036] X1 = A or Q

[0037] X2=E, K, Y, T, F, L, W, I, M, V, A, H or R

[0038] X3 = H or K

[0039] X4 = A or N

[0040] X5 = A or G

[0041] X6 = Q or E

[0042] X7 = S or K

[0043] X8 = E or D

[0044] X9 = Q or V

[0045] X 10 =K, R, or A

[0046] X 11 =A, E or N

[0047] X 12 =I or L

[0048] X 13 =K or R

[0049] X 14 =L or Y

[0050] The N11 (X2) mutation (such as the N11E or N11K mutation) may be a unique mutation, or the polypeptide may contain additional mutations, such as substitutions at at least one position corresponding to positions 3, 6, 9, 10, 15, 18, 23, 28, 29, 32, 33, 36, 37, 40, 42, 43, 44, 47, 50, 51, 55, and 57 of SEQ ID NO: 4-7. At one or more of these positions, the original amino acid residue may be substituted, for example, with an amino acid that is not asparagine, proline, or cysteine. The original amino acid residue may be substituted, for example, with alanine, valine, threonine, serine, lysine, glutamic acid, or aspartic acid. Furthermore, one or more amino acid residues may be deleted, for example, from positions 1-6 and / or from positions 56-58.

[0051] In some embodiments, the amino acid residue (X1) corresponding to position 9 in SEQ ID NO:4-7 is an amino acid that is not glutamine, asparagine, proline, or cysteine, such as alanine. The combination of mutations at positions 9 and 11 provides particularly good base stability, as shown by the examples. In a particular embodiment, in SEQ ID NO:7, the amino acid residue at position 9 is alanine and the amino acid residue at position 11 is lysine or glutamic acid, such as lysine. The mutation at position 9 is also discussed in the concurrently pending application PCT / SE2014 / 050872, which is incorporated herein by reference in its entirety.

[0052] In some embodiments, the amino acid residue (X) corresponding to position 50 in SEQ ID NO:4-7 13 It is arginine or glutamic acid.

[0053] In some embodiments, the amino acid residue at position 3 of SEQ ID NO:4-7 is alanine and / or the amino acid residue at position 6 of SEQ ID NO:4-7 is aspartic acid. One of the amino acid residues at positions 3 and 6 may be asparagine, while in alternative embodiments, both amino acid residues at positions 3 and 6 may be asparagine.

[0054] In some embodiments, the amino acid residue (X) corresponding to position 43 in SEQ ID NO:4-7 11 The amino acid residue at position 9 and 11 of SEQ ID NO: 7 is alanine or glutamic acid, for example, alanine. In a particular embodiment, the amino acid residues at position 9 and 11 of SEQ ID NO: 7 are alanine and lysine / glutamic acid, respectively, while the amino acid residue at position 43 is alanine or glutamic acid.

[0055] In some embodiments, the amino acid residue (X5) at position 28 corresponding to SEQ ID NO:4-7 is alanine or asparagine, such as alanine.

[0056] In some embodiments, the amino acid residue (X9) corresponding to position 40 in SEQ ID NO:4-7 is selected from asparagine, alanine, glutamic acid, and valine, or selected from glutamic acid and valine. In a particular embodiment, the amino acid residues at positions 9 and 11 of SEQ ID NO:7 are alanine and glutamic acid, respectively, while the amino acid residue at position 40 is valine. Optionally, the amino acid residue at position 43 may be either alanine or glutamic acid.

[0057] In some embodiments, the amino acid residue (X) corresponding to position 42 in SEQ ID NO:4-7 10 () is alanine, lysine, or arginine.

[0058] In some embodiments, the amino acid residue (X3) corresponding to position 18 in SEQ ID NO:4-7 is lysine or histidine, for example, lysine.

[0059] In some embodiments, the amino acid residue (X7) corresponding to position 33 in SEQ ID NO:4-7 is lysine or serine, for example, lysine.

[0060] In some embodiments, the amino acid residue (X8) corresponding to position 37 in SEQ ID NO:4-7 is glutamic acid or aspartic acid, such as glutamic acid.

[0061] In some embodiments, the amino acid residue (X) corresponding to position 51 in SEQ ID NO:4-7 14 ) is tyrosine or leucine, for example, tyrosine.

[0062] In some embodiments, the amino acid residue (X) corresponding to position 44 in SEQ ID NO:4-7 12 The amino acid residue at position 9 of SEQ ID NO: 7 is leucine or isoleucine. In a specific embodiment, the amino acid residues at positions 9 and 11 of SEQ ID NO: 7 are alanine and lysine / glutamic acid, respectively, while the amino acid residue at position 44 is isoleucine. Optionally, the amino acid residue at position 43 may be alanine or glutamic acid.

[0063] In some embodiments, amino acid residues at positions 1, 2, 3, and 4, or positions 3, 4, 5, and 6, corresponding to SEQ ID NO: 4-7, have been deleted. In certain variations of these embodiments, the parent polypeptide is the C domain of protein A (SEQ ID NO: 5). The effects of these deletions on the native C domain are described in US9018305 and US8329860, which are incorporated herein by reference in their entirety.

[0064] In some embodiments, the mutations in SEQ ID NO 4-7 (e.g., in SEQ ID NO 7) are selected from: N11K; N11E; N11Y; N11T; N11F; N11L; N11W; N11I; N11M; ​​N11V; N11A; N11H; N11R; N11E,Q32A; N11E,Q32E,Q40E; N11E,Q32E,K50R; Q9A,N11E,N43A; Q9A,N11E,N28A,N43A; Q9A,N11E,Q40V,A42K,N43E,L44I; Q9A,N11E,Q40V,A42K,N43A,L44I; N11K, H18K, S33K, D37E, A42R, N43A, L44I, K50R, L51Y; Q9A, N11E, N28A, Q40V, A4 2K, N43A, L44I; Q9A, N11K, H18K, S33K, D37E, A42R, N43A, L44I, K50R, L51Y; N11K, H18K, D37E, A42R, N43A, L44I; Q9A, N11K, H18K,D37E, A42R, N43A, L44I; Q9A, N11K, H18K, D37E, A42R, N43A, L44I, K50R; Q9A,N11K,H18K,D37E,A42R; Q9A,N11E,D37E,Q40V,A42K,N43A,L44I and Q9A,N11E,D37E,Q40V,A42R,N43A,L44I. These mutations provide exceptionally high base stability.Mutations in SEQ ID NO 4-7, such as those in SEQ ID NO 7, may also be selected from N11K; N11Y; N11F; N11L; N11W; N11I; N11M; ​​N11V; N11A; N11H; N11R; Q9A, N11E, N43A; Q9A, N11E, N28A, N43A; Q9A, N11E, Q40V, A42K, N43E, L44I; Q9A, N11E, Q40V, A42 K, N43A, L44I; Q9A, N11E, N28A, Q40V, A42K, N43A, L44I; N11K, H18K, S33K, D37E, A42R, N 43A, L44I, K50R, L51Y; Q9A, N11K, H18K, S33K, D37E, A42R, N43A, L44I, K50R, L51Y; N11K, H18K, D37E, A42R, N43A, L44I; Q9A, N11K, H18K, D37E, A42R, N43A, L44I and Q9A, N11K, H18K, D37E, A42R, N43A, L44I, K50R.

[0065] In some embodiments, the polypeptide comprises or is substantially composed of sequences selected from the following: SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41, SEQ ID NO 42, SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49 and SEQ ID NO 50. It may, for example, comprise or consist essentially of sequences selected from the following: SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 16, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, and SEQ ID NO 29. It may also comprise or consist essentially of sequences selected from the following: SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 16, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 38, SEQ ID NO 40; SEQ ID NO 41; SEQ ID NO 42; SEQ NO 43, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, and SEQ ID NO 48. The polypeptide may be defined, for example, by sequences selected from the above groups or subsets of these groups, but it may also contain additional N- and / or C-terminal amino acid residues, such as a leader sequence at the N-terminus and / or a tail sequence at the C-terminus.

[0066] SEQ ID NO 8 Zvar(Q9A,N11E,N43A)

[0067] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SAALLAEAKK LNDAQAPK

[0068] SEQ ID NO 9 Zvar(Q9A,N11E,N28A,N43A)

[0069] VDAKFDKEAQ EAFYEILHLP NLTEEQRAAF IQSLKDDPSQ SAALLAEAKK LNDAQAPK

[0070] SEQ ID NO 10 Zvar(Q9A,N11E,Q40V,A42K,N43E,L44I)

[0071] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKEILAEAKK LNDAQAPK

[0072] SEQ ID NO 11 Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)

[0073] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPK

[0074] SEQ ID NO 12 Zvar(N11E,Q32A)

[0075] VDAKFDKEQQ EAFYEILHLP NLTEEQRNAF IASLKDDPSQ SANLLAEAKK LNDAQAPK

[0076] SEQ ID NO 13 Zvar(N11E)

[0077] VDAKFDKEQQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPK

[0078] SEQ ID NO 14 Zvar(N11E,Q32E,Q40E)

[0079] VDAKFDKEQQ EAFYEILHLP NLTEEQRNAF IESLKDDPSE SANLLAEAKK LNDAQAPK

[0080] SEQ ID NO 15 Zvar(N11E,Q32E,K50R)

[0081] VDAKFDKEQQ EAFYEILHLP NLTEEQRNAF IESLKDDPSQ SANLLAEAKR LNDAQAPK

[0082] SEQ ID NO 16 Zvar(N11K)

[0083] VDAKFDKEQQ KAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPK

[0084] SEQ ID NO 23 Zvar(N11K,H18K,S33K,D37E,A42R,N43A,L44I,K50R,L51Y)

[0085] VDAKFDKEQQ KAFYEILKLP NLTEEQRNAF IQKLKDEPSQ SRAILAEAKR YNDAQAPK

[0086] SEQ ID NO 24 Zvar(Q9A,N11E,N28A,Q40V,A42K,N43A,L44I)

[0087] VDAKFDKEAQ EAFYEILHLP NLTEEQRAAF IQSLKDDPSV SKAILAEAKK LNDAQAPK

[0088] SEQ ID NO 25 Zvar(Q9A,N11K,H18K,S33K,D37E,A42R,N43A,L44I,K50R,L51Y)

[0089] VDAKFDKEAQ KAFYEILKLP NLTEEQRAAF IQKLKDEPSQ SRAILAEAKR YNDAQAPK

[0090] SEQ ID NO 26 Zvar(N11K, H18K, D37E, A42R, N43A, L44I)

[0091] VDAKFDKEQQ KAFYEILKLP NLTEEQRNAF IQSLKDEPSQ SRAILAEAKK LNDAQAPK

[0092] SEQ ID NO 27 Weld(Q9A, N11K, H18K, D37E, A42R, N43A, L44I)

[0093] VDAKFDKEAQ KAFYEILKLP NLTEEQRNAF IQSLKDEPSQ SRAILAEAKK LNDAQAPK

[0094] SEQ ID NO 28 Weld(Q9A, N11K, H18K, D37E, A42R, N43A, L44I, K50R)

[0095] VDAKFDKEAQ KAFYEILKLP NLTEEQRNAF IQSLKDEPSQ SRAILAEAKR LNDAQAPK

[0096] SEQ ID NO 29 Weld(Q9A,N11K,H18K,D37E,A42R)

[0097] VDAKFDKEAQ KAFYEILKLP NLTEEQRNAF IQSLKDEPSQ SRNLLAEAKK LNDAQAPK

[0098] SEQ ID NO 36 B(Q9A,N11E,Q40V,A42K,N43A,L44I)

[0099] ADNKFNKEAQ EAFYEILHLP NNLEEQRNGF IQSLKDDPSV SKAILAEAKK LNDAQAPK

[0100] SEQ ID NO: 37 C(Q9A,N11E,E43A)

[0101] ADNKFNKEAQ EAFYEILHLP NLTEEQRNGF IQSLKDDPSV SKAILAEAKK LNDAQAPK

[0102] SEQ ID NO 38 Weld(N11Y)

[0103] VDAKFDKEQQ YAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPK

[0104] SEQ ID NO 39 Zvar(N11T)

[0105] VDAKFDKEQQ TAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPK

[0106] SEQ ID NO 40 Zvar(N11F)

[0107] VDAKFDKEQQ FAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPK

[0108] SEQ ID NO 41 Zvar(N11L)

[0109] VDAKFDKEQQ LAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPK

[0110] SEQ ID NO 42 Zvar(N11W)

[0111] VDAKFDKEQQ WAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPK

[0112] SEQ ID NO 43 Zvar(N11I)

[0113] VDAKFDKEQQ IAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPK

[0114] SEQ ID NO 44 Zvar(N11M)

[0115] VDAKFDKEQQ MAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPK

[0116] SEQ ID NO 45 Zvar(N11V)

[0117] VDAKFDKEQQ VAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPK

[0118] SEQ ID NO 46 Zvar(N11A)

[0119] VDAKFDKEQQ AAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPK

[0120] SEQ ID NO 47 Zvar(N11H)

[0121] VDAKFDKEQQ HAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPK

[0122] SEQ ID NO 48 Zvar(N11R)

[0123] VDAKFDKEQQ RAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPK

[0124] SEQ ID NO 49 Zvar(Q9A,N11E,D37E,Q40V,A42K,N43A,L44I)

[0125] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDEPSV SKAILAEAKK LNDAQAPK

[0126] SEQ ID NO 50 Zvar(Q9A,N11E,D37E,Q40V,A42R,N43A,L44I)

[0127] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDEPSV SRAILAEAKK LNDAQAPK

[0128] In a second aspect, the present invention discloses a polymer comprising, or substantially composed of, a plurality of polypeptide units as defined in any of the embodiments disclosed above. The polymer can be, for example, a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, or nonamer. It can be a homopolymer, wherein all units in the polymer are identical, or it can be a heteropolymer, wherein at least one unit differs from the others. Advantageously, all units in the polymer, for example by incorporating the mutations disclosed above, are base-stable. The polypeptides can be directly linked to each other via peptide bonds between the C-terminus and N-terminus of the polypeptide. Alternatively, two or more units in the polymer can be linked by a linker comprising an oligomer or polymeric type, for example, comprising up to 15 or 30 amino acids, such as 1-5, 1-10, or 5-10 amino acids. This is particularly true for the mutations in SEQ ID NO 51 and 52 and for the polypeptide in SEQ ID NO 53, where specific examples of the linker can be, for example, VDAKFD or ADNKFN, such as VDAKFD. The properties of such a linker should preferably not disrupt the stability of the spatial conformation of the protein unit. This can be achieved, for example, by avoiding the presence of proline in the linker. Furthermore, the linker should preferably be sufficiently stable in an alkaline environment without impairing the properties of the mutant protein unit. For this purpose, it is advantageous if the linker is free of asparagine. It may be additionally advantageous if the linker is free of glutamine. The multimer may further include multiple amino acid residues at the N-terminus, which, for example, originate from the cloning process or constitute a signal transduction sequence from cleavage. The number of additional amino acid residues may be, for example, 15 or fewer, such as 10 or fewer, or 5 or fewer. As a specific example, the multimer may contain an AQ sequence at the N-terminus.

[0129] In some embodiments, the multimer may comprise, or consist substantially of, sequences selected from, the following sequences: SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 30, SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34, and SEQ ID NO 35. These sequences are listed below and referred to as parent (mutant) n, where n is the number of monomer units in the multimer.

[0130] SEQ ID NO 17 Zvar(Q9A,N11E,N43A)4

[0131] AQGT VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SAALLEAAK LNDAQAPKVDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SAALLEAAK LNDAQAPK VDAKFDKEAQEAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SAALLEAAK LNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SAALLEAKK LNDAQAPK

[0132] SEQ ID NO 18 Weld(Q9A,N11E,N28A,N43A)4

[0133] AQGT VDAKFDKEAQ EAFYEILHLP NLTEEQRAAF IQSLKDDPSQ SAALLEAAK LNDAQAPKVDAKFDKEAQ EAFYEILHLP NLTEEQRAAF IQSLKDDPSQ SAALLEAAK LNDAQAPK VDAKFDKEAQEAFYEILHLP NLTEEQRAAF IQSLKDDPSQ SAALLEAAK LNDAQAPK VDAKFDKEAQ EAFYEILHLPNLTEEQRAAF IQSLKDDPSQ SAALLEAKK LNDAQAPK

[0134] SEQ ID NO 19 Weld(Q9A,N11E,Q40V,A42K,N43E,L44I)4

[0135] AQGT VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKEILAEAKK LNDAQAPKVDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKEILAEAKK LNDAQAPK VDAKFDKEAQEAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKEILAEAKK LNDAQAPK VDAKFDKEAQ EAFYEILHLPNLTEEQRNAF IQSLKDDPSV SKEILAEAKK LNDAQAPK

[0136] SEQ ID NO 20 Weld(Q9A,N11E,Q40V,A42K,N43A,L44I)4

[0137] AQGT VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPKVDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPK VDAKFDKEAQEAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPK VDAKFDKEAQ EAFYEILHLPNLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPK

[0138] SEQ ID NO 30 Weld(N11K,H18K,S33K,D37E,A42R,N43A,L44I,K50R,L51Y)4

[0139] AQGT VDAKFDKEQQ KAFYEILKLP NLTEEQRNAF IQKLKDEPSQ SRAILAEAKR YNDAQAPKVDAKFDKEQQ KAFYEILKLP NLTEEQRNAF IQKLKDEPSQ SRAILAEAKR NDAQAPKVDAKFDKEQQ KAFYEILKLP NLTEEQRNAF IQKLKDEPSQ SRAILEAKR YNDAQAPKVDAKFDKEQQ KAFYEILKLP NLTEEQRNAF IQKLKDEPSQ SRAILEAKR YNDAQAPKC

[0140] SEQ ID NO 31 Weld(Q9A,N11K,H18K,D37E,A42R)4

[0141] AQGT VDAKFDKEAQ KAFYEILKLP NLTEEQRNAF IQSLKDEPSQ SRNLLAEAKKNLDAQAPK VDAKFDKEAQ KAFYEILKLP NLTEEQRNAF IQSLKDEPSQ SRNLLAEAKK LNDAQAPKVDAKFDKEAQ KAFYEILKLP NLTEEQRNAF IQSLKDEPSQ SRNLLAEAKK LNDAQAPKVDAKFDKEAQ KAFYEILKLP NLTEEQRNAF IQSLKDEPSQ SRNLLAEAKK LNDAQAPKC

[0142] SEQ ID NO 32 Weld(Q9A,N11E,N28A,Q40V,A42K,N43A,L44I)4

[0143] AQGT VDAKFDKEAQ EAFYEILHLP NLTEEQRAAF IQSLKDDPSV SKAILEAAKKLNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRAAF IQSLKDDPSV SKAILAEAKK LNDAQAPKVDAKFDKEAQ EAFYEILHLP NLTEEQRAAF IQSLKDDPSV SKAILEAKK LNDAQAPKVDAKFDKEAQ EAFYEILHLP NLTEEQRAAF IQSLKDDPSV SKAILEAKK LNDAQAPKC

[0144] SEQ ID NO 33 Weld(Q9A,N11E,Q40V,A42K,N43A,L44I)6

[0145] AQGT VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPKVDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPK VDAKFDKEAQEAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILEAKK LNDAQAPK VDAKFDKEAQ EAFYEILHLPNLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRNAFIQSLKDDPSV SKAILAEAKK LNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSVSKAILAEAKK LNDAQAPKC

[0146] SEQ ID NO 34 Weld(Q9A,N11E,D37E,Q40V,A42K,N43A,L44I)4

[0147] AQGT VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDEPSV SKAILEAAKKLNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDEPSV SKAILAEAKK LNDAQAPKVDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDEPSV SKAILEAKK LNDAQAPKVDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDEPSV SKAILEAKK LNDAQAPKC

[0148] SEQ ID NO 35 Weld(Q9A,N11E,D37E,Q40V,A42R,N43A,L44I)4

[0149] AQGT VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDEPSV SRAILAEAKKLNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDEPSV SRAILAEAKK LNDAQAPKVDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDEPSV SRAILAEAKK LNDAQAPKVDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDEPSV SRAILAEAKK LNDAQAPKC

[0150] In some embodiments, the polypeptides and / or polymers disclosed above further include one or more coupling elements at the C-terminus or N-terminus, selected from one or more cysteine ​​residues, multiple lysine residues, and multiple histidine residues. The coupling element may also be located within 1-5 amino acid residues, for example, 1-3 or 1-2 amino acid residues, away from the C-terminus or N-terminus. The coupling element may, for example, be a single cysteine ​​residue at the C-terminus. The coupling element may be directly attached to the C- or N-terminus, or it / they may be attached via a stretch consisting of up to 15 amino acids, for example 1-5, 1-10, or 5-10 amino acids. This stretch preferably should also be sufficiently stable in a basic environment to not impair the properties of the mutant protein. For this purpose, it is advantageous if the stretch does not contain asparagine. It may be additionally advantageous if the stretch does not contain glutamine. The advantage of having a C-terminal cysteine ​​is that the terminal coupling of the protein can be accomplished by the reaction of the cysteine ​​thiol with an electrophilic group on the support. This provides superior mobility for coupling proteins, for which binding capacity is important.

[0151] The alkaline stability of peptides or polymers can be evaluated by coupling them to an SPR chip, for example, using an NHS- or maleimide coupling chemistry to a Biacore CM5 sensor chip, as described in the examples, and before and after incubation in an alkaline solution at a specified temperature, such as 22 ± 2 °C, typically using the immunoglobulin-binding capacity of a polyclonal human IgG measurement chip. Incubation can be performed, for example, in 0.5 M NaOH for numerous 10-min cycles, such as 100, 200, or 300 cycles. After 100 cycles of incubation in 0.5 M NaOH at 22 ± 2 °C, the IgG capacity of the matrix can be at least 55%, for example, at least 60, at least 80, or at least 90% of the pre-incubation IgG capacity. Alternatively, the remaining IgG capacity of a specific mutant after 100 cycles, as measured above, can be compared to the remaining IgG capacity for the parent peptide / polymer. In this case, the remaining IgG capacity for the mutant can be at least 105%, such as at least 110%, at least 125%, at least 150%, or at least 200% of the parent peptide / polymer.

[0152] In a third aspect, the present invention discloses nucleic acids encoding polypeptides or polymers according to any of the embodiments disclosed above. Therefore, the present invention covers all forms of the nucleic acid sequence encoding polypeptides or polymers, such as RNA and DNA. The present invention covers vectors, such as plasmids, which, in addition to the coding sequence, contain a signal sequence required for expressing the polypeptide or polymer according to the present invention. In one embodiment, the vector contains a nucleic acid encoding a polymer according to the present invention, wherein the individual nucleic acids encoding the respective units may have homologous or heterologous DNA sequences.

[0153] In a fourth aspect, the present invention discloses an expression system comprising the nucleic acid or vector disclosed above. The expression system can be, for example, a Gram-positive or Gram-negative prokaryotic host cell system, such as *Escherichia coli* (E. coli). E. coli ) or Bacillus ( Bacillus sp The expression system has been modified to express the polypeptide or polymer of the present invention. In alternative embodiments, the expression system is a eukaryotic host cell system, such as yeast, for example, Pichia pastoris (…). Pichia pastoris ) or brewer's yeast ( Saccharomyces cerevisiae ), or mammalian cells, such as CHO cells.

[0154] In a fifth aspect, the present invention discloses a separation matrix in which multiple polypeptides or polymers according to any of the embodiments disclosed above have been coupled to a solid support. Such a matrix can be used to separate immunoglobulins or other Fc-containing proteins, and due to the improved alkaline stability of the polypeptides / polymers, the matrix will withstand highly alkaline conditions during cleaning, which is crucial for long-term repeated use in biological treatment separation devices. The alkaline stability of the matrix can be evaluated by measuring the immunoglobulin-binding capacity using polyclonal human IgG, typically before and after incubation in an alkaline solution at a specific temperature, for example, 22 ± 2 °C. Incubation can be performed, for example, in 0.5 M or 1.0 M NaOH for a certain number of 15-min cycles, such as 100, 200, or 300 cycles, corresponding to a total incubation time of 25, 50, or 75 h. After incubation at 22 ± 2°C in 0.5 M NaOH for 96-100 cycles of 15 min each, or a total incubation time of 24 or 25 h, the IgG capacity of the matrix can be at least 80%, for example, at least 85%, at least 90%, or at least 95% of the pre-incubation IgG capacity. After incubation at 22 ± 2°C in 1.0 M NaOH for a total incubation time of 24 h, the capacity of the matrix can be at least 70%, for example, at least 80%, or at least 90% of the pre-incubation IgG capacity.

[0155] As those skilled in the art should understand, expressed peptides or polymers should be purified to an appropriate degree before being immobilized onto a support. Such purification methods are well known in the art, and the immobilization of protein-based ligands onto a support is readily performed using standard methods. Suitable methods and supports will be discussed in more detail below.

[0156] The solid support for the matrix according to the invention can be any suitable and well-known type. Conventional affinity separation matrices typically have organic properties and are based on polymers with hydrophilic surfaces exposed to the aqueous medium used, i.e., hydroxyl (-OH), carboxyl (-COOH), formamide (-CONH2, possibly in N-substituted form), amino (-NH2, possibly in substituted form), oligo- or polyoxyethylene groups exposed on their outer surfaces and (if present) also on their inner surfaces. The solid support can be suitable to be porous. Porosity can be expressed as a Kav or Kd value (the fraction of pore volume obtainable by a probe molecule of a specific size), which is measured by reverse size exclusion chromatography, for example according to the method described in Gel Filtration Principles and Methods, Pharmacia LKB Biotechnology 1991, pp 6-13. By definition, both Kd and Kav values ​​are always in the range of 0-1. The Kav value can advantageously be 0.6–0.95, for example 0.7–0.90 or 0.6–0.8, as measured using a 110 kDa dextran as a probe molecule. Its advantage is that the support has a large fraction of pores capable of accommodating the polypeptides / multimers of the invention and the immunoglobulins bound to the polypeptides / multimers, and providing mass transport of immunoglobulins toward and away from the binding site.

[0157] Peptides or polymers can be attached to a support via conventional coupling techniques, utilizing groups such as thiol, amino, and / or carboxyl groups present in the ligand. Bisepoxides, epichlorohydrin, CNBr, and N-hydroxysuccinimide (NHS) are well-known coupling agents. A molecule called a spacer region can be introduced between the support and the peptide / polymer, which improves the availability of the peptide / polymer and promotes its chemical coupling to the support. Depending on the nature of the peptide / polymer and the coupling conditions, coupling can be multi-site coupling (e.g., via multiple lysine residues) or single-site coupling (e.g., via a single cysteine ​​residue). Alternatively, peptides / polymers can be attached to the support via non-covalent bonding, such as physical adsorption or biospecific adsorption.

[0158] In some embodiments, the matrix comprises 5-25, for example 5-20 mg / ml, 5-15 mg / ml, 5-11 mg / ml, or 6-11 mg / ml, of a peptide or polymer coupled to the support. The amount of coupled peptide / polymer can be controlled by the concentration of the peptide / polymer used during coupling, by the activation and coupling conditions used, and / or by the pore structure of the support used. As a general rule, the absolute binding capacity of the matrix increases with the amount of coupled peptide / polymer until at least a point where the pores become significantly restricted by the coupled peptide / polymer. The relative binding capacity per mg of coupled peptide / polymer will decrease at high coupling levels, resulting in optimal cost-effectiveness within the range specified above.

[0159] In some embodiments, the peptide or polymer is coupled to the support via a thioether bond. Methods for performing such coupling are well known in the art and readily performed by those skilled in the art using standard techniques and equipment. Thioether bonds are flexible and stable and are generally suitable for affinity chromatography. In particular, when the thioether bond is via a terminal or near-terminal cysteine ​​residue on the peptide or polymer, the mobility of the coupled peptide / polymer increases, providing improved binding capacity and binding kinetics. In some embodiments, the peptide / polymer is coupled via a C-terminal cysteine ​​residue provided on the protein as described above. This allows the cysteine ​​thiol to be efficiently coupled to electrophilic groups on the support, such as epoxide groups, haloalcohol groups, etc., resulting in thioether-bridged coupling.

[0160] In some embodiments, the support comprises a polyhydroxy polymer, such as a polysaccharide. Examples of polysaccharides include, for example, dextran, starch, cellulose, amylopectin, agar, agarose, etc. Polysaccharides are inherently hydrophilic, have a low degree of nonspecific interactions, provide a high content of reactive (activatable) hydroxyl groups, and are generally stable to alkaline cleaning solutions used for biological treatment.

[0161] In some embodiments, the support comprises agar or agarose. The support used in this invention can be readily prepared according to standard methods, such as inverse suspension gelation (SHjertén: BiochimBiophys Acta 79(2), 393-398 (1964)). Alternatively, the alkaline matrix is ​​a commercially available product, such as cross-linked agarose beads commercially available under the trade name SEPHAROSE™ FF (GE Healthcare). In embodiments particularly advantageous for large-scale separation, the support is modified to increase its rigidity using the methods described in US6602990 or US7396467 (incorporated herein by reference in its entirety).

[0162] In some embodiments, the support, such as a polysaccharide or agarose support, is cross-linked, for example, with hydroxyalkyl ethers. The cross-linking agent that produces such cross-links can be, for example, an epihaloalcohol like epichlorohydrin, a diepoxide like butanediol diglycidyl ether, or an alkylating agent like an allyl halide or allyl glycidyl ether. Cross-linking is beneficial for the rigidity of the support and improves chemical stability. Hydroxyalkyl ether cross-linking is base-stable and does not cause significant nonspecific adsorption.

[0163] Alternatively, the solid support is based on a synthetic polymer, such as polyvinyl alcohol, polyhydroxyalkyl acrylate, polyhydroxyalkyl methacrylate, polyacrylamide, polymethacrylamide, etc. In the case of hydrophobic polymers, such as those based on divinyl and monovinyl-substituted benzene matrices, the surface of the matrix is ​​often hydrophilized to expose the hydrophilic groups as defined above to the surrounding aqueous liquid. Such polymers are readily produced according to standard methods, see, for example, "Styrene based polymer supports developed by suspension polymerization" (R Arshady: Chimicae L'Industria 70(9), 70-75 (1988)). Alternatively, commercially available products such as SOURCE™ (GE Healthcare) can be used. In another alternative, the solid support according to the invention comprises an inorganic support, such as silica, zirconium oxide, etc.

[0164] In yet another embodiment, the solid support takes the form of another material, such as a surface, a chip, a capillary, or a filter (e.g., a membrane or a deep filtration matrix).

[0165] Regarding the shape of the matrix according to the invention, in one embodiment, the matrix is ​​presented as a porous, monolithic material. In alternative embodiments, the matrix is ​​presented as beads or particles, which may be porous or non-porous. Matrix presented as beads or particles can be used as a packed bed or in suspension. Suspension forms include those referred to as expanded beds and pure suspensions, in which the particles or beads move freely. In the cases of monolithic, packed, and expanded beds, the separation procedure typically follows conventional chromatography with a concentration gradient. In the case of pure suspensions, a batch mode is used.

[0166] In a sixth aspect, the present invention discloses a method for isolating immunoglobulins, wherein the isolation matrix as disclosed above is used.

[0167] In some implementations, the method includes the following steps:

[0168] a) Contact the liquid sample containing immunoglobulins with the separation matrix as disclosed above.

[0169] b) Wash the separation matrix with a washing liquid.

[0170] c) Elute immunoglobulins from the separation matrix with elution buffer, and

[0171] d) Clean the separation matrix with a cleaning liquid, which may alternatively be called an in-situ cleaning (CIP) liquid, for example, by contacting (incubating) for at least 10 minutes.

[0172] The method may further include the following steps: prior to step a), providing an affinity separation matrix according to any embodiment described above and providing a solution containing immunoglobulins and at least one other substance as a liquid sample; and after step c), recovering the eluent and optionally subjecting the eluent to further separation steps, such as by anion or cation exchange chromatography, multi-peak chromatography, and / or hydrophobic interaction chromatography. Suitable compositions of the liquid sample, washing liquid, and eluent, as well as general conditions for performing the separation, are well known in the field of affinity chromatography, and particularly in the field of protein A chromatography. The liquid sample containing Fc-containing proteins and at least one other substance may contain host cell proteins (HCPs), such as CHO cell, E. coli, or yeast proteins. The content of CHO cell and E. coli proteins can be conveniently determined by immunoassays against these proteins, such as the CHO HCP or E. coli HCP ELISA kit from Cygnus Technologies. The host cell protein or CHO cell / E. coli protein may be desorbed during step b).

[0173] Elution can be performed using any suitable solution for eluting from protein A media. This can be, for example, a solution or buffer having a pH of 5 or lower, such as pH 2.5-5 or 3-5. In some cases, it can also be a solution or buffer having a pH of 11 or higher, such as pH 11-14 or pH 11-13. In some embodiments, the elution buffer or elution buffer gradient contains at least one mono-, di-, or trifunctional carboxylic acid or a salt of such a carboxylic acid. In some embodiments, the elution buffer or elution buffer gradient contains at least one anionic species selected from acetate, citrate, glycine, succinate, phosphate, and formate.

[0174] In some implementations, the cleaning liquid is alkaline, for example, having a pH of 13-14. Such a solution provides effective cleaning of the matrix, especially at the upper end of the zone.

[0175] In some embodiments, the cleaning liquid contains 0.1-2.0 M NaOH or KOH, for example 0.5-2.0 or 0.5-1.0 M NaOH or KOH. These are effective cleaning solutions, especially when the NaOH or KOH concentration is above 0.1 M or at least 0.5 M. The high stability of the peptides of the present invention makes the use of such strongly alkaline solutions possible.

[0176] The method may also include the step of disinfecting the matrix with a disinfectant liquid, which may, for example, contain peroxides, such as hydrogen peroxide, and / or peracids, such as peracetic acid or performic acid.

[0177] In some implementations, steps a)-d) are repeated at least 10 times, for example at least 50 times, 50-200 times, 50-300 times, or 50-500 times. This is important for process economy because the substrate can be reused multiple times.

[0178] Steps a)-c) may also be repeated at least 10 times, for example at least 50 times, 50-200 times, 50-300 times, or 50-500 times, and step d) may be performed after multiple steps c), such that step d) is performed at least 10 times, for example at least 50 times. Step d) may be performed, for example, in the case of every 2nd to 20th step c). Example

[0179] Protein mutagenesis

[0180] Site-directed mutagenesis was performed via two-step PCR using oligonucleotides encoding the mutation. A plasmid containing a single Z, B, or C domain was used as a template. The PCR fragment was ligated into an *E. coli* expression vector. DNA sequencing was used to verify the correct sequence of the inserted fragment.

[0181] To form the mutant multimer, the Acc I site, located at the start codon (GTA GAC) in the B, C, or Z domain, corresponding to amino acid VD, is used. The vector for the monomeric domain is digested with Acc I and phosphatase. Sticky-end primers specific to each variant are designed for Acc I, and two overlapping PCR products are generated from each template. The PCR products are purified and their concentrations estimated by comparing them on a 2% agarose gel. Equal volumes of paired PCR products are hybridized in ligation buffer (90°C -> 25°C, 45 min). The resulting product composition may be approximately ¼ of the fragment ligated to the Acc I site (the correct PCR fragment and / or the digested vector). After ligation and transformation, colonies are screened by PCR to identify constructs containing the desired mutant. Positive clones are validated by DNA sequencing.

[0182] Construct expression and purification

[0183] The construct was expressed in the periplasm of bacteria via fermentation of *E. coli* K12 in standard medium. Following fermentation, the cells were heat-treated to release the periplasmic contents into the medium. The constructed construct released into the medium was recovered by microfiltration using a membrane with 0.2 µm pores.

[0184] Each construct is now purified by affinity in the permeate from the filtration step. The permeate is loaded into a chromatographic medium containing immobilized IgG (IgG Sepharose 6FF, GE Healthcare). The loaded product is washed with phosphate-buffered saline and eluted by lowering the pH.

[0185] The elution cell was adjusted to neutral pH (pH 8) and reduced by the addition of dithiothreitol. The sample was then loaded into an anion exchanger. Following the washing step, the construct was eluted in a NaCl gradient to separate it from any contaminants. The elution cell was concentrated to 40–50 mg / ml by ultrafiltration. It should be noted that the successful affinity purification of the construct on the immobilized IgG medium indicates that the construct involved has a high affinity for IgG.

[0186] The purified ligands were analyzed by RPC LC-MS to determine purity and molecular weight corresponding to the expected value (based on amino acid sequence).

[0187] Example 1

[0188] The purified monomeric ligands listed in Table 1 (also containing an N-terminal AQGT leader sequence and a C-terminal cysteine) were immobilized on a Biacore CM5 sensor chip (GE Healthcare, Sweden) using an amine coupling kit from GE Healthcare (for carbodiimide coupling of amines on carboxymethyl groups on the chip) in an amount sufficient to give a signal intensity of approximately 200–1500 RU in a Biacore surface plasmon resonance (SPR) instrument (GE Healthcare, Sweden). To track IgG binding capacity on the immobilized surface, 1 mg / ml of human polyclonal IgG (Gammanorm) was passed through the chip and the signal intensity (proportional to the amount bound) was recorded. The surface was then cleaned in situ (CIP), i.e., rinsed with 500 mM NaOH for 10 min at room temperature (22 ± 2 °C). This was repeated for 96–100 cycles, and the base stability of the immobilized ligands was tracked at the end of each cycle based on the remaining IgG binding capacity (signal intensity).The results are shown in Table 1 and indicate that at least the ligands Zvar(N11K)1, Zvar(N11E)1, Zvar(N11Y)1, Zvar(N11T)1, Zvar(N11F)1, Zvar(N 11L)1, Zvar(N11W)1, ZN11I)1, Zvar(N11M)1, Zvar(N11V)1, Zvar(N11A)1, Zvar(N11H1), Zvar(N1 1R)1, Zvar(N11E,Q32A)1, Zvar(N11E,Q32E,Q40E)1 and Zvar(N11E,Q32E,K50R)1, Zvar(Q9A,N11E,N 43A)1, Zvar(Q9A,N11E,N28A,N43A)1, Zvar(Q9A,N11E,Q40V,A42K,N43E,L44I)1, Zvar(Q9A,N11E ,Q40V,A42K,N43A,L44I)1,Zvar(Q9A,N11E,N28A,Q40V,A42K,N43A,L44I)1,Zvar(N11K,H18K,S3 3K,D37E,A42R,N43A,L44I,K50R,L51Y)1, Zvar(Q9A,N11K,H18K,S33K,D37E,A42R,N43A,L44I,K5 Zvar(Q9A,N11K,H18K,D37E,A42R,N43A,L44I)1, Zvar(Q9A,N11K,H18K,D37E,A42R,N43A,L44I)1, and Zvar(Q9A,N11K,H18K,D37E,A42R,N43A,L44I,K50R)1 exhibit improved base stability compared to the parental structure Zvar1 (used as a reference). Furthermore, ligands B(Q9A,N11E,Q40V,A42K,N43A,L44I)1 and C(Q9A,N11E,E43A)1 exhibit improved stability compared to the parental B and C domains used as a reference.

[0189] Table 1. Monomer ligands evaluated using Biacore (0.5 M NaOH).

[0190]

[0191]

[0192] Example 2

[0193] The purified tetrameric and hexameric ligands listed in Table 2 were immobilized on a Biacore CM5 sensor chip (GE Healthcare, Sweden) in an amount sufficient to produce a signal intensity of approximately 200–1500 RU in a Biacore instrument (GE Healthcare, Sweden) using a GE Healthcare amine coupling kit (for carbodiimide coupling of amines on carboxymethyl groups on a chip). To track IgG binding capacity on the immobilized surface, 1 mg / ml of human polyclonal IgG (Gammanorm) was passed through the chip and the signal intensity (proportional to the amount bound) was recorded. The surface was then cleaned in situ (CIP), i.e., rinsed with 500 mM NaOH for 10 min at room temperature (22 ± 2 °C). 300 cycles were repeated, and the base stability of the immobilized ligands was tracked at the end of each cycle based on the remaining IgG binding capacity (signal intensity). The results are shown in Table 2 and Figure 2 The results also show that at least the ligands Zvar(Q9A,N11E,N43A)4, Zvar(Q9A,N11E,N28A,N43A)4, Zvar(Q9A,N11E,Q40V,A42K,N43E,L44I)4, Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)4, Zvar(Q9A,N11E,D37E,Q40V,A42K,N43A,L44I)4, and Zvar(Q9A,N11E,D37E,Q40V,A42R,N43A,L44I)4 have improved base stability compared to the parental structure Zvar4 (which is used as a reference). The hexameric ligand Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)6 also exhibits improved base stability compared to the parental structure Zvar6 used as a reference.

[0194] Table 2. Tetrameric and hexameric ligands evaluated using Biacore (0.5M NaOH)

[0195]

[0196] Example 3

[0197] Example 2 was repeated with 100 CIP cycles using 1 M NaOH instead of the 500 mM used in Example 2. The results are shown in Table 3 and indicate that all three ligands also exhibit improved base stability in 1 M NaOH compared to the parental structure Zvar4 used as a reference.

[0198] Table 3. Tetrameric ligands evaluated using Biacore (1M NaOH).

[0199]

[0200] Example 4

[0201] The purified tetrameric ligands in Table 2 (all with an additional N-terminal cysteine) were immobilized on agarose beads using the method described below, and their potency and stability were evaluated. The results are shown in Table 4 and... Figure 3 .

[0202] Table 4. Matrix with tetrameric ligands evaluated in column (0.5 M NaOH).

[0203]

[0204] activation

[0205] The base matrix used was rigid cross-linked agarose beads with a median diameter of 85 micrometers (volume-weighted, d50V) prepared according to the method in US6602990, and with a pore size corresponding to a Kav value of 0.70 for dextran with a Mw of 110 kDa, prepared according to the method described in Gel Filtration Principles and Methods, Pharmacia LKB Biotechnology 1991, pp 6-13.

[0206] At 25°C, 25 mL (g) of drained base matrix, 10.0 mL of distilled water, and 2.02 g of NaOH were mixed in a 100 mL flask equipped with a mechanical stirrer for 10 min. 4.0 mL of epichlorohydrin was added, and the reaction proceeded for 2 h. The activated gel was washed with 10 gel settling volumes (GV) of water.

[0207] Coupling

[0208] To a 20 mL ligand solution (50 mg / mL) in a 50 mL Falcon tube, add 169 mg NaHCO3, 21 mg Na2CO3, 175 mg NaCl, and 7 mg EDTA. Place the Falcon tube on a roller shaker for 5–10 min, then add 77 mg DTE. Reduction proceeds for >45 min. The ligand solution is then desalted on a PD10 column packed with Sephadex G-25. The ligand content in the desalted solution is determined by measuring the 276 nm UV absorbance.

[0209] The activated gel was washed with 3–5 GV {0.1 M phosphate / 1 mM EDTA pH 8.6} and then ligands were coupled according to the method described in US6399750. All buffers used in the experiments were degassed with nitrogen for at least 5–10 min. The ligand content of the gel can be controlled by varying the volume and concentration of the ligand solution.

[0210] After fixation, the gel was washed 3xGV with distilled water. The mixed gel was then incubated overnight at room temperature with 1 GV {0.1 M phosphate / 1 mM EDTA / 10% thioglycerol pH 8.6} on a shaking table. The gel was then washed alternately with 3xGV {0.1 M TRIS / 0.15 M NaCl pH 8.6} and 0.5 M HAc, followed by washing with 8–10xGV distilled water. Gel samples were sent to an external laboratory for amino acid analysis, and the ligand content (mg / ml gel) was calculated from the total amino acid content.

[0211] protein

[0212] Dilute Gammanorm 165 mg / ml (Octapharma) to 2 mg / ml in equilibration buffer.

[0213] Balanced buffer

[0214] PBS phosphate buffer 10 mM + 0.14 M NaCl + 0.0027 M KCl, pH 7.4 (Medicago)

[0215] Adsorption buffer

[0216] PBS phosphate buffer 10 mM + 0.14 M NaCl + 0.0027 M KCl, pH 7.4 (Medicago)

[0217] Elution buffer

[0218] 100 mM acetate pH 2.9

[0219] Dynamic integration capability

[0220] Pack 2 ml of resin into a TRICORN™ 5 100 column. Penetration was determined using an ÄKTA Explorer 10 system with a residence time of 6 minutes (0.33 ml / min flow rate). Pass equilibration buffer through the bypass column until a stable baseline is obtained. This is performed before automatic zeroing. Apply the sample to the column until 100% UV signal is obtained. Then, apply equilibration buffer again until a stable baseline is obtained.

[0221] Load the sample onto the column until a UV signal of 85% of maximum absorbance is reached. Then wash the column with 5 column volumes (CV) of equilibration buffer at a flow rate of 0.5 ml / min. Elute the protein with 5 CV of elution buffer at a flow rate of 0.5 ml / min. Then clean the column with 0.5 M NaOH at a flow rate of 0.2 ml / min and reequilibrate with equilibration buffer.

[0222] To calculate the penetration capacity at 10%, the following equation is used. That is, the amount of IgG loaded into the column until the column effluent concentration is 10% of the feed IgG concentration.

[0223]

[0224] A 100% = 100% UV signal;

[0225] A sub = Contribution of absorbance from non-binding IgG subclasses;

[0226] A(V) = Absorbance in a given application volume;

[0227] V c = Column volume;

[0228] V app = Up to 10% of the application volume penetrated;

[0229] V sys = System dead volume;

[0230] C0 = Feed concentration.

[0231] Calculate the dynamic bonding capacity (DBC) at 10% penetration. Calculate the dynamic bonding capacity (DBC) for 10% and 80% penetration.

[0232] CIP - 0.5 M NaOH

[0233] 10% breakthrough DBC (Qb10) was determined before and after repeated exposure to an alkaline cleaning solution. Each cycle consisted of a CIP step with 0.5 M NaOH pumped through the column at a rate of 0.5 ml / min for 20 min, followed by column incubation for 4 h. Exposure occurred at room temperature (22 ± 2 °C). After incubation, the column was washed with equilibration buffer at a flow rate of 0.5 ml / min for 20 min. Table 4 shows the residual capacity after six 4-h cycles (i.e., the cumulative 24-h exposure to 0.5 M NaOH), expressed in absolute and relative values ​​to the initial capacity.

[0234] Example 5

[0235] Example 4 was repeated using the tetrameric ligands shown in Table 5, but instead of 0.5 M, 1.0 M NaOH was used for the CIP step. The results are shown in Table 5 and Figure 4 middle.

[0236] Table 5. Evaluation of matrices with tetrameric ligands in column-1.0 M NaOH

[0237]

[0238] This written specification uses examples to disclose the invention, including the best mode, and enables any person skilled in the art to practice the invention, including making and using any device or system and performing any combined methods. The patentability of the invention is defined by the claims and may include other embodiments conceivable to those skilled in the art. Such other embodiments are intended to fall within the scope of the claims, provided that they have the same structural elements as the verbal language of the claims, or that they include equivalent structural elements that are not substantially different from the verbal language of the claims. All patents and patent applications mentioned herein are incorporated herein by reference in their entirety as if they were individually incorporated herein.

Claims

1. An Fc-binding polypeptide, wherein the amino acid sequence of said polypeptide is defined by any one of SEQ ID NO. 8-11, 24-25 and 27-28.

2. The polypeptide of claim 1, wherein the amino acid sequence of the polypeptide is defined by any one of SEQ ID NO. 8-11 and 27-28.

3. A polymer comprising or composed of a plurality of polypeptides as defined in any one of the preceding claims.

4. The polymer of claim 3, wherein the polypeptide is linked by a linker comprising up to 15 amino acids.

5. The polymer of claim 3 or 4, further comprising one or more coupling elements at the C-terminus of the polymer, or one or more coupling elements located 1-5 amino acid residues away from the C-terminus of the polymer, said coupling element being selected from one or more cysteine ​​residues, a plurality of lysine residues, and a plurality of histidine residues.

6. A separation matrix comprising a plurality of polypeptides of claim 1 or 2 or a plurality of polymers of any one of claims 3-5 and a solid support, wherein the plurality of polypeptides or polymers are coupled to the solid support.

7. The separation matrix of claim 6, wherein the polypeptide or polymer is coupled to the solid support via a thioether bond.

8. The separation matrix of claim 6 or 7, wherein the solid support is a polysaccharide.

9. The separation matrix of claim 6 or 7, wherein the IgG-binding capacity of the matrix after incubation at 22 + / - 2°C in 0.5 M NaOH for 24 hours is at least 80% of the IgG-binding capacity before incubation.

10. The separation matrix of claim 6 or 7, wherein the IgG-binding capacity of the matrix after incubation at 22 + / - 2°C in 1.0 M NaOH for 24 hours is at least 70% of the IgG-binding capacity before incubation.

11. A method for isolating immunoglobulins, wherein the method includes the step of using an isolation matrix according to any one of claims 6-10.

12. A method for isolating immunoglobulins, comprising the following steps: a) Contacting a liquid sample containing immunoglobulins with the separation matrix of any one of claims 6-10, b) Wash the separation matrix with a washing liquid. c) Elute immunoglobulins from the separation matrix with elution buffer, and d) Clean the separation matrix with an alkaline cleaning liquid.

13. The method of claim 12, wherein the cleaning liquid comprises 0.1-1.0 M NaOH or KOH.

14. The method of claim 13, wherein the cleaning liquid contains at least 0.5 M NaOH or KOH.

15. The method of any one of claims 12-14, wherein steps a)-d) are repeated at least 10 times.

16. The method of any one of claims 12-14, wherein steps a)-c) are repeated at least 10 times and wherein step d) is performed after a plurality of steps c).

17. The method of any one of claims 12-14, wherein the liquid sample further comprises host cell proteins.

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