(19)
(11)EP 3 455 243 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
24.03.2021 Bulletin 2021/12

(21)Application number: 17727808.2

(22)Date of filing:  10.05.2017
(51)Int. Cl.: 
C07K 14/31  (2006.01)
(86)International application number:
PCT/EP2017/061164
(87)International publication number:
WO 2017/194597 (16.11.2017 Gazette  2017/46)

(54)

SEPARATION MATRIX

TRENNMATRIX

MATRICE DE SÉPARATION


(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

(30)Priority: 11.05.2016 GB 201608229
11.05.2016 GB 201608232
30.09.2016 US 201615282367

(43)Date of publication of application:
20.03.2019 Bulletin 2019/12

(73)Proprietor: Cytiva BioProcess R&D AB
751 84 Uppsala (SE)

(72)Inventors:
  • RODRIGO, Gustav, José
    751 84 Uppsala (SE)
  • BJORKMAN, Tomas
    751 84 Uppsala (SE)
  • ANDER, Mats
    75184 Uppsala (SE)
  • HANSSON, Jesper, Ulf
    751 84 Uppsala (SE)

(74)Representative: Larsson, Jan Anders 
GE Healthcare Bio-Sciences AB Björkgatan 30
751 84 Uppsala
751 84 Uppsala (SE)


(56)References cited: : 
WO-A1-2012/083425
WO-A1-2016/079033
WO-A1-2015/005859
  
      
    Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


    Description

    Technical field of the invention



    [0001] The present invention relates to the field of affinity chromatography, and more specifically to mutated immunoglobulin-binding domains of Protein A, which are useful in affinity chromatography of immunoglobulins. The invention also relates to multimers of the mutated domains and to separation matrices containing the mutated domains or multimers.

    Background of the invention



    [0002] Immunoglobulins represent the most prevalent biopharmaceutical products in either manufacture or development worldwide. The high commercial demand for and hence value of this particular therapeutic market has led to the emphasis being placed on pharmaceutical companies to maximize the productivity of their respective mAb manufacturing processes whilst controlling the associated costs.

    [0003] Affinity chromatography is used in most cases, as one of the key steps in the purification of these immunoglobulin molecules, such as monoclonal or polyclonal antibodies. A particularly interesting class of affinity reagents is proteins capable of specific binding to invariable parts of an immunoglobulin molecule, such interaction being independent on the antigen-binding specificity of the antibody. Such reagents can be widely used for affinity chromatography recovery of immunoglobulins from different samples such as but not limited to serum or plasma preparations or cell culture derived feed stocks. An example of such a protein is staphylococcal protein A, containing domains capable of binding to the Fc and Fab portions of IgG immunoglobulins from different species. These domains are commonly denoted as the E-, D-, A-, B- and C-domains.

    [0004] Staphylococcal protein A (SpA) based reagents have due to their high affinity and selectivity found a widespread use in the field of biotechnology, e.g. in affinity chromatography for capture and purification of antibodies as well as for detection or quantification. At present, SpA-based affinity medium probably is the most widely used affinity medium for isolation of monoclonal antibodies and their fragments from different samples including industrial cell culture supernatants. Accordingly, various matrices comprising protein A-ligands are commercially available, for example, in the form of native protein A (e.g. Protein A SEPHAROSE™, GE Healthcare, Uppsala, Sweden) and also comprised of recombinant protein A (e.g. rProtein A-SEPHAROSE™, GE Healthcare). More specifically, the genetic manipulation performed in the commercial recombinant protein A product is aimed at facilitating the attachment thereof to a support and at increasing the productivity of the ligand.

    [0005] These applications, like other affinity chromatography applications, require comprehensive attention to definite removal of contaminants. Such contaminants can for example be non-eluted molecules adsorbed to the stationary phase or matrix in a chromatographic procedure, such as non-desired biomolecules or microorganisms, including for example proteins, carbohydrates, lipids, bacteria and viruses. The removal of such contaminants from the matrix is usually performed after a first elution of the desired product, in order to regenerate the matrix before subsequent use. Such removal usually involves a procedure known as cleaning-in-place (CIP), wherein agents capable of eluting contaminants from the stationary phase are used. One such class of agents often used is alkaline solutions that are passed over said stationary phase. At present the most extensively used cleaning and sanitizing agent is NaOH, and the concentration thereof can range from 0.1 up to e.g. 1 M, depending on the degree and nature of contamination. This strategy is associated with exposing the matrix to solutions with pH-values above 13. For many affinity chromatography matrices containing proteinaceous affinity ligands such alkaline environment is a very harsh condition and consequently results in decreased capacities owing to instability of the ligand to the high pH involved.

    [0006] An extensive research has therefore been focused on the development of engineered protein ligands that exhibit an improved capacity to withstand alkaline pH-values. For example, Gülich et al. (Susanne Gülich, Martin Linhult, Per-Åke Nygren, Mathias Uhlén, Sophia Hober, Journal of Biotechnology 80 (2000), 169-178) suggested protein engineering to improve the stability properties of a Streptococcal albumin-binding domain (ABD) in alkaline environments. Gülich et al. created a mutant of ABD, wherein all the four asparagine residues have been replaced by leucine (one residue), aspartate (two residues) and lysine (one residue). Further, Gülich et al. report that their mutant exhibits a target protein binding behavior similar to that of the native protein, and that affinity columns containing the engineered ligand show higher binding capacities after repeated exposure to alkaline conditions than columns prepared using the parental non-engineered ligand. Thus, it is concluded therein that all four asparagine residues can be replaced without any significant effect on structure and function.

    [0007] Recent work shows that changes can also be made to protein A (SpA) to effect similar properties. US patent application publication US 2005/0143566 discloses that when at least one asparagine residue is mutated to an amino acid other than glutamine or aspartic acid, the mutation confers an 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). The authors show that when these mutated proteins are used as affinity ligands, the separation media as expected can better withstand cleaning procedures using alkaline agents. Further mutations of protein A domains with the purpose of increasing the alkali stability have also been published in US 8,329,860, JP 2006304633A, US 8,674,073, US 2010/0221844, US 2012/0208234, US 9,051,375, US 2014/0031522, US 2013/0274451 and WO 2014/146350. However, the currently available mutants are still sensitive to alkaline pH and the NaOH concentration during cleaning is usually limited to 0.1 M, which means that complete cleaning is difficult to achieve. Higher NaOH concentrations, which would improve the cleaning, lead to unacceptable capacity losses. WO 2015/005859 discloses mutated Protein A domains with improved alkali stability.

    [0008] There is thus still a need in this field to obtain a separation matrix containing protein ligands having a further improved stability towards alkaline cleaning procedures. There is also a need for such separation matrices with an improved binding capacity to allow for economically efficient purification of therapeutic antibodies.

    Summary of the invention



    [0009] One aspect of the invention is to provide a separation matrix capable of selectively binding immunoglobulins and other Fc-containing proteins and exhibiting an improved alkaline stability. This is achieved with a separation matrix as defined in claim 1. The matrix comprises at least 11 mg/ml Fc-binding ligands covalently coupled to a porous support, wherein:
    1. a) the ligands comprise multimers of alkali-stabilized Protein A domains,
    2. b) the porous support comprises cross-linked polymer particles having a volume-weighted median diameter (d50,v) of 56-70 micrometers and a dry solids weight of 55-80 mg/ml.


    [0010] One advantage is that a high dynamic binding capacity is provided. A further advantage is that a high degree of alkali stability is achieved.

    [0011] A second aspect of the invention is to provide an efficient and economical method of isolating an immunoglobulin or other Fc-containing protein. This is achieved with a method as defined in claim 13, comprising the steps of:
    1. a) contacting a liquid sample comprising an immunoglobulin with a separation matrix as disclosed above,
    2. b) washing the separation matrix with a washing liquid,
    3. c) eluting the immunoglobulin from the separation matrix with an elution liquid, and
    4. d) cleaning the separation matrix with a cleaning liquid.


    [0012] Further suitable embodiments of the invention are described in the dependent claims.

    Definitions



    [0013] The terms "antibody" and "immunoglobulin" are used interchangeably herein, and are understood to include also fragments of antibodies, fusion proteins comprising antibodies or antibody fragments and conjugates comprising antibodies or antibody fragments.

    [0014] The terms an "Fc-binding polypeptide" and "Fc-binding protein" mean a polypeptide or protein respectively, capable of binding to the crystallisable part (Fc) of an antibody and includes e.g. Protein A and Protein G, or any fragment or fusion protein thereof that has maintained said binding property.

    [0015] The term "linker" herein means an element linking two polypeptide units, monomers or domains to each other in a multimer.

    [0016] The term "spacer" herein means an element connecting a polypeptide or a polypeptide multimer to a support.

    [0017] The term "% identity" with respect to comparisons of amino acid sequences is determined by standard alignment algorithms such as, for example, Basic Local Alignment Tool (BLAST™) described in Altshul et al. (1990) J. Mol. Biol., 215: 403-410. A web-based software for this is freely available from the US National Library of Medicine at http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE TYPE=BlastSearch&LINK LOC=blasthome . Here, the algorithm "blastp (protein-protein BLAST)" is used for alignment of a query sequence with a subject sequence and determining i.a. the % identity.

    [0018] As used herein, the terms "comprises," "comprising," "containing," "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean "includes," "including," and the like; "consisting essentially of or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

    Brief description of figures



    [0019] 

    Fig. 1 shows an alignment of the Fc-binding domains as defined by SEQ ID NO:1-7 and 51-52.

    Fig. 2 shows results from Example 2 for the alkali stability of parental and mutated tetrameric Zvar (SEQ ID NO 7) polypeptide variants coupled to an SPR biosensor chip.

    Fig. 3 shows results from Example 4 for the alkali stability (0.5 M NaOH) of parental and mutated tetrameric Zvar (SEQ ID NO 7) polypeptide variants coupled to agarose beads.

    Fig. 4 shows results from Example 4 for the alkali stability (1.0 M NaOH) of parental and mutated tetrameric Zvar (SEQ ID NO 7) polypeptide variants coupled to agarose beads.

    Fig. 5 shows results from Example 7 for the alkali stability (1.0 M NaOH) of agarose beads with different amounts of mutated multimer variants (SEQ ID NO. 20) coupled. The results are plotted as the relative remaining dynamic capacity (Qbl0%, 6 min residence time) vs. incubation time in 1 M NaOH.

    Fig. 6 shows results from Example 7 for the alkali stability (1.0 M NaOH) of agarose beads with different amounts of mutated multimer variants (SEQ ID NO. 20) coupled. The results are plotted as the relative remaining dynamic capacity (Qb10%, 6 min residence time) after 31 h incubation in 1 M NaOH vs. the ligand content of the prototypes.


    Detailed description of embodiments



    [0020] The Fc-binding ligands in the matrix of the invention may comprise an Fc-binding polypeptide, which comprises, or consists essentially of, a mutant of an Fc-binding domain of Staphylococcus Protein A (SpA), as defined by, or having at least 90%, at least 95% or at least 98% identity to, 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 the linker region amino acids 1-8 and 56-58) or SEQ ID NO 52 (C-domain without the linker region amino acids 1-8 and 56-58) as illustrated in Fig. 1, wherein at least the asparagine (or serine, in the case of SEQ ID NO 2) residue at the 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. Protein Z (SEQ ID NO:6) is a mutated B-domain as disclosed in US5143844, while SEQ ID NO 7 denotes a further mutated variant of Protein Z, here called Zvar, with the mutations N3A,N6D,N23T. SEQ ID NO:22 is a natural variant of the A-domain in Protein A from Staphylococcus aureus strain N315, having an A46S mutation, using the position terminology of Fig. 1. The mutation of N11 in these domains confers an improved alkali stability in comparison with the parental domain/polypeptide, without impairing the immunoglobulin-binding properties. Hence, the polypeptide can also be described as an Fc- or immunoglobulin-binding polypeptide, or alternatively as an Fc- or immunoglobulin-binding polypeptide unit.

    [0021] *Throughout this description, the amino acid residue position numbering convention of Fig 1 is used, and the position numbers are designated as corresponding to those in SEQ ID NO 4-7. This applies also to multimers, where the position numbers designate the positions in the polypeptide units or monomers according to the convention of Fig. 1.

    SEQ ID NO 51 (truncated Zvar)
    QQ NAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQ

    SEQ ID NO 52 (truncated C domain)
    QQ NAFYEILHLP NLTEEQRNGF IQSLKDDPSV SKEILAEAKK LNDAQ



    [0022] In alternative language, the Fc-binding ligands may comprise an Fc-binding polypeptide which comprises a sequence as defined by, or having at least 90%, at least 95% or at least 98% identity to SEQ ID NO 53.

    wherein individually of each other:

    X1=A, Q or is deleted

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

    X3=H or K

    X4=A or N

    X5=A, G, S,Y,Q,T,N,F,L,W,I,M,V,D,E,H,R or K, such as S,Y,Q,T,N,F,L,W,I,M,V,D,E,H,R or K

    X6=Q or E

    X7=S or K

    X8=E or D

    X9=Q, V or is deleted

    X10=K, R, A or is deleted

    X11=A, E, N or is deleted

    X12=I or L

    X13=K or R

    X14=L or Y

    X15=D, F,Y,W,K or R



    [0023] Specifically, the amino acid residues in SEQ ID NO 53 may individually of each other be:

    X1 = A or is deleted

    X2 = E

    X3 = H

    X4 = N

    X6 = Q

    X7 = S

    X8 = D

    X9 = V or is deleted

    X10 = K or is deleted

    X11 = A or is deleted

    X12 = I

    X13 = K

    X14 = L.



    [0024] In certain embodiments, the amino acid residues in SEQ ID NO 53 may be: X1=A, X2 = E, X3 = H, X4 = N, X6 = Q, X7 = S, X8 = D, X9 = V, X10 = K, X11 = A, X12 = I, X13 = K, X14 = L. In some embodiments X2 = E, X3 = H, X4 = N, X5 = A, X6 = Q, X7 = S, X8 = D, X12 = I, X13 = K, X14 = L and X15 = D and one or more of X1, X9, X10 and X11 is deleted. In further embodiments, X1=A, X2 = E, X3 = H, X4 = N, X5 = S,Y,Q,T,N,F,L,W,I,M,V,D,E,H,R or K, X6 = Q, X7 = S, X8 = D, X9 = V, X10 = K, X11 = A, X12 = I, X13 = K, X14 = L and X15=D, or alternatively X1=A, X2 = E, X3 = H, X4 = N, X5 = A, X6 = Q, X7 = S, X8 = D, X9 = V, X10 = K, X11 = A, X12 = I, X13 = K, X14 = L and X15 = F,Y,W,K or R.

    [0025] The N11 (X2) mutation (e.g. a N11E or N11K mutation) may be the only mutation or the polypeptide may also comprise further mutations, such as substitutions in at least one of the positions 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 in SEQ ID NO:4-7. In one or more of these positions, the original amino acid residue may e.g. be substituted with an amino acid which is not asparagine, proline or cysteine. The original amino acid residue may e.g. be substituted with an alanine, a valine, a threonine, a serine, a lysine, a glutamic acid or an aspartic acid. Further, one or more amino acid residues may be deleted, e.g. from positions 1-6 and/or from positions 56-58.

    [0026] In some embodiments, the amino acid residue at the position corresponding to position 9 in SEQ ID NO:4-7 (X1) is an amino acid other than glutamine, asparagine, proline or cysteine, such as an alanine or it can be deleted. The combination of the mutations at positions 9 and 11 provides particularly good alkali stability, as shown by the examples. In specific embodiments, in SEQ ID NO: 7 the amino acid residue at position 9 is an alanine and the amino acid residue at position 11 is a lysine or glutamic acid, such as a lysine. Mutations at position 9 are also discussed in copending application PCT/SE2014/050872.

    [0027] In some embodiments, the amino acid residue at the position corresponding to position 50 in SEQ ID NO:4-7 (X13) is an arginine or a glutamic acid.

    [0028] In certain embodiments, the amino acid residue at the position corresponding to position 3 in SEQ ID NO:4-7 is an alanine and/or the amino acid residue at the position corresponding to position 6 in SEQ ID NO:4-7 is an aspartic acid. One of the amino acid residues at positions 3 and 6 may be an asparagine and in an alternative embodiment both amino acid residues at positions 3 and 6 may be asparagines.

    [0029] In some embodiments the amino acid residue at the position corresponding to position 43 in SEQ ID NO:4-7 (X11) is an alanine or a glutamic acid, such as an alanine or it can be deleted. In specific embodiments, the amino acid residues at positions 9 and 11 in SEQ ID NO: 7 are alanine and lysine/glutamic acid respectively, while the amino acid residue at position 43 is alanine or glutamic acid.

    [0030] In certain embodiments the amino acid residue at the position corresponding to position 28 in SEQ ID NO:4-7 (X5) is an alanine or an asparagine, such as an alanine.

    [0031] In some embodiments the amino acid residue at the position corresponding to position 40 in SEQ ID NO:4-7 (X9) is selected from the group consisting of asparagine, alanine, glutamic acid and valine, or from the group consisting of glutamic acid and valine or it can be deleted. In specific embodiments, the amino acid residues at positions 9 and 11 in 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 then be alanine or glutamic acid.

    [0032] In certain embodiments, the amino acid residue at the position corresponding to position 42 in SEQ ID NO:4-7 (X10) is an alanine, lysine or arginine or it can be deleted.

    [0033] In some embodiments the amino acid residue at the position corresponding to position 18 in SEQ ID NO:4-7 (X3) is a lysine or a histidine, such as a lysine.

    [0034] In certain embodiments the amino acid residue at the position corresponding to position 33 in SEQ ID NO:4-7 (X7) is a lysine or a serine, such as a lysine.

    [0035] In some embodiments the amino acid residue at the position corresponding to position 37 in SEQ ID NO:4-7 (X8) is a glutamic acid or an aspartic acid, such as a glutamic acid.

    [0036] In certain embodiments the amino acid residue at the position corresponding to position 51 in SEQ ID NO:4-7 (X14) is a tyrosine or a leucine, such as a tyrosine.

    [0037] In some embodiments, the amino acid residue at the position corresponding to position 44 in SEQ ID NO:4-7 (X12) is a leucine or an isoleucine. In specific embodiments, the amino acid residues at positions 9 and 11 in 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 then be alanine or glutamic acid.

    [0038] In some embodiments, the amino acid residues at the positions corresponding to positions 1, 2, 3 and 4 or to positions 3, 4, 5 and 6 in SEQ ID NO: 4-7 have been deleted. In specific variants of these embodiments, the parental 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.

    [0039] In certain embodiments, the mutation in SEQ ID NO 4-7, such as in SEQ ID NO 7, is selected from the group consisting of: 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,NIIE,Q40V,A42K,N43E,L441; Q9A,N11E,Q40V,A42K,N43A,L44I; N11K,H18K,S33K,D37E,A42R,N43A,L44I,K50R,L51Y; Q9A,N11E,N28A,Q40V,A42K,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,NIIE,D37E,Q40V,A42K,N43A,L44I and Q9A,N11E,D37E,Q40V,A42R,N43A,L44I. These mutations provide particularly high alkaline stabilities. The mutation in SEQ ID NO 4-7, such as in SEQ ID NO 7, can also be selected from the group consisting of 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,A42K,N43A,L44I; Q9A,N11E,N28A,Q40V,A42K,N43A,L44I; N11K,H18K,S33K,D37E,A42R,N43A,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.

    [0040] In some embodiments, the polypeptide comprises or consists essentially of a sequence defined by or having at least 90%, 95% or 98% identity to an amino acid sequence selected from the group consisting of: 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 e.g. comprise or consist essentially of a sequence defined by or having at least 90%, 95% or 98% identity to an amino acid sequence selected from the group consisting of: 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 can also comprise or consist essentially of a sequence defined by or having at least 90%, 95% or 98% identity to an amino acid sequence selected from the group consisting of: 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.

    [0041] In certain embodiments, the polypeptide comprises or consists essentially of a sequence defined by or having at least 90%, 95% or 98% identity to an amino acid sequence selected from the group consisting of SEQ ID NO 54-70. comprises or consists essentially of a sequence defined by or having at least 90%, 95% or 98% identity to an amino acid sequence selected from the group consisting of SEQ ID NO 71-75, or it may comprise or consist essentially of a sequence defined by or having at least 90%, 95% or 98% identity to an amino acid sequence selected from the group consisting of SEQ ID NO 76-79. It may further comprise or consist essentially of a sequence defined by or having at least 90%, 95% or 98% identity to an amino acid sequence selected from the group consisting of SEQ ID NO 89-95.

    [0042] The polypeptide may e.g. be defined by a sequence selected from the groups above or from subsets of these groups, but it may also comprise additional amino acid residues at the N- and/or C-terminal end, e.g. a leader sequence at the N-terminal end and/or a tail sequence at the C-terminal end.





































































































































    [0043] The Fc-binding ligands may be multimers comprising, or consisting essentially of, a plurality of polypeptide units as defined by any embodiment disclosed above. The use of multimers may increase the immunoglobulin binding capacity and multimers may also have a higher alkali stability than monomers. The multimer can e.g. be a dimer, a trimer, a tetramer, a pentamer, a hexamer, a heptamer, an octamer or a nonamer. It can be a homomultimer, where all the units in the multimer are identical or it can be a heteromultimer, where at least one unit differs from the others. Advantageously, all the units in the multimer are alkali stable, such as by comprising the mutations disclosed above. The polypeptides can be linked to each other directly by peptide bonds between the C-terminal and N-terminal ends of the polypeptides. Alternatively, two or more units in the multimer can be linked by linkers comprising oligomeric or polymeric species, such as linkers comprising peptides with up to 25 or 30 amino acids, such as 3-25 or 3-20 amino acids. The linkers may e.g. comprise or consist essentially of a peptide sequence defined by, or having at least 90% identity or at least 95% identity, with an amino acid sequence selected from the group consisting of APKVDAKFDKE, APKVDNKFNKE, APKADNKFNKE, APKVFDKE, APAKFDKE, AKFDKE, APKVDA, VDAKFDKE, APKKFDKE, APK, APKYEDGVDAKFDKE and YEDG or alternatively selected from the group consisting of APKADNKFNKE, APKVFDKE, APAKFDKE, AKFDKE, APKVDA, VDAKFDKE, APKKFDKE, APKYEDGVDAKFDKE and YEDG. They can also consist essentially of a peptide sequence defined by or having at least 90% identity or at least 95% identity with an amino acid sequence selected from the group consisting of APKADNKFNKE, APKVFDKE, APAKFDKE, AKFDKE, APKVDA, VDAKFDKE, APKKFDKE, APK and APKYEDGVDAKFDKE. In some embodiments the linkers do not consist of the peptides APKVDAKFDKE or APKVDNKFNKE, or alternatively do not consist of the peptides APKVDAKFDKE, APKVDNKFNKE , APKFNKE, APKFDKE, APKVDKE or APKADKE.

    [0044] The nature of such a linker should preferably not destabilize the spatial conformation of the protein units. This can e.g. be achieved by avoiding the presence of proline in the linkers. Furthermore, said linker should preferably also be sufficiently stable in alkaline environments not to impair the properties of the mutated protein units. For this purpose, it is advantageous if the linkers do not contain asparagine. It can additionally be advantageous if the linkers do not contain glutamine. The multimer may further at the N-terminal end comprise a plurality of amino acid residues e.g. originating from the cloning process or constituting a residue from a cleaved off signaling sequence. The number of additional amino acid residues may e.g. be 20 or less, such as 15 or less, such as 10 or less or 5 or less. As a specific example, the multimer may comprise an AQ, AQGT, VDAKFDKE, AQVDAKFDKE or AQGTVDAKFDKE sequence at the N-terminal end.

    [0045] In certain embodiments, the multimer may comprise, or consist essentially, of a sequence selected from the group consisting of: SEQ ID NO 80-87. These and additional sequences are listed below and named as Parent(Mutations)n, where n is the number of monomer units in a multimer.







































    [0046] In some embodiments, the polypeptide and/or multimer, as disclosed above, further comprises at the C-terminal or N-terminal end one or more coupling elements, selected from the group consisting of one or more cysteine residues, a plurality of lysine residues and a plurality of histidine residues. The coupling element(s) may also be located within 1-5 amino acid residues, such as within 1-3 or 1-2 amino acid residues from the C-terminal or N-terminal end. The coupling element may e.g. be a single cysteine at the C-terminal end. The coupling element(s) may be directly linked to the C- or N-terminal end, or it/they may be linked via a stretch comprising up to 15 amino acids, such as 1-5, 1-10 or 5-10 amino acids. This stretch should preferably also be sufficiently stable in alkaline environments not to impair the properties of the mutated protein. For this purpose, it is advantageous if the stretch does not contain asparagine. It can additionally be advantageous if the stretch does not contain glutamine. An advantage of having a C-terminal cysteine is that endpoint coupling of the protein can be achieved through reaction of the cysteine thiol with an electrophilic group on a support. This provides excellent mobility of the coupled protein which is important for the binding capacity.

    [0047] The alkali stability of the polypeptide or multimer can be assessed by coupling it to an SPR chip, e.g. to Biacore CM5 sensor chips as described in the examples, using e.g. NHS- or maleimide coupling chemistries, and measuring the immunoglobulin-binding capacity of the chip, typically using polyclonal human IgG, before and after incubation in alkaline solutions at a specified temperature, e.g. 22 +/- 2 °C. The incubation can e.g. be performed in 0.5 M NaOH for a number of 10 min cycles, such as 100, 200 or 300 cycles. The IgG capacity of the matrix after 100 10 min incubation cycles in 0.5 M NaOH at 22 +/- 2 °C can be at least 55, such as at least 60, at least 80 or at least 90% of the IgG capacity before the incubation. Alternatively, the remaining IgG capacity after 100 cycles for a particular mutant measured as above can be compared with the remaining IgG capacity for the parental polypeptide/multimer. In this case, the remaining IgG capacity for the mutant may be at least 105%, such as at least 110%, at least 125%, at least 150% or at least 200% of the parental polypeptide/multimer.

    [0048] The present invention discloses a separation matrix, wherein a plurality of multimers according to any embodiment disclosed above have been coupled to a solid support. The separation matrix comprises at least 11, such as 11-21, 15-21 or 15-18 mg/ml Fc-binding ligands covalently coupled to a porous support, wherein:
    1. a) the ligands comprise multimers of alkali-stabilized Protein A domains,
    2. b) the porous support comprises cross-linked polymer particles having a volume-weighted median diameter (d50,v) of 56-70, such as 56-66, micrometers and a dry solids weight of 55-80, such as 60-78 or 65-78, mg/ml. The cross-linked polymer particles have a pore size corresponding to an inverse gel filtration chromatography Kd value of 0.69-0.85, such as 0.70-0.85 or 0.69-0.80, for dextran of Mw 110 kDa. Suitably, the cross-linked polymer particles can have a high rigidity, to be able to withstand high flow rates. The rigidity can be measured with a pressure-flow test as further described in Example 8, where a column packed with the matrix is subjected to increasing flow rates of distilled water. The pressure is increased stepwise and the flow rate and back pressure measured, until the flow rate starts to decrease with increasing pressures. The maximum flow rate achieved and the maximum pressure (the back pressure corresponding to the maximum flow rate) are measured and used as measures of the rigidity. When measured in a FineLine™ 35 column (GE Healthcare Life Sciences) at a bed height of 300 +/- 10 mm, the max pressure can suitably be at least 0.58 MPa, such as at least 0.60 MPa. This allows for the use of smaller particle diameters, which is beneficial for the dynamic capacity. The multimers may e.g. comprise tetramers, pentamers, hexamers or heptamers of alkali-stabilized Protein A domains, such as hexamers of alkali-stabilized Protein A domains. The combination of the high ligand contents with the particle size range, the dry solids weight range and the optional Kd range provides for a high binding capacity, e.g. such that the 10% breakthrough dynamic binding capacity for IgG is at least 45 mg/ml, such as at least 50 or at least 55 mg/ml at 2.4 min residence time. Alternatively, or additionally, the 10% breakthrough dynamic binding capacity for IgG may be at least 60 mg/ml, such as at least 65, at least 70 or at least 75 mg/ml at 6 min residence time.
    The alkali-stabilized Protein A multimers are highly selective for IgG and the separation matrix can suitably have a dissociation constant for human IgG2 of below 0.2 mg/ml, such as below 0.1 mg/ml, in 20 mM phosphate buffer, 180 mM NaCl, pH 7.5. This can be determined according to the adsorption isotherm method described in N Pakiman et al: J Appl Sci 12, 1136-1141 (2012).

    [0049] In certain embodiments the alkali-stabilized Protein A domains comprise mutants of a parental Fc-binding domain of Staphylococcus Protein A (SpA), as defined by, or having at least 80% such as at least 90%, 95% or 98% identity to, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:22, SEQ ID NO 51 or SEQ ID NO 52, wherein at least the asparagine or serine residue at the 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, such as an amino acid selected from the group consisting of glutamic acid and lysine. The amino acid residue at the position corresponding to position 40 in SEQ ID NO:4-7 may further be, or be mutated to, a valine. The alkali-stabilized Protein A domains may also comprise any mutations as described in the polypeptide and/or multimer embodiments above.

    [0050] In some embodiments the alkali-stabilized Protein A domains comprise an Fc-binding polypeptide having an amino acid sequence as defined by, or having at least 80% or at least 90, 95% or 98% identity to SEQ ID NO 53.

    wherein individually of each other:

    X1=A or Q or is deleted

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

    X3=H or K

    X4=A or N

    X5=A, G, S,Y,Q,T,N,F,L,W,I,M,V,D,E,H,R or K

    X6=Q or E

    X7=S or K

    X8=E or D

    X9=Q or V or is deleted

    X10=K,R or A or is deleted

    X11=A,E or N or is deleted

    X12=I or L

    X13=K or R

    X14=L or Y

    X15=D, F,Y,W,K or R



    [0051] In some embodiments, the amino acid residues may individually of each other be:
    1. a) X1 = A or is deleted, X2 = E, X3 = H, X4 = N, X6 = Q, X7 = S, X8 = D, X9 = V or is deleted, X10 = K or is deleted, X11 = A or is deleted, X12 = I, X13 = K, X14 = L.
    2. b) X1=A, X2 = E, X3 = H, X4 = N, X5 = A, X6 = Q, X7 = S, X8 = D, X9 = V, X10 = K, X11 = A, X12 = I, X13 = K, X14 = L and X15=D.
    3. c) X1 is A, X2 = E, X3 = H, X4 = N, X6 = Q, X7 = S, X8 = D, X9 = V, X10 = K, X11 = A, X12 = I, X13 = K, X14 = L and X15=D or
    4. d) X1 is A, X3 = H, X4 = N, X5 = A, X6 = Q, X7 = S, X8 = D, X9 = V, X10 = K, X11 = A, X12 = I, X13 = K, X14 = L and X15=D.


    [0052] In certain embodiments the invention discloses a separation matrix comprising at least 15, such as 15-21 or 15-18 mg/ml Fc-binding ligands covalently coupled to a porous support, wherein the ligands comprise multimers of alkali-stabilized Protein A domains. These multimers can suitably be as disclosed in any of the embodiments described above or as specified below.

    [0053] Such a matrix is useful for separation of immunoglobulins or other Fc-containing proteins and, due to the improved alkali stability of the polypeptides/multimers, the matrix will withstand highly alkaline conditions during cleaning, which is essential for long-term repeated use in a bioprocess separation setting. The alkali stability of the matrix can be assessed by measuring the immunoglobulin-binding capacity, typically using polyclonal human IgG, before and after incubation in alkaline solutions at a specified temperature, e.g. 22 +/- 2 °C. The incubation can e.g. be performed in 0.5 M or 1.0 M NaOH for a number of 15 min cycles, such as 100, 200 or 300 cycles, corresponding to a total incubation time of 25, 50 or 75 h. The IgG capacity of the matrix after 96-100 15 min incubation cycles or a total incubation time of 24 or 25 h in 0.5 M NaOH at 22 +/- 2 °C can be at least 80, such as at least 85, at least 90 or at least 95% of the IgG capacity before the incubation. The capacity of the matrix after a total incubation time of 24 h in 1.0 M NaOH at 22 +/- 2 °C can be at least 70, such as at least 80 or at least 90% of the IgG capacity before the incubation. The the 10% breakthrough dynamic binding capacity (Qb10%) for IgG at 2.4 min or 6 min residence time may e.g. be reduced by less than 20 % after incubation 31 h in 1.0 M aqueous NaOH at 22 +/- 2 C.

    [0054] As the skilled person will understand, the expressed polypeptide or multimer should be purified to an appropriate extent before being immobilized to a support. Such purification methods are well known in the field, and the immobilization of protein-based ligands to supports is easily carried out using standard methods. Suitable methods and supports will be discussed below in more detail.

    [0055] A conventional affinity separation matrix is often of organic nature and based on polymers that expose a hydrophilic surface to the aqueous media used, i.e. expose hydroxy (-OH), carboxy (-COOH), carboxamido (-CONH2, possibly in N- substituted forms), amino (-NH2, possibly in substituted form), oligo- or polyethylenoxy groups on their external and, if present, also on internal surfaces. The solid support is porous. The porosity can be expressed as a Kav or Kd value (the fraction of the pore volume available to a probe molecule of a particular size) measured by inverse size exclusion chromatography, e.g. according to the methods described in Gel Filtration Principles and Methods, Pharmacia LKB Biotechnology 1991, pp 6-13. Kav is determined as the ratio (Ve-Vo)/(Vt-Vo), where Ve is the elution volume of a probe molecule (e.g. Dextran 110 kD), Vo is the void volume of the column (e.g. the elution volume of a high Mw void marker, such as raw dextran) and Vt is the total volume of the column. Kd can be determined as (Ve-V0)/Vi, where Vi is the elution volume of a salt (e.g. NaCl) able to access all the volume except the matrix volume (the volume occupied by the matrix polymer molecules). By definition, both Kd and Kav values always lie within the range 0 - 1. The Kav value can advantageously be 0.6 - 0.95, e.g. 0.7 - 0.90 or 0.6 - 0.8, as measured with dextran of Mw 110 kDa as a probe molecule. The Kd value as measured with dextran of Mw 110 kDa can suitably be 0.68-0.90, such as 0.68-0.85 or 0.70-0.85. An advantage of this is that the support has a large fraction of pores able to accommodate both the polypeptides/multimers of the invention and immunoglobulins binding to the polypeptides/multimers and to provide mass transport of the immunoglobulins to and from the binding sites.

    [0056] The polypeptides or multimers may be attached to the support via conventional coupling techniques utilising e.g. thiol, amino and/or carboxy groups present in the ligand. Bisepoxides, epichlorohydrin, CNBr, N-hydroxysuccinimide (NHS) etc are well-known coupling reagents. Between the support and the polypeptide/multimer, a molecule known as a spacer can be introduced, which improves the availability of the polypeptide/multimer and facilitates the chemical coupling of the polypeptide/multimer to the support. Depending on the nature of the polypeptide/multimer and the coupling conditions, the coupling may be a multipoint coupling (e.g. via a plurality of lysines) or a single point coupling (e.g. via a single cysteine). Alternatively, the polypeptide/multimer may be attached to the support by noncovalent bonding, such as physical adsorption or biospecific adsorption.

    [0057] The amount of coupled polypeptide/multimer can be controlled by the concentration of polypeptide/multimer used in the coupling process, 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 polypeptide/multimer, at least up to a point where the pores become significantly constricted by the coupled polypeptide/multimer. Without being bound by theory, it appears though that for the Kd values recited for the support, the constriction of the pores by coupled ligand is of lower significance. The relative binding capacity per mg coupled polypeptide/multimer will decrease at high coupling levels, resulting in a cost-benefit optimum within the ranges specified above.

    [0058] In certain embodiments the polypeptides or multimers are coupled to the support via thioether bonds. Methods for performing such coupling are well-known in this field and easily performed by the skilled person in this field using standard techniques and equipment. Thioether bonds are flexible and stable and generally suited for use in affinity chromatography. In particular when the thioether bond is via a terminal or near-terminal cysteine residue on the polypeptide or multimer, the mobility of the coupled polypeptide/multimer is enhanced which provides improved binding capacity and binding kinetics. In some embodiments the polypeptide/multimer is coupled via a C-terminal cysteine provided on the protein as described above. This allows for efficient coupling of the cysteine thiol to electrophilic groups, e.g. epoxide groups, halohydrin groups etc. on a support, resulting in a thioether bridge coupling.

    [0059] The support comprises agarose. The supports used in the present invention can easily be prepared according to standard methods, such as inverse suspension gelation (S Hjertén: Biochim Biophys Acta 79(2), 393-398 (1964). Alternatively, the base matrices are commercially available products, such as crosslinked agarose beads sold under the name of SEPHAROSE™ FF (GE Healthcare). In an embodiment, which is especially advantageous for large-scale separations, the support has been adapted to increase its rigidity using the methods described in US6602990 or US7396467, and hence renders the matrix more suitable for high flow rates.

    [0060] In certain embodiments the agarose support, is crosslinked, such as with hydroxyalkyl ether crosslinks. Crosslinker reagents producing such crosslinks can be e.g. epihalohydrins like epichlorohydrin, diepoxides like butanediol diglycidyl ether, allylating reagents like allyl halides or allyl glycidyl ether. Crosslinking is beneficial for the rigidity of the support and improves the chemical stability. Hydroxyalkyl ether crosslinks are alkali stable and do not cause significant nonspecific adsorption.

    [0061] Matrices in beaded or particle form can be used as a packed bed or in a suspended form. Suspended forms include those known as expanded beds and pure suspensions, in which the particles or beads are free to move. In case of monoliths, packed bed and expanded beds, the separation procedure commonly follows conventional chromatography with a concentration gradient. In case of pure suspension, batch-wise mode will be used.

    [0062] In a second aspect, the present invention discloses a method of isolating an immunoglobulin, wherein a separation matrix as disclosed above is used. The method may comprise the steps of:
    1. a) contacting a liquid sample comprising an immunoglobulin with a separation matrix as disclosed above,
    2. b) washing the separation matrix with a washing liquid,
    3. c) eluting the immunoglobulin from the separation matrix with an elution liquid, and
    4. d) cleaning the separation matrix with a cleaning liquid, which may comprise 0.1 - 1.0 M NaOH or KOH, such as 0.4 - 1.0 M NaOH or KOH.
    Steps a) - d) may be repeated at least 10 times, such as at least 50 times or 50 - 200 times.

    [0063] In certain embodiments, the method comprises the steps of:
    1. a) contacting a liquid sample comprising an immunoglobulin with a separation matrix as disclosed above,
    2. b) washing said separation matrix with a washing liquid,
    3. c) eluting the immunoglobulin from the separation matrix with an elution liquid, and
    4. d) cleaning the separation matrix with a cleaning liquid, which can alternatively be called a cleaning-in-place (CIP) liquid, e.g. with a contact (incubation) time of at least 10 min.
    The method may also comprise steps of, before step a), providing an affinity separation matrix according to any of the embodiments described above and providing a solution comprising an immunoglobulin and at least one other substance as a liquid sample and of, after step c), recovering the eluate and optionally subjecting the eluate to further separation steps, e.g. by anion or cation exchange chromatography, multimodal chromatography and/or hydrophobic interaction chromatography. Suitable compositions of the liquid sample, the washing liquid and the elution liquid, as well as the general conditions for performing the separation are well known in the art of affinity chromatography and in particular in the art of Protein A chromatography. The liquid sample comprising an Fc-containing protein and at least one other substance may comprise host cell proteins (HCP), such as CHO cell, E Coli or yeast proteins. Contents of CHO cell and E Coli proteins can conveniently be determined by immunoassays directed towards these proteins, e.g. the CHO HCP or E Coli HCP ELISA kits from Cygnus Technologies. The host cell proteins or CHO cell/E Coli proteins may be desorbed during step b).

    [0064] The elution may be performed by using any suitable solution used for elution from Protein A media. This can e.g. be a solution or buffer with pH 5 or lower, such as pH 2.5 - 5 or 3 - 5. It can also in some cases be a solution or buffer with pH 11 or higher, such as pH 11 - 14 or pH 11 - 13. In some embodiments the elution buffer or the elution buffer gradient comprises at least one mono- di- or trifunctional carboxylic acid or salt of such a carboxylic acid. In certain embodiments the elution buffer or the elution buffer gradient comprises at least one anion species selected from the group consisting of acetate, citrate, glycine, succinate, phosphate, and formiate.

    [0065] In some embodiments, the cleaning liquid is alkaline, such as with a pH of 13 - 14. Such solutions provide efficient cleaning of the matrix, in particular at the upper end of the interval

    [0066] In certain embodiments, the cleaning liquid comprises 0.1 - 2.0 M NaOH or KOH, such as 0.5 - 2.0 or 0.5 - 1.0 M NaOH or KOH. These are efficient cleaning solutions, and in particular so when the NaOH or KOH concentration is above 0.1 M or at least 0.5 M. The high stability of the polypeptides of the invention enables the use of such strongly alkaline solutions.

    [0067] The method may also include a step of sanitizing the matrix with a sanitization liquid, which may e.g. comprise a peroxide, such as hydrogen peroxide and/or a peracid, such as peracetic acid or performic acid.

    [0068] In some embodiments, steps a) - d) are repeated at least 10 times, such as at least 50 times, 50 - 200, 50-300 or 50-500 times. This is important for the process economy in that the matrix can be re-used many times.

    [0069] Steps a) - c) can also be repeated at least 10 times, such as at least 50 times, 50 - 200, 50-300 or 50-500 times, with step d) being performed after a plurality of instances of step c), such that step d) is performed at least 10 times, such as at least 50 times. Step d) can e.g. be performed every second to twentieth instance of step c).

    Examples


    Mutagenesis of protein



    [0070] Site-directed mutagenesis was performed by a two-step PCR using oligonucleotides coding for the mutations. As template a plasmid containing a single domain of either Z, B or C was used. The PCR fragments were ligated into an E. coli expression vector. DNA sequencing was used to verify the correct sequence of inserted fragments.
    To form multimers of mutants an Acc I site located in the starting codons (GTA GAC) of the B, C or Z domain was used, corresponding to amino acids VD. The vector for the monomeric domain was digested with Acc I and phosphatase treated. Acc I sticky-ends primers were designed, specific for each variant, and two overlapping PCR products were generated from each template. The PCR products were purified and the concentration was estimated by comparing the PCR products on a 2% agarose gel. Equal amounts of the pair wise PCR products were hybridized (90°C -> 25°C in 45min) in ligation buffer. The resulting product consists approximately to ¼ of fragments likely to be ligated into an Acc I site (correct PCR fragments and/or the digested vector). After ligation and transformation colonies were PCR screened to identify constructs containing the desired mutant. Positive clones were verified by DNA sequencing.

    Construct expression and purification



    [0071] The constructs were expressed in the bacterial periplasm by fermentation of E. coli K12 in standard media. After fermentation the cells were heat-treated to release the periplasm content into the media. The constructs released into the medium were recovered by microfiltration with a membrane having a 0.2 µm pore size.

    [0072] Each construct, now in the permeate from the filtration step, was purified by affinity. The permeate was loaded onto a chromatography medium containing immobilized IgG (IgG Sepharose 6FF, GE Healthcare). The loaded product was washed with phosphate buffered saline and eluted by lowering the pH.

    [0073] The elution pool was adjusted to a neutral pH (pH 8) and reduced by addition of dithiothreitol. The sample was then loaded onto an anion exchanger. After a wash step the construct was eluted in a NaCl gradient to separate it from any contaminants. The elution pool was concentrated by ultrafiltration to 40-50 mg/ml. It should be noted that the successful affinity purification of a construct on an immobilized IgG medium indicates that the construct in question has a high affinity to IgG.

    [0074] The purified ligands were analyzed with RPC LC-MS to determine the purity and to ascertain that the molecular weight corresponded to the expected (based on the amino acid sequence).

    Example 1



    [0075] The purified monomeric ligands listed in Table 1, further comprising for SEQ ID NO 8-16, 23-28 and 36-48 an AQGT leader sequence at the N-terminus and a cysteine at the C terminus, were immobilized on Biacore CM5 sensor chips (GE Healthcare, Sweden), using the amine coupling kit of GE Healthcare (for carbodiimide coupling of amines on the carboxymethyl groups on the chip) in an amount sufficient to give a signal strength of about 200-1500 RU in a Biacore surface plasmon resonance (SPR) instrument (GE Healthcare, Sweden) . To follow the IgG binding capacity of the immobilized surface 1mg/ml human polyclonal IgG (Gammanorm) was flowed over the chip and the signal strength (proportional to the amount of binding) was noted. The surface was then cleaned-in-place (CIP), i.e. flushed with 500mM NaOH for 10 minutes at room temperature (22 +/- 2°C). This was repeated for 96-100 cycles and the immobilized ligand alkaline stability was followed as the remaining IgG binding capacity (signal strength) after each cycle. 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(N11L)1, Zvar(N11W)1, ZN11I)1, Zvar(N11M)1, Zvar(N11V)1, Zvar(N11A)1, Zvar(N11H1), Zvar(N11R)1, Zvar(N11E,Q32A)1, Zvar(N11E,Q32E,Q40E)1 and Zvar(N11E,Q32E,K50R)1, Zvar(Q9A,N11E,N43A)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,S33K,D37E,A42R,N43A,L44I,K50R,L51Y)1, Zvar(Q9A,N11K,H18K,S33K,D37E,A42R,N43A,L44I,K50R,L51Y)1, Zvar(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, as well as the varieties of Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)1 having G,S,Y,Q,T,N,F,L,W,I,M,V,D,E,H,R or K in position 29, the varieties of Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)1 having F,Y,W,K or R in position 53 and the varieties of Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)1 where Q9, Q40, A42 or N43 has been deleted, have an improved alkali stability compared to the parental structure Zvar1, used as the reference. Further, the ligands B(Q9A,N11E,Q40V,A42K,N43A,L44I)1 and C(Q9A,N11E,E43A)1 have an improved stability compared to the parental B and C domains, used as references.
    Table 1. Monomeric ligands, evaluated by Biacore (0.5 M NaOH).
    LigandSequenceCapacity after 96-100 cyclesReference capacity after 96-100 cyclesCapacity relative to reference
    Zvar(N11E,Q32A)1 SEQ ID NO 12 57% 55% 1.036
    Zvar(N11E)1 SEQ ID NO 13 59% 55% 1.073
    Zvar(N1 1E,Q32E,Q40E)1 SEQ ID NO 14 52% 51% 1.020
    Zvar(N1 1E,Q32E,K50R)1 SEQ ID NO 15 53% 51% 1.039
    Zvar(N11K)1 1 SEQ ID NO 16 62% 49% 1.270
    Zvar(N11Y)1 1 SEQ ID NO 38 55% 46% 1.20
    Zvar(N11T)1 SEQ ID NO 39 50% 46% 1.09
    Zvar(N11F)1 SEQ ID NO 40 55% 46% 1.20
    Zvar(N11L)1 SEQ ID NO 41 57% 47% 1.21
    Zvar(N11W)1 SEQ ID NO 42 57% 47% 1.21
    Zvar(N11I)1 SEQ ID NO 43 57% 47% 1.21
    Zvar(N11M) 1 SEQ ID NO 44 58% 46% 1.26
    Zvar(N11V)1 SEQ ID NO 45 56% 46% 1.22
    Zvar(N11A) 1 SEQ ID NO 46 58% 46% 1.26
    Zvar(N11H) 1 SEQ ID NO 47 57% 46% 1.24
    Zvar(N11R)1 SEQ ID NO 48 59% 46% 1.28
    Zvar(Q9A,N11E,N43A)1 SEQ ID NO 8 70% 47% 1.49
    Zvar(Q9A,N1 1E,N28A,N43A)1 SEQ ID NO 9 68% 47% 1.45
    Zvar(Q9A,N11E,Q40V,A42K,N43E,L44I)1 SEQ ID NO 10 67% 47% 1.43
    Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)1 1 SEQ ID NO 11 66% 47% 1.40
    Zvar(Q9A,N11E,N28A,Q40V,A42K,N43A,L44I)1 SEQ ID NO 24 65% 48% 1.35
    Zvar(N11K,H18K,S33K,D37E,A42R,N43A,L44I,K50R,L51Y)1 SEQ ID NO 23 67% 46% 1.46
    Zvar(Q9A,N 11K,H18K,S33K,D37E,A42R,N43 A,L44I,K50R,L5 1Y)1 SEQ ID NO 25 59% 46% 1.28
    Zvar(N11K,H18K, D37E, A42R, N43A, L44I)1 SEQ ID NO 26 59% 45% 1.31
    Zvar(Q9A, N11K, H18K, D37E, A42R, N43A, L44I)1 SEQ ID NO 27 63% 45% 1.40
    Zvar(Q9A, N11K, H18K, D37E, A42R, N43A, L44I, K50R)1 SEQ ID NO 28 67% 45% 1.49
    B(Q9A,N11E,Q40V,A42K,N43A,L44I)1 SEQ ID NO 36 39% 35% 1.11
    C(Q9A,NIIE,E43A)1 SEQ ID NO 37 60% 49% 1.22
    Zvar(Q9A,N11E,A29G,Q40V,A42K,N43 A,L44I)1 SEQ ID NO 54 69% 48% 1.44
    Zvar(Q9A,N11E,A29S,Q40V,A42K,N43A,L44I)1 SEQ ID NO 55 66% 48% 1.38
    Zvar(Q9A,N11E,A29Y,Q40V,A42K,N43A,L44I)1 SEQ ID NO 56 61% 48% 1.27
    Zvar(Q9A,N11E,A29Q,Q40V,A42K,N43A,L44I)1 SEQ ID NO 57 60% 47% 1.28
    Zvar(Q9A,N11E,A29T,Q40V,A42K,N43A,L44I)1 1 SEQ ID NO 58 60% 47% 1.28
    Zvar(Q9A,N11E,A29N,Q40V,A42K,N43A,L44I)1 SEQ ID NO 59 61% 47% 1.30
    Zvar(Q9A,N11E,A29F,Q40V,A42K,N43A,L44I)1 SEQ ID NO 60 62% 46% 1.35
    Zvar(Q9A,N11E,A29L,Q40V,A42K,N43A,L44I)1 SEQ ID NO 61 61% 46% 1.33
    Zvar(Q9A,N11E,A29W,Q40V,A42K,N43A,L44I)1 SEQ ID NO 62 60% 46% 1.30
    Zvar(Q9A,N11E,A29I,Q40V,A42K,N43A,L44I)1 SEQ ID NO 63 58% 47% 1.23
    Zvar(Q9A,N11E,A29M,Q40V,A42K,N43A,L44I)1 SEQ ID NO 64 62% 47% 1.32
    Zvar(Q9A,N11E,A29V,Q40V,A42K,N43A,L44I)1 SEQ ID NO 65 62% 47% 1.32
    Zvar(Q9A,N11E,A29D,Q40V,A42K,N43A,L44I)1 SEQ ID NO 66 56% 47% 1.19
    Zvar(Q9A,N11E,A29E,Q40V,A42K,N43A,L44I)1 SEQ ID NO 67 57% 47% 1.21
    Zvar(Q9A,N11E,A29H,Q40V,A42K,N43A,L44I)1 1 SEQ ID NO 68 57% 47% 1.21
    Zvar(Q9A,N11E,A29R,Q40V,A42K,N43A,L44I)1 SEQ ID NO 69 58% 46% 1.26
    Zvar(Q9A,N11E,A29K,Q40V,A42K,N43A,L44I)1 SEQ ID NO 70 59% 46% 1.28
    Zvar(Q9A,N 11E,Q40V,A42K,N43A,L44I,D53F) 1 SEQ ID NO 71 58% 46% 1.26
    Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I,D53Y)1 SEQ ID NO 72 59% 46% 1.28
    Zvar(Q9A,N 11E,Q40V,A42K,N43A,L44I,D53W) 1 SEQ ID NO 73 62% 46% 1.35
    Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I,D53K)1 SEQ ID NO 74 65% 46% 1.41
    Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I,D53R)1 SEQ ID NO 75 60% 46% 1.30
    Zvar(Q9del,N11E,Q40V,A42K,N43A,L44I)1 SEQ ID NO 76 60% 46% 1.30
    Zvar(Q9A,N11E,Q40del,A42K,N43A,L44I)1 SEQ ID NO 77 59% 46% 1.28
    Zvar(Q9A,N11E,Q40V,A42del,N43A,L44I)1 SEQ ID NO 78 57% 46% 1.24
    Zvar(Q9A,N11E,Q40V,A42K,N43del,L44I) 1 SEQ ID NO 79 55% 46% 1.20


    [0076] The Biacore experiment can also be used to determine the binding and dissociation rates between the ligand and IgG. This was used with the set-up as described above and with an IgG1 monoclonal antibody as probe molecule. For the reference Zvar1, the on-rate (105 M-1S-1) was 3.1 and the off-rate (105 s-1) was 22.1, giving an affinity (off-rate/on-rate) of 713 pM. For Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)1 (SEQ ID NO. 11), the on-rate was 4.1 and the off-rate 43.7, with affinity 1070 pM. The IgG affinity was thus somewhat higher for the mutated variant.

    Example 2



    [0077] The purified dimeric, tetrameric and hexameric ligands listed in Table 2 were immobilized on Biacore CM5 sensor chips (GE Healthcare, Sweden), using the amine coupling kit of GE Healthcare (for carbodiimide coupling of amines on the carboxymethyl groups on the chip) in an amount sufficient to give a signal strength of about 200-1500 RU in a Biacore instrument (GE Healthcare, Sweden) . To follow the IgG binding capacity of the immobilized surface 1mg/ml human polyclonal IgG (Gammanorm) was flowed over the chip and the signal strength (proportional to the amount of binding) was noted. The surface was then cleaned-in-place (CIP), i.e. flushed with 500mM NaOH for 10 minutes at room temperature (22 +/- 2°C). This was repeated for 300 cycles and the immobilized ligand alkaline stability was followed as the remaining IgG binding capacity (signal strength) after each cycle. The results are shown in Table 2 and in Fig. 2 and indicate that at least the ligands Zvar(Q9A,N11E,N43A)4, Zvar(Q9A,N11E,N28A,N43A)4, Zvar(Q9A,N11E,Q40V,A42K,N43E,L44I)4 and 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 an improved alkali stability compared to the parental structure Zvar4, which was used as a reference. The hexameric ligand Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)6 also has improved alkali stability compared to the parental structure Zvar6, used as a reference. Further, Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I) dimers with deletions of a) D2,A3,K4; b) K58,V1,D2; c) P57,K58,V1,D2,A3; d) K4,F5,D6,K7,E8; e) A56,P57,K58; V1,D2,A3 or f) V1,D2,A3,K4,F5,D6,K7,E8 from the linker region between the two monomer units have improved alkali stability compared to the parental structure Zvar2, used as a reference. Also Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I) dimers with an insertion of YEDG between K58 and VI in the linker region have improved alkali stability compared to Zvar2.
    Table 2. Dimeric, tetrameric and hexameric ligands, evaluated by Biacore (0.5M NaOH).
    LigandSEQ ID NO:Remaining capacity 100 cycles (%)Capacity relative to ref. 100 cyclesRemaining capacity 200 cycles (%)Capacity relative to ref. 200 cyclesRemaining capacity 300 cycles (%)Capacity relative to ref. 300 cycles
    Zvar4 21 67 1 36 1 16 1
    Zvar(Q9A,N11E,N43A)4 17 81 1.21 62 1.72 41 2.56
    Zvar(Q9A,N11E,N28A,N 43A)4 18 80 1.19 62 1.72 42 2.62
    Zvar(Q9A,N11E,Q40V,A 42K,N43E,L44I)4 19 84 1.25 65 1.81 48 3.00
    Zvar(Q9A,N11E,Q40V,A 42K,N43A,L44I)4 20 90 1.34 74 2.06 57 3.56
    Zvar(Q9A,N11E,N28A,Q 40V,A42K,N43A,L44I)4 32 84 1.24 Not tested Not tested Not tested Not tested
    Zvar(Q9A,N11E,Q40V,A 42K,N43A,L44I)6 33 87 1.30 Not tested Not tested Not tested Not tested
    Zvar(Q9A,N11E,D37E,Q 40V,A42K,N43A,L44I)4 34 81 1.13 Not tested Not tested Not tested Not tested
    Zvar(Q9A,N11E,D37E,Q 40V,A42R,N43A,L44I)4 35 84 1.17 Not tested Not tested Not tested Not tested
    Zvar(Q9A,N11E,Q40V,A 42K,N43A,L44I)2 with D2, A3 and K4 in linker deleted 80 70 1.27 Not tested Not tested Not tested Not tested
    Zvar(Q9A,N11E,Q40V,A 42K,N43A,L44I)2 with K58, V1 and D2 in linker deleted 81 76 1.38 Not tested Not tested Not tested Not tested
    Zvar(Q9A,N11E,Q40V,A 42K,N43A,L44I)2 with P57, K58, V1, D2 and A3 in linker deleted 82 74 1.35 Not tested Not tested Not tested Not tested
    Zvar(Q9A,N11E,Q40V,A 42K,N43A,L44I)2 with K4, F5, D6, K7 and E8 in linker deleted 83 70 1.30 Not tested Not tested Not tested Not tested
    Zvar(Q9A,N11E,Q40V,A 42K,N43A,L44I)2 with A56, P57 and K58 in linker deleted 84 68 1.26 Not tested Not tested Not tested Not tested
    Zvar(Q9A,N11E,Q40V,A 42K,N43A,L44I)2 with V1, D2 and A3 in linker deleted 85 75 1.39 Not tested Not tested Not tested Not tested
    Zvar(Q9A,N11E,Q40V,A 42K,N43A,L44I)2 with V1, D2, A3, K4, F5, D6, K7 and E8 in linker deleted 86 62 1.13 Not tested Not tested Not tested Not tested
    Zvar(Q9A,N11E,Q40V,A 42K,N43A,L44I)2 with YEDG inserted in linker between K58 and VI 87 72 1.31 Not tested Not tested Not tested Not tested
    Zvar2 88 55 1 Not tested Not tested Not tested Not tested

    Example 3



    [0078] Example 2 was repeated with 100 CIP cycles of three ligands using 1 M NaOH instead of 500 mM as in Example 2. The results are shown in Table 3 and show that all three ligands have an improved alkali stability also in 1M NaOH, compared to the parental structure Zvar4 which was used as a reference.
    Table 3. Tetrameric ligands, evaluated by Biacore (1M NaOH).
    LigandSequenceRemaining capacity 100 cycles (%)Capacity relative to ref. 100 cycles
    Zvar4 SEQ ID NO 21 27 1
    Zvar(Q9A,N11E,N28A,N43A)4 SEQ ID NO 18 55 2.04
    Zvar(Q9A,N11E,Q40V,A42K,N43E,L44I)4 SEQ ID NO 19 54 2.00
    Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)4 SEQ ID NO 20 56 2.07

    Example 4



    [0079] The purified tetrameric ligands of Table 2 (all with an additional N-terminal cysteine) were immobilized on agarose beads using the methods described below and assessed for capacity and stability. The results are shown in Table 4 and Fig. 3.
    Table 4. Matrices with tetrametric ligands, evaluated in columns (0.5 M NaOH).
    LigandSEQ ID NO.Ligand content (mg/ml)Initial IgG capacity Qb10 (mg/ml)Remainin g IgG capacity Qb10 after six 4 h cycles (mg/ml)Remainin g IgG capacity after six 4 h cycles (%)Capaci ty retenti on relative to ref. after six 4 h cycles
    Zvar4 21 7 52.5 36.5 60 1
    Zvar4 21 12 61.1 43.4 71 1
    Zvar(Q9A,N11E,N28A,N43A)4 18 7.0 49.1 44.1 90 1.50
    Zvar(Q9A,N11E,N28A,N43A)4 18 12.1 50.0 46.2 93 1.31
    Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)4 20 7.2 49.0 44.2 90 1.50
    Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)4 20 12.8 56.3 53.6 95 1.34
    Zvar(N11K,H18K,S33K,D37E,A42R,N43A ,L44I,K50R,L51Y)4 30 9.7 56.3 52.0 92 1.53
    Zvar(Q9A,N11K,H18K,D37E,A42R)4 31 10.8 56.9 52.5 92 1.30

    Activation



    [0080] The base matrix used was rigid cross-linked agarose beads of 85 micrometers (volume-weighted, d50V) median diameter, prepared according to the methods of US6602990, and with a pore size corresponding to an inverse gel filtration chromatography Kav value of 0.70 for dextran of Mw 110 kDa, according to the methods described in Gel Filtration Principles and Methods, Pharmacia LKB Biotechnology 1991, pp 6-13.

    [0081] 25 mL (g) of drained base matrix, 10.0 mL distilled water and 2.02 g NaOH (s) was mixed in a 100 mL flask with mechanical stirring for 10 min at 25°C. 4.0 mL of epichlorohydrin was added and the reaction progressed for 2 hours. The activated gel was washed with 10 gel sediment volumes (GV) of water.

    Coupling



    [0082] To 20 mL of ligand solution (50 mg/mL) in a 50 ml Falcon tube, 169 mg NaHCO3, 21 mg Na2CO3, 175 mg NaCl and 7 mg EDTA, was added. The Falcon tube was placed on a roller table for 5-10 min, and then 77 mg of DTE was added. Reduction proceeded for >45 min. The ligand solution was then desalted on a PD10 column packed with Sephadex G-25. The ligand content in the desalted solution was determined by measuring the 276 nm UV absorption.

    [0083] The activated gel was washed with 3-5 GV {0.1 M phosphate/1 mM EDTA pH 8.6} and the ligand was then coupled according to the method described in US6399750. All buffers used in the experiments had been degassed by nitrogen gas for at least 5-10 min. The ligand content of the gels could be controlled by varying the amount and concentration of the ligand solution.

    [0084] After immobilization the gels were washed 3xGV with distilled water. The gels + 1 GV {0.1 M phosphate/1 mM EDTA/10% thioglycerol pH 8.6} was mixed and the tubes were left in a shaking table at room temperature overnight. The gels were then washed alternately with 3xGV {0.1 M TRIS/0.15 M NaCl pH 8.6} and 0.5 M HAc and then 8-10xGV with 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.

    Protein



    [0085] Gammanorm 165 mg/ml (Octapharma), diluted to 2mg/ml in Equilibration buffer.

    Equilibration buffer



    [0086] PBS Phosphate buffer 10 mM + 0.14 M NaCl + 0.0027 M KCl, pH 7,4 (Medicago)

    Adsorption buffer



    [0087] PBS Phosphate buffer 10 mM + 0.14 M NaCl + 0.0027 M KCl, pH 7,4 (Medicago)

    Elution buffers



    [0088] 100 mM acetate pH 2.9

    Dynamic binding capacity



    [0089] 2 ml of resin was packed in TRICORN™ 5 100 columns. The breakthrough capacity was determined with an ÄKTAExplorer 10 system at a residence time of 6 minutes (0.33 ml/min flow rate). Equilibration buffer was run through the bypass column until a stable baseline was obtained. This was done prior to auto zeroing. Sample was applied to the column until a 100% UV signal was obtained. Then, equilibration buffer was applied again until a stable baseline was obtained.

    [0090] Sample was loaded onto the column until a UV signal of 85% of maximum absorbance was reached. The column was then washed with 5 column volumes (CV) equilibration buffer at flow rate 0.5ml/min. The protein was eluted with 5 CV elution buffer at a flow rate of 0.5 ml/min. Then the column was cleaned with 0.5M NaOH at flow rate 0.2 ml/min and re-equilibrated with equilibration buffer.

    [0091] For calculation of breakthrough capacity at 10%, the equation below was used. That is the amount of IgG that is loaded onto the column until the concentration of IgG in the column effluent is 10% of the IgG concentration in the feed.

    A100% = 100% UV signal;

    Asub = absorbance contribution from non-binding IgG subclass;

    A(V) = absorbance at a given applied volume;

    Vc = column volume;

    Vapp = volume applied until 10% breakthrough;

    Vsys = system dead volume;

    C0 = feed concentration.



    [0092] The dynamic binding capacity (DBC) at 10% breakthrough was calculated. The dynamic binding capacity (DBC) was calculated for 10 and 80% breakthrough.

    CIP - 0.5 M NaOH



    [0093] The 10% breakthrough DBC (Qb10) was determined both before and after repeated exposures to alkaline cleaning solutions. Each cycle included a CIP step with 0.5 M NaOH pumped through the column at a rate of 0.5/min for 20 min, after which the column was left standing for 4 h. The exposure took place at room temperature (22 +/- 2°C). After this incubation, the column was washed with equilibration buffer for 20 min at a flow rate of 0.5 ml/min. Table 4 shows the remaining capacity after six 4 h cycles (i.e. 24 h cumulative exposure time to 0.5 M NaOH), both in absolute numbers and relative to the initial capacity.

    Example 5



    [0094] Example 4 was repeated with the tetrameric ligands shown in Table 5, but with 1.0 M NaOH used in the CIP steps instead of 0.5 M. The results are shown in Table 5 and in Fig. 4.
    Table 5. Matrices with tetrametric ligands, evaluated in columns - 1.0 M NaOH.
    LigandSEQ ID NO.Ligand content (mg/ml)Initial IgG capacity Qb10 (mg/ml)Remainin g IgG capacity Qb10 after six 4 h cycles (mg/ml)Remainin g IgG capacity after six 4 h cycles (%)Capaci ty retenti on relative to ref. after six 4 h cycles
    Zvar4 21 12 60.1 33.5 56 1
    Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)4 20 12.8 60.3 56.0 93 1.67
    Zvar(N11K,H18K,S33K,D37E,A42R,N43A ,L44I,K50R,L51Y)4 30 9.7 62.1 48.1 77 1.44

    Example 6


    Base matrices



    [0095] The base matrices used were a set of rigid cross-linked agarose bead samples of 59-93 micrometers (volume-weighted, d50V) median diameter (determined on a Malvern Mastersizer 2000 laser diffraction instrument), prepared according to the methods of US6602990 and with a pore size corresponding to an inverse gel filtration chromatography Kd value of 0.62-0.82 for dextran of Mw 110 kDa, according to the methods described above, using HR10/30 columns (GE Healthcare) packed with the prototypes in 0.2 M NaCl and with a range of dextran fractions as probe molecules (flow rate 0.2 ml/min). The dry weight of the bead samples ranged from 53 to 86 mg/ml, as determined by drying 1.0 ml drained filter cake samples at 105 °C over night and weighing.
    Table 6. Base matrix samples
    Base matrixKdd50v (µm)Dry weight (mg/ml)
    A18 0.704 59.0 56.0
    A20 0.70 69.2 55.8
    A27 0.633 87.2 74.2
    A28 0.638 67.4 70.2
    A29 0.655 92.6 57.5
    A32 0.654 73.0 70.5
    A33 0.760 73.1 55.5
    A38 0.657 70.9 56.2
    A39 0.654 66.0 79.1
    A40 0.687 64.9 74.9
    A41 0.708 81.7 67.0
    A42 0.638 88.0 59.4
    A43 0.689 87.5 77.0
    A45 0.670 56.6 66.0
    A52 0.620 53.10 63.70
    A53 0.630 52.6 86.0
    A54 0.670 61.3 75.3
    A55 0.640 62.0 69.6
    A56 0.740 61.0 56.0
    A56-2 0.740 51.0 56.0
    A62a 0.788 48.8 70.1
    A62b 0.823 50.0 46.9
    A63a 0.790 66.8 59.6
    A63b 0.765 54.0 79.0
    A65a 0.796 58.0 60.0
    A65b 0.805 57.3 46.0
    B5 0.793 69.0 84.4
    C1 0.699 71.0 73.4
    C2 0.642 66.5 81.1
    C3 0.711 62.0 82.0
    C4 0.760 62.0 82.0
    H31 0.717 82.0 59.0
    H35 0.710 81.1 61.0
    H40 0.650 52.8 65.0
    I1 0.640 50.0 67.0
    41 0.702 81.6 60.6
    517 0.685 87.9 64.4
    106 0.692 86.7 64.6
    531C 0.661 51.7 63.8
    P10 0.741 59.3 70.0
    S9 0.736 64.1 72.2

    Coupling



    [0096] 100 ml base matrix was washed with 10 gel volumes distilled water on a glass filter. The gel was weighed (1 g = 1 ml) and mixed with 30 ml distilled water and 8.08 g NaOH (0.202 mol) in a 250 ml flask with an agitator. The temperature was adjusted to 27 +/- 2 °C in a water bath. 16 ml epichlorohydrin (0.202 mol) was added under vigorous agitation (about 250 rpm) during 90 +/- 10 minutes. The reaction was allowed to continue for another 80 +/- 10 minutes and the gel was then washed with >10 gel volumes distilled water on a glass filter until neutral pH was reached. This activated gel was used directly for coupling as below.

    [0097] To 16.4 mL of ligand solution (50 mg/mL) in a 50 ml Falcon tube, 139 mg NaHCO3, 17.4 mg Na2CO3, 143.8 mg NaCl and 141 mg EDTA, was added. The Falcon tube was placed on a roller table for 5-10 min, and then 63 mg of DTE was added. Reduction proceeded for >45 min. The ligand solution was then desalted on a PD10 column packed with Sephadex G-25. The ligand content in the desalted solution was determined by measuring the 276 nm UV absorption.

    [0098] The activated gel was washed with 3-5 GV {0.1 M phosphate/1 mM EDTA pH 8.6} and the ligand was then coupled according to the method described in US6399750 5.2.2, although with considerably higher ligand amounts (see below). All buffers used in the experiments had been degassed by nitrogen gas for at least 5-10 min. The ligand content of the gels was controlled by varying the amount and concentration of the ligand solution, adding 5-20 mg ligand per ml gel. The ligand was either a tetramer (SEQ ID NO. 20) or a hexamer (SEQ ID NO. 33) of an alkali-stabilized mutant.

    [0099] After immobilization the gels were washed 3xGV with distilled water. The gels + 1 GV {0.1 M phosphate/1 mM EDTA/10% thioglycerol pH 8.6} was mixed and the tubes were left in a shaking table at room temperature overnight. The gels were then washed alternately with 3xGV {0.1 M TRIS/0.15 M NaCl pH 8.6} and 0.5 M HAc and then 8-10xGV with 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.

    Evaluation



    [0100] The Qb10 % dynamic capacity for polyclonal human IgG at 2.4 and 6 min residence time was determined as outlined in Example 4.
    Table 7. Prototype results
    PrototypeBase matrixLigand content (mg/ml)MultimerQb10% 2.4 min (mg/ml)Qb10% 6 min (mg/ml)
    N1 A38 7.45 tetramer 44.4 58.25
    N2 A20 7.3 tetramer 45.12 57.21
    N3 A42 6.72 tetramer 33.56 50.02
    N4 A29 7.3 tetramer 36.34 51.8
    N5 A28 7.9 tetramer 42.38 58.25
    N6 A39 6.96 tetramer 41.88 54.67
    N7 A27 7.5 tetramer 29.19 48.73
    N8 A43 6.99 tetramer 33.43 49.79
    N9 A38 11.34 tetramer 48.1 72.78
    N10 A20 10.6 tetramer 50.66 70.07
    N11 A42 11.1 tetramer 32.25 57.78
    N12 A29 11 tetramer 34.85 64.68
    N13 A28 11.9 tetramer 39.92 63.75
    N14 A39 10.48 tetramer 44.37 64.79
    N15 A27 12.1 tetramer 24.8 55.56
    N16 A43 10.51 tetramer 31.82 58.04
    N17 A41 8.83 tetramer 38.5 56.8
    N18 A41 8.83 tetramer 37.84 58.6
    N19 A41 8.83 tetramer 35.06 57.23
    N20 A41 5.0 tetramer 35.64 46.04
    N21 A41 13.0 tetramer 34.95 62.23
    N22 A40 13.15 tetramer 56.85 71.09
    N23 A33 7.33 tetramer 48.69 55.76
    N24 A40 11.03 tetramer 54.96 73.8
    033A A38 7.5 tetramer 44 58
    033B A38 11.3 tetramer 48 73
    097A A20 7.3 tetramer 45 57
    097B A20 10.6 tetramer 51 70
    003A A28 7.9 tetramer 42 58
    003B A28 11.9 tetramer 40 64
    003C A28 15.8 tetramer 37 67
    038A A39 7.0 tetramer 42 55
    038B A39 10.5 tetramer 44 65
    074 A40 13.2 tetramer 57 71
    093 A33 7.3 tetramer 49 56
    058A A40 11.0 tetramer 55 74
    077 A18 8.2 tetramer 52 59
    010 A32 10.7 tetramer 40 57
    099 A32 13.3 tetramer 37 66
    030A B5 6.3 tetramer 32 38
    030B B5 9.6 tetramer 45 47
    293A C1 5.4 tetramer 38 47
    293B C1 10.8 tetramer 43 60
    294A C2 5.1 tetramer 39 46
    294B C2 10.5 tetramer 42 57
    336A H40 5.6 tetramer 47 52
    336B H40 9.1 tetramer 52 67
    091 A18 13.4 tetramer N/A 63
    092 A20 12.8 tetramer 49 67
    080 A33 9.4 tetramer 51 58
    089 A40 6.1 tetramer 49 59
    688A A62a 6.6 tetramer 41 46
    688B A62a 14.8 tetramer 55 62
    871 A62a 9.7 tetramer 48 60
    934A A63a 6.6 tetramer 40 44
    934B A63a 14.0 tetramer 48 56
    017B A65a 13.1 tetramer 56 64
    041A A62b 5.2 tetramer 40 N/A
    041B A62b 11.1 tetramer 52 N/A
    116A A65b 5.8 tetramer 42 46
    116B A65b 8.8 tetramer 49 56
    017A A65a 6.1 tetramer 40 44
    387A A62a 6.4 tetramer 43 45
    387B A62a 7.5 tetramer 47 56
    432 A63a 6.1 tetramer 39 44
    433A A65a 6.6 tetramer 42 47
    433B A65a 13.6 tetramer 52 61
    579A I1 6.1 tetramer 45 51
    579B I1 11.2 tetramer 57 68
    064A C3 5.9 tetramer 44 52
    064B C3 9.0 tetramer 49 62
    064C C3 14.3 tetramer 51 70
    352A C4 10.1 tetramer 55 63
    352B C4 14.4 tetramer 59 67
    066A C3 6.8 hexamer 48 59
    066B C3 11.9 hexamer 51 73
    066C C3 15.1 hexamer 43 61
    353A C4 11.2 hexamer 62 74
    353B C4 15.2 hexamer 57 82
    872A A62a 9.6 hexamer 56 72
    872B A62a 14.5 hexamer 62 84
    869A H40 6.9 hexamer 50 56
    869B H40 14.3 hexamer 56 75
    869C H40 23.0 hexamer 41 65
    962A H35 6.8 hexamer 36 49
    962B H35 12.3 hexamer 31 54
    962C H35 20.3 hexamer 20 43
    112A A56 7.9 hexamer 47 55
    112B A56 12.4 hexamer 57 73
    112C A56 19.2 hexamer 55 80
    113A A56 7.1 hexamer 48 57
    113B A56 12.4 hexamer 53 73
    113C A56 15.2 hexamer 48 76
    212A H31 6.5 hexamer 37 38
    212B H31 10.4 hexamer 50 61
    212C H31 20.0 hexamer 31 52
    213A A33 6.5 hexamer 44 53
    213B A33 10.9 hexamer 50 65
    213C A33 11.1 hexamer 50 68
    432A A20 6.4 hexamer 41 56
    432B A20 12.4 hexamer 38 64
    432C A20 21.1 hexamer 44 43
    433A A38 5.9 hexamer 47 57
    433B A38 11.6 hexamer 48 72
    433C A38 15.8 hexamer 36 62
    742A A54 6.7 hexamer 38 46
    742B A54 12.6 hexamer 45 52
    742C A54 21.1 hexamer 38 65
    726A A63b 6.4 hexamer 42 46
    726B A63b 10.6 hexamer 49 60
    726C A63b 16.7 hexamer 53 69
    793A A56-2 6.8 hexamer 50 58
    793B A56-2 12.5 hexamer 59 72
    793C A56-2 19.2 hexamer 61 82
    517 517 12.0 tetramer* 35 56
    106 106 5.8 tetramer* 33 45
    531C 531C 11.2 tetramer* 54 65
    P10 P10 19.0 hexamer   76
    S9 S9 18.4 hexamer 56 75
    *SEQ ID NO 21

    Example 7



    [0101] A series of prototypes, prepared as above, with different ligand content (tetramer, SEQ ID NO:20) were incubated in 1 M NaOH for 4, 8 and 31 hours at 22 +/- 2 °C and the dynamic IgG capacity (Qb10%, 6 min residence time) was measured before and after incubation. The prototypes are shown in Table 8 and the results in Figs. 5 and 6. It can be seen that the stability towards this harsh alkali treatment increases with increasing ligand content.
    Table 8 Samples for incubation in 1 M NaOH
    PrototypeLigand content (mg/ml)Qb10%, 6 min, before incubation (mg/ml)
    N1 12 78
    LE28 13 79
    N17 16 73
    N16 20 73

    Example 8


    Pressure-flow testing of matrices



    [0102] 300 ml sedimented matrix was packed in a FineLine™ 35 column (GE Healthcare Life Sciences, Uppsala, Sweden), with 35 mm inner diameter and 330 mm tube height. The gel was suspended in distilled water to produce a slurry volume of 620 ml and the height of the packed bed was 300 +/- 10 mm. The packing pressure was 0.10 +/- 0.02 bar (10 +/- 2 kPa).

    [0103] Distilled water was then pumped through the column at increasing pump rates and the flow rate (expressed as the linear flow velocity, cm/h) and back pressure (MPa) was measured after 5 min for each pump setting. The measurements were continued until a max flow rate and a max pressure was reached, i.e. the flow rate and back pressure achieved when the flow rate starts to diminish at increasing back pressures.
    Table 9 Pressure flow performance of matrices
    MatrixMax flow velocity, cm/hMax pressure (MPa)
    517 1343 0.56
    106 1306 0.56
    531C 513 0.51
    P10 862 0.60
    S9 1172 0.64


    [0104] The P10 and S9 matrices have a higher rigidity, as indicated by the max pressure, and can thus sustain comparatively high flow velocities despite their low (59-64 micrometers) median particle diameters.

    SEQUENCE LISTING



    [0105] 

    <110> GE Healthcare Bioprocess R&D AB

    <120> MUTATED IMMUNOGLOBULIN-BINDING POLYPEPTIDES

    <130> 313838A

    <160> 95

    <170> PatentIn version 3.5

    <210> 1
    <211> 51
    <212> PRT
    <213> Staphylococcus aureus

    <400> 1

    <210> 2
    <211> 61
    <212> PRT
    <213> Staphylococcus aureus

    <400> 2

    <210> 3
    <211> 58
    <212> PRT
    <213> Staphylococcus aureus

    <400> 3

    <210> 4
    <211> 58
    <212> PRT
    <213> Staphylococcus aureus

    <400> 4

    <210> 5
    <211> 58
    <212> PRT
    <213> Staphylococcus aureus

    <400> 5

    <210> 6
    <211> 58
    <212> PRT
    <213> Escherichia coli

    <400> 6

    <210> 7
    <211> 58
    <212> PRT
    <213> Escherichia coli

    <400> 7

    <210> 8
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    <400> 8



    <210> 9
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    <400> 9

    <210> 10
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    <400> 10

    <210> 11
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    <400> 11



    <210> 12
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    <400> 14

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    <210> 18
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    <210> 20
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    <210> 21
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    <400> 21



    <210> 22
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    <400> 22

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    <400> 23



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    <210> 27
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    <210> 31
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    <210> 32
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    <210> 33
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    <210> 35
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    <210> 36
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    <400> 41

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    <400> 43



    <210> 44
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    <210> 47
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    <210> 49
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    <400> 49

    <210> 50
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    <400> 50

    <210> 51
    <211> 47
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    <400> 51

    <210> 52
    <211> 47
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    <400> 52

    <210> 53
    <211> 47
    <212> PRT
    <213> Escherichia coli

    <220>
    <221> misc-feature
    <222> (1)..(1)
    <223> Xaa can be any naturally occurring amino acid

    <220>
    <221> misc-feature
    <222> (3)..(3)
    <223> Xaa can be any naturally occurring amino acid

    <220>
    <221> misc-feature
    <222> (10)..(10)
    <223> Xaa can be any naturally occurring amino acid

    <220>
    <221> misc-feature
    <222> (20) .. (21)
    <223> Xaa can be any naturally occurring amino acid

    <220>
    <221> misc-feature
    <222> (24) .. (25)
    <223> Xaa can be any naturally occurring amino acid

    <220>
    <221> misc-feature
    <222> (29) .. (29)
    <223> Xaa can be any naturally occurring amino acid

    <220>
    <221> misc-feature
    <222> (32)..(32)
    <223> Xaa can be any naturally occurring amino acid

    <220>
    <221> misc-feature
    <222> (34)..(36)
    <223> Xaa can be any naturally occurring amino acid

    <220>
    <221> misc-feature
    <222> (42)..(43)
    <223> Xaa can be any naturally occurring amino acid

    <220>
    <221> misc-feature
    <222> (45)..(45)
    <223> Xaa can be any naturally occurring amino acid

    <400> 53

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    <210> 78
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    <400> 95




    Claims

    1. A separation matrix comprising at least 11, such as at least 15, 15-21, 17-21 or 18-20 mg/ml Fc-binding ligands covalently coupled to a porous support, wherein:

    a) said ligands comprise multimers of alkali-stabilized Protein A domains,

    b) said porous support comprises cross-linked agarose particles having a volume-weighted median diameter (d50,v) of 56-70 micrometers, a dry solids weight of 55-80 mg/ml, and

    a pore size corresponding to an inverse gel filtration chromatography Kd value of 0.69-0.85 for dextran of Mw 110 kDa.
     
    2. The separation matrix of any preceding claim, wherein said multimers comprise tetramers, pentamers, hexamers or heptamers of alkali-stabilized Protein A domains, such as hexamers of alkali-stabilized Protein A domains.
     
    3. The separation matrix of any preceding claim, having a 10% breakthrough dynamic binding capacity for IgG of at least 45 mg/ml, such as at least 50 or at least 55 mg/ml at 2.4 min residence time, and/or having a 10% breakthrough dynamic binding capacity for IgG of at least 60 mg/ml, such as at least 65, at least 70 or at least 75 mg/ml at 6 min residence time.
     
    4. The separation matrix of any preceding claim, wherein the 10% breakthrough dynamic binding capacity for IgG at 2.4 min or 6 min residence time is reduced by less than 20 % after incubation 31 h in 1.0 M aqueous NaOH at 22 +/- 2 C.
     
    5. The separation matrix of any preceding claim, having a dissociation constant for IgG2 of below 0.2 mg/ml, such as below 0.1 mg/ml, in 20 mM phosphate buffer, 180 mM NaCl, pH 7.5.
     
    6. The separation matrix of any preceding claim, which has a max pressure of at least 0.58, such as at least 0.60, MPa when packed at 300 +/-10 mm bed height in a FineLine™ 35 column.
     
    7. The separation matrix of any preceding claim, wherein said alkali-stabilized Protein A domains comprise mutants of a parental Fc-binding domain of Staphylococcus Protein A (SpA), as 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 SEQ ID NO 52, wherein at least the asparagine or serine residue at the 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.
     
    8. The separation matrix of claim 7, wherein:

    i) the amino acid residue at the position corresponding to position 11 in SEQ ID NO:4-7 is, or has been mutated to, a glutamic acid or a lysine;

    ii) the amino acid residue at the position corresponding to position 9 in SEQ ID NO:4-7 is an alanine;

    iii) the amino acid residue at the position corresponding to position 9 in SEQ ID NO:4-7 has been deleted;

    iv) the amino acid residue at the position corresponding to position 40 in SEQ ID NO:4-7 is, or has been mutated to, a valine;

    v) one or more of the amino acid residues at the positions corresponding to positions 1, 2, 3, 4, 5, 6, 7, 8, 56, 57 or 58 in SEQ ID NO: 4-7 have been deleted.


     
    9. The separation matrix of any one of claims 1-6, wherein said alkali-stabilized Protein A domains comprise an Fc-binding polypeptide having an amino acid sequence as defined by, or having at least 80% or at least 90, 95% or 98% identity to SEQ ID NO 53

    wherein individually of each other:

    X1=A or Q or is deleted

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

    X3=H or K

    X4=A or N

    X5=A, G, S,Y,Q,T,N,F,L,W,I,M,V,D,E,H,R or K X6=Q or E

    X7=S or K

    X8=E or D

    X9=Q or V or is deleted

    X10=K,R or A or is deleted X11=A,E or N or is deleted

    X12=I or L

    X13=K or R

    X14=L or Y

    X15=D, F,Y,W,K or R


     
    10. The separation matrix of claim 9, wherein individually of each other:

    X1 = A or is deleted, X2 = E, X3 = H, X4 = N, X6 = Q, X7 = S, X8 = D, X9 = V or is deleted,

    X10 = K or is deleted, X11 = A or is deleted, X12 = I, X13 = K, X14 = L.


     
    11. The separation matrix of any preceding claim, wherein the polypeptides are linked by linkers comprising up to 25 amino acids, such as 3-25 or 3-20 amino acids.
     
    12. The separation matrix of any preceding claim, wherein at least two polypeptides are linked by linkers comprising or consisting essentially of a sequence having at least 90% identity with an amino acid sequence selected from the group consisting of APKVDAKFDKE, APKVDNKFNKE, APKADNKFNKE, APKVFDKE, APAKFDKE, AKFDKE, APKVDA, VDAKFDKE, APKKFDKE, APK, APKYEDGVDAKFDKE and YEDG.
     
    13. A method of isolating an immunoglobulin, comprising the steps of:

    a) contacting a liquid sample comprising an immunoglobulin with a separation matrix according to any preceding claim,

    b) washing said separation matrix with a washing liquid,

    c) eluting the immunoglobulin from the separation matrix with an elution liquid, and

    d) cleaning the separation matrix with a cleaning liquid.


     
    14. The method of claim 13, wherein the cleaning liquid comprises 0.1 - 1.0 M NaOH or KOH, such as 0.4 - 1.0 M NaOH or KOH, and/or wherein steps a) - d) are repeated at least 10 times, such as at least 50 times or 50 - 200 times.
     


    Ansprüche

    1. Trennmatrix, umfassend mindestens 11, zum Beispiel mindestens 15, 15-21, 17-21 oder 18-20 mg/ml, Fc-bindende Liganden, die kovalent an einen porösen Träger gebunden sind, wobei:

    a) die Liganden Multimere von alkalisch stabilisierten Protein-A-Domänen umfassen,

    b) der poröse Träger vernetzte Agarosepartikel umfasst, die einen volumengewichteten mittleren Durchmesser (d50,v) von 56-70 Mikrometern, ein Trockenfeststoffgewicht von 55-80 mg/ml und eine Porengröße, die einem Kd-Wert bei inverser Gelfiltrationschromatografie von 0,69-0,85 für Dextran eines Mw von 110 kDa entspricht, aufweisen.


     
    2. Trennmatrix nach Anspruch 1, wobei die Multimere Tetramere, Pentamere, Hexamere oder Heptamere von alkalisch stabilisierten Protein-A-Domänen, zum Beispiel Hexamere von alkalisch stabilisierten Protein-A-Domänen, umfassen.
     
    3. Trennmatrix nach einem vorstehenden Anspruch, aufweisend eine dynamische Bindungskapazität für IgG bei 10 % Durchbruch von mindestens 45 mg/ml, zum Beispiel mindestens 50 oder mindestens 55 mg/ml, bei einer Verweilzeit von 2,4 min und/oder aufweisend eine dynamische Bindungskapazität für IgG bei 10% Durchbruch von mindestens 60 mg/ml, zum Beispiel mindestens 65, mindestens 70 oder mindestens 75 mg/ml, bei einer Verweilzeit von 6 min.
     
    4. Trennmatrix nach einem vorstehenden Anspruch, wobei die dynamische Bindungskapazität für lgG bei 10 % Durchbruch bei einer Verweilzeit von 2,4 min oder 6 min nach Inkubation für 31 h in 1,0 M wässrigem NaOH bei 22 ± 2 °C um weniger als 20 % reduziert ist.
     
    5. Trennmatrix nach einem vorstehenden Anspruch, aufweisend eine Dissoziationskonstante für lgG2 von unter 0,2 mg/ml, zum Beispiel bis zu 0,1 mg/ml, in 20 mM Phosphatpuffer, 180 mM NaCl, pH-Wert 7,5.
     
    6. Trennmatrix nach einem vorstehenden Anspruch, die einen maximalen Druck von mindestens 0,58, zum Beispiel mindestens 0,60 MPa, aufweist, wenn sie in einer Betthöhe von 300 ± -10 mm in einer FineLine™-35-Säule gepackt ist.
     
    7. Trennmatrix nach einem vorstehenden Anspruch, wobei die alkalisch stabilisierten Protein-A-Domänen Mutanten einer parenteralen Fc-bindenden Domäne von Staphylococcus-Protein-A (SpA), wie durch 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 oder SEQ ID NO: 52 definiert, umfassen, wobei mindestens der Asparagin- oder Serinrest an der Position, die Position 11 in SEQ ID NO: 4-7 entspricht, zu einer Aminosäure mutiert ist, die ausgewählt ist aus der Gruppe, bestehend aus Glutaminsäure, Lysin, Tyrosin, Threonin, Phenylalanin, Leucin, Isoleucin, Tryptophan, Methionin, Valin, Alanin, Histidin und Arginin.
     
    8. Trennmatrix nach Anspruch 7, wobei:

    i) der Aminosäurerest an der Position, die Position 11 in SEQ ID NO: 4-7 entspricht, eine Glutaminsäure oder ein Lysin ist oder dazu mutiert ist;

    ii) der Aminosäurerest an der Position, die Position 9 in SEQ ID NO: 4-7 entspricht, ein Alanin ist;

    iii) der Aminosäurerest an der Position, die Position 9 in SEQ ID NO: 4-7 entspricht, deletiert ist;

    iv) der Aminosäurerest an der Position, die Position 40 in SEQ ID NO: 4-7 entspricht, ein Valin ist oder dazu mutiert ist;

    iv) ein oder mehrere der Aminosäurereste an den Positionen, die den Positionen 1, 2, 3, 4, 5, 6, 7, 8, 56, 57 oder 58 in SEQ ID NO: 4-7 entsprechen, deletiert sind.


     
    9. Trennmatrix nach einem der Ansprüche 1-6, wobei die alkalisch stabilisierten Protein-A-Domänen ein Fc-bindendes Polypeptid umfassen, das eine Aminosäuresequenz aufweist, die wie definiert ist oder zu mindestens 80 % oder mindestens 90 %, 95 % oder 98% mit SEQ ID NO: 53 (X1Q X2AFYEILX3LP NLTEEQRX4X5F IXsX7LKDX8PSX9 SX10X11X12LAEAKX13 X14NX15AQ) identisch ist,
    wobei einzeln jedes andere:

    X1 = A oder Q oder deletiert

    X2 = E, K, Y, T, F, L, W, I, M, V, A, H oder R

    X3 = H oder K

    X4 = A oder N

    X5 = A, G, S, Y, Q, T, N, F, L, W, I, M, V, D, E, H, R oder K

    X6 = Q oder E

    X7 = S oder K

    Xg = E oder D

    X9 = Q oder V oder deletiert

    X10 = K, R oder A oder deletiert

    X11 = A, E oder N oder deletiert

    X12 = I oder L

    X13 = K oder R

    X14 = L oder Y

    X15 = D, F, Y, W, K oder R


     
    10. Trennmatrix nach Anspruch 9, wobei einzeln jedes andere:
    X1 = A oder deletiert, X2 = E, X3 = H, X4 = N, X6 = Q, X7 = S, X8 = D, X9 = V oder deletiert, X10 = K oder deletiert, X11 = A oder deletiert, X12 = I, X13 = K, X14 = L.
     
    11. Trennmatrix nach einem vorstehenden Anspruch, wobei die Polypeptide durch Linker, die bis zu 25 Aminosäuren, zum Beispiel 3-25 oder 3-20 Aminosäuren, umfassen, verbunden sind.
     
    12. Trennmatrix nach einem vorstehenden Anspruch, wobei mindestens zwei Polypeptide durch Linker verbunden sind, die eine Sequenz umfassen oder daraus bestehen, die zu mindestens 90% mit einer Aminosäuresequenz identisch ist, die ausgewählt ist aus der Gruppe, bestehend aus APKVDAKFDKE, APKVDNKFNKE, APKADNKFNKE, APKVFDKE, APAKFDKE, AKFDKE, APKVDA, VDAKFDKE, APKKFDKE, APK, APKYEDGVDAKFDKE und YEDG.
     
    13. Verfahren zum Isolieren eines Immunglobulins, wobei das Verfahren die folgenden Schritte umfasst:

    a) In-Kontakt-Bringen einer flüssigen Probe, die ein Immunglobulin umfasst, mit einer Trennmatrix nach einem vorstehenden Anspruch,

    b) Waschen der Trennmatrix mit einer Waschflüssigkeit,

    c) Eluieren des Immunglobulins aus der Trennmatrix mit einer Elutionsflüssigkeit und

    d) Reinigen der Trennmatrix mit einer Reinigungsflüssigkeit.


     
    14. Verfahren nach Anspruch 13, wobei die Reinigungsflüssigkeit 0,1-1,0 M NaOH oder KOH, zum Beispiel 0,4-1,0 M NaOH oder KOH, umfasst und/oder wobei die Schritte a) bis d) mindestens 10 Mal, zum Beispiel mindestens 50 Mal oder 50-200 Mal, wiederholt werden.
     


    Revendications

    1. Matrice de séparation comprenant au moins 11, comme au moins 15, 15-21, 17-21 ou 18-20 mg/ml de ligands se liant à Fc couplés de manière covalente à un support poreux, dans laquelle :

    a) lesdits ligands comprennent des multimères de domaines de protéine A stabilisés par alcalin,

    b) ledit support poreux comprend des particules d'agarose réticulé présentant un diamètre médian pondéré en volume (d50, v) de 56-70 micromètres, un poids de solides secs de 55-80 mg/ml, et une taille de pore correspondant à une valeur de Kd par chromatographie par filtration sur gel inverse de 0,69-0,85 pour un dextrane de Mw de 110 kDa.


     
    2. Matrice de séparation selon la revendication 1, dans laquelle lesdits multimères comprennent des tétramères, des pentamères, des hexamères ou des heptamères de domaines de protéine A stabilisés par alcalin, tels que des hexamères de domaines de protéine A stabilisés par alcalin.
     
    3. Matrice de séparation selon une quelconque revendication précédente, présentant une capacité de liaison dynamique à une restitution de 10% pour une IgG d'au moins 45 mg/ml, comme au moins 50 ou au moins 55 mg/ml à un temps de séjour de 2,4 min, et/ou présentant une capacité de liaison dynamique à une restitution de 10 % pour une IgG d'au moins 60 mg/ml, comme au moins 65, au moins 70 ou au moins 75 mg/ml à un temps de séjour de 6 min.
     
    4. Matrice de séparation selon une quelconque revendication précédente, dans laquelle la capacité de liaison dynamique à une restitution de 10% pour une IgG à un temps de séjour de 2,4 min ou 6 min est réduite de moins de 20 % après 31 h d'incubation dans du NaOH aqueux 1,0 M à 22 +/- 2 °C.
     
    5. Matrice de séparation selon une quelconque revendication précédente, présentant une constante de dissociation pour une lgG2 inférieure à 0,2 mg/ml, comme inférieure à 0,1 mg/ml, dans un tampon au phosphate à 20 mM, NaCl à 180 mM, pH 7,5.
     
    6. Matrice de séparation selon une quelconque revendication précédente, qui présente une pression maximale d'au moins 0,58 MPa, comme au moins 0,60 MPa, quand elle est introduite à une hauteur de lit de 300 +/- 10 mm dans une colonne FineLine™ 35.
     
    7. Matrice de séparation selon une quelconque revendication précédente, dans laquelle lesdits domaines de protéine A stabilisés par alcalin comprennent des mutants d'un domaine parental se liant à Fc d'une protéine A de Staphylococcus (SpA), comme défini par 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 ou SEQ ID NO 52, dans laquelle au moins le résidu asparagine ou sérine à la position correspondant à la position 11 dans SEQ ID NO : 4-7 a été muté en un acide aminé sélectionné dans le groupe consistant en l'acide glutamique, la lysine, la tyrosine, la thréonine, la phénylalanine, la leucine, l'isoleucine, le tryptophane, la méthionine, la valine, l'alanine, l'histidine et l'arginine.
     
    8. Matrice de séparation selon la revendication 7, dans laquelle :

    i) le résidu d'acide aminé à la position correspondant à la position 11 dans SEQ ID NO : 4-7 est, ou a été muté en, un acide glutamique ou une lysine ;

    ii) le résidu d'acide aminé à la position correspondant à la position 9 dans SEQ ID NO : 4-7 est une alanine ;

    iii) le résidu d'acide aminé à la position correspondant à la position 9 dans SEQ ID NO : 4-7 a été supprimé ;

    iv) le résidu d'acide aminé à la position correspondant à la position 40 dans SEQ ID NO : 4-7 est, ou a été muté en, une valine ;

    v) un ou plusieurs des résidus d'acide aminé aux positions correspondant aux positions 1, 2, 3, 4, 5, 6, 7, 8, 56, 57 ou 58 dans SEQ ID NO : 4-7 ont été supprimés.


     
    9. Matrice de séparation selon l'une quelconque des revendications 1-6, dans laquelle lesdits domaines de protéine A stabilisés par alcalin comprennent un polypeptide se liant à Fc présentant une séquence d'acides aminés telle que définie par, ou présentant au moins 80 % ou au moins 90, 95 % ou 98 % d'identité avec SEQ ID NO 53

    dans lequel individuellement les uns des autres :

    X1 = A ou Q ou est supprimé

    X2 = E, K, Y, T, F, L, W, I, M, V, A, H ou R

    X3 = H ou K

    X4 = A ou N

    X5 = A, G, S, Y, Q, T, N, F, L, W, I, M, V, D, E, H, R ou K

    X6 = Qou E

    X7 = S ou K

    Xg = E ou D

    X9 = Q ou V ou est supprimé

    X10 = K, R ou A ou est supprimé

    X11 = A, E ou N ou est supprimé

    X12 = I ou L

    X13 = K ou R

    X14 = L ou Y

    X15 = D, F, Y, W, K ou R.


     
    10. Matrice de séparation selon la revendication 9, dans laquelle individuellement les uns des autres :
    X1 = A ou est supprimé, X2 = E, X3 = H, X4 = N, X6 = Q, X7 = S, X5 = D, X9 = V ou est supprimé, X10 = K ou est supprimé, X11 = A ou est supprimé, X12 = I, X13 = K, X14 = L.
     
    11. Matrice de séparation selon une quelconque revendication précédente, dans laquelle les polypeptides sont liés par des lieurs comprenant jusqu'à 25 acides aminés, comme 3-25 ou 3-20 acides aminés.
     
    12. Matrice de séparation selon une quelconque revendication précédente, dans laquelle au moins deux polypeptides sont liés par des lieurs comprenant ou consistant essentiellement en une séquence présentant au moins 90 % d'identité avec une séquence d'acides aminés sélectionnée dans le groupe consistant en APKVDAKFDKE, APKVDNKFNKE, APKADNKFNKE, APKVFDKE, APAKFDKE, AKFDKE, APKVDA, VDAKFDKE, APKKFDKE, APK, APKYEDGVDAKFDKE et YEDG.
     
    13. Procédé d'isolement d'une immunoglobuline, comprenant les étapes suivantes :

    a) la mise en contact d'un échantillon liquide comprenant une immunoglobuline avec une matrice de séparation selon une quelconque revendication précédente,

    b) le lavage de ladite matrice de séparation avec un liquide de lavage,

    c) l'élution de l'immunoglobuline à partir de la matrice de séparation avec un liquide d'élution, et

    d) le nettoyage de la matrice de séparation avec un liquide de nettoyage.


     
    14. Procédé selon la revendication 13, dans lequel le liquide de nettoyage comprend 0,1-1,0 M de NaOH ou de KOH, comme 0,4-1,0 M de NaOH ou de KOH, et/ou dans lequel les étapes a)-d) sont répétées au moins 10 fois, comme au moins 50 fois ou 50-200 fois.
     




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    REFERENCES CITED IN THE DESCRIPTION



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    Patent documents cited in the description




    Non-patent literature cited in the description