Patent Description:
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 immunoglobulin manufacturing processes whilst controlling the associated costs.

Affinity chromatography is used in most cases, as one of the key steps in the purification of these immunoglobulin molecules, such as monoclonal antibodies (mAbs) or polyclonal antibodies (pAbs). 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.

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.

An ongoing trend in the biopharmaceutical industry is the use of versatile multi-product production facilities instead of single-product production facilities, allowing production-on-demand of biopharmaceuticals and a greater product variety, e.g. personalized or orphan biopharmaceuticals. Production campaigns in such multi-product facilities are shorter and there is a need to effectively store the affinity separation matrix between campaigns.

A common medium for storing separation matrices between campaigns is sodium hydroxide. According to the PDA Biotechology Cleaning Validation Committee, concentrations of <NUM> to <NUM> sodium hydroxide are common for storing packed chromatography columns. However, such storage conditions are associated with exposing the matrix to solutions with pH-values above <NUM> for long periods. For many affinity chromatography matrices containing proteinaceous affinity ligands such alkaline environment is a very harsh condition and consequently results in decreased capacity of the affinity separation matrix owing to instability of the ligand to the high pH involved.

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. (<NPL>) 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, Gülichet 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.

Recent work shows that changes can also be made to protein A (SpA) to effect similar properties. US patent application publication <CIT>, which is hereby incorporated by reference in its entirety, 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 <NUM>-<NUM> 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 (<CIT>, incorporated by reference in its entirety). 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 <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT> and <CIT>. However, the currently available mutants are still sensitive to alkaline pH and the corresponding affinity separation matrices are therefore typically stored in <NUM>% ethanol solution or <NUM>% benzyl alcohol solution.

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

The inventors of the present invention have recognised that alcohol solutions are suboptimal for the storage of affinity separation matrices. Alcohols are flammable, subject to regulation, and difficult to dispose of. The inventors have recognised that aqueous alkali metal hydroxide solutions have a number of advantages as storage solutions. Alkali metal hydroxide solutions are bactericidal or bacteriostatic depending on concentration. They can inactive most viruses, bacteria, yeasts, fungi and endotoxins. They are relatively cheap, easily disposed and removal from the separation matrix is simple to detect using pH and/or conductivity measurements.

It is therefore an object of the present invention to provide an affinity separation matrix for the purification of immunoglobulins that is stored in alkali metal hydroxide solution. It is a further object of the present invention to provide a method for storing an affinity separation matrix for the purification of immunoglobulins in an alkali metal hydroxide solution.

These objects are achieved by the method according to the appended claims of storing a separation matrix comprising multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support. The alkali-stabilized Protein A domains comprise mutants of a parental Fc-binding domain of Staphylococcus Protein A (SpA), as defined by SEQ ID NO <NUM> or SEQ ID NO <NUM>, wherein the amino acid residues at positions <NUM> and <NUM> of SEQ ID NO <NUM> or <NUM> are asparagines and wherein at least the asparagine residue at position <NUM> of SEQ ID NO <NUM> or <NUM> 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. The method comprises the steps of:.

By using a separation matrix comprising multimers of immunoglobulin-binding alkali-stabilized Protein A domains as defined above, a highly alkali-stable separation matrix having a high dynamic binding capacity is obtained. The inventors of the present invention have observed that such separation matrices are stable upon immersion in aqueous alkali metal hydroxide solution for extended periods such as five days or more, and substantially retain dynamic binding capacity after such prolonged immersion. This means that such a separation matrices are suitable for storage in alkali metal hydroxide solutions.

Further mutations to the immunoglobulin-binding alkali-stabilized Protein A domains may provide further enhancement of properties such as enhanced alkali stability. For example, the glutamine residue at position <NUM> of SEQ ID NO <NUM> or <NUM> may be mutated to an alanine; and/or the asparagine or glutamic acid residue at position <NUM> of SEQ ID NO <NUM> or <NUM> may be mutated to an alanine.

The multimers of immunoglobulin-binding alkali-stabilized Protein A domains may be homomultimers selected from the group consisting of dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers or nonamers. By using an appropriate multimer, the immunoglobulin binding capacity and alkali stability of the separation matrix may be increased.

The multimers of immunoglobulin-binding alkali-stabilized Protein A domains may each comprise a C-terminal cysteine residue for covalent coupling to the porous support. The multimers of immunoglobulin-binding alkali-stabilized Protein A domains may be coupled to the porous support via thioether links. This provides a robust, alkali-stable and well-proven method of attaching the ligands to the solid support.

The separation matrix may comprise at least <NUM>/ml, such as at least <NUM>/ml, of the multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to the porous support. This ensures a separation matrix with a good binding capacity.

The porous support may comprise cross-linked polymer particles having a volume-weighted median diameter (d50,v) of <NUM>-<NUM> micrometers and a dry solids weight of <NUM>-<NUM>/ml. The porous support may for example be highly cross-linked agarose beads.

The aqueous alkali metal hydroxide solution used in the storage liquid may be sodium hydroxide solution, potassium hydroxide solution or a mixture thereof, preferably sodium hydroxide solution. Sodium hydroxide solution is relatively cheap, readily available and widely accepted for use as a storage solution. The aqueous alkali metal hydroxide solution may have a molarity of from <NUM> to <NUM>, such as from <NUM> to <NUM>. This ensures a solution with a stable pH and good bacteriostatic or bactericidal properties.

The storage liquid may in some instances further comprise a C<NUM> - C<NUM> alcohol, such as ethanol, isopropanol or benzyl alcohol. A storage liquid combining an alcohol and an alkali metal hydroxide may be more effective in inactivating certain microorganisms, such as some spore-forming bacteria.

The storage liquid may comprise at least <NUM>% by volume aqueous alkali metal hydroxide solution, such as at least <NUM>% by volume aqueous alkali metal hydroxide solution, preferably at least <NUM>% by volume aqueous alkali metal hydroxide solution.

In some instances the storage liquid may consist of, or consist essentially of, aqueous alkali metal hydroxide solution.

The minimum storage time for the separation matrix may be as short a time as storage is required, such as at least <NUM> days, such as at least <NUM> days, such as at least <NUM> days, such as at least <NUM> days, or such as at least <NUM> days. The maximum storage time for the separation matrix may be as long a time as storage is required, such as up to <NUM> days, or such as up to <NUM> days.

Prior to storing, the separation matrix may be cleaned and/or sanitized with a cleaning fluid, wherein the cleaning fluid comprises at least <NUM>% by volume of an aqueous alkali metal hydroxide solution and wherein the aqueous alkali metal hydroxide solution has a molarity of from <NUM> to <NUM>, such as from <NUM> to <NUM>. The cleaning fluid may consist of, or consist essentially of, aqueous alkali metal hydroxide solution. Thus, the separation matrix may be cleaned, sanitized and stored with little or no requirement for using alcohols.

The separation matrix retains at least <NUM>% of its original dynamic binding capacity, such as at least <NUM>% of its original dynamic binding capacity, after step b), i.e. after prolonged storage. Thus, the separation matrix may be stored in aqueous alkali metal hydroxide solution without subsequent excessive negative impact on its ability to purify immunoglobulins.

According to the present disclosure the objects of the present invention are achieved by use of a storage liquid, said use of a storage liquid not being comprised in the claimed invention. That is to say, use of a storage liquid comprising at least <NUM>% by volume of an aqueous alkali metal hydroxide solution for the storage of a separation matrix comprising multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support.

The storage liquid may be the same as the storage liquid previously described above in relation to the method of storing a separation matrix. For example, it may comprise, consist essentially of, or consist of, sodium hydroxide solution having a molarity of from <NUM> to <NUM>, such as from <NUM> to <NUM>.

The objects of the present invention may be achieved by a separation matrix product, said separation matrix product not being comprised in the claimed invention. The separation matrix product comprises a storage receptacle, a separation matrix and a storage liquid. The storage receptacle contains the separation matrix permeated with the storage liquid. The separation matrix comprises multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support, wherein 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 <NUM>% such as at least <NUM>%, <NUM>% or <NUM>% identity to, SEQ ID NO <NUM> or SEQ ID NO <NUM>, wherein the amino acid residues at positions <NUM> and <NUM> of SEQ ID NO <NUM> or <NUM> are asparagines and wherein at least the asparagine residue at position <NUM> of SEQ ID NO <NUM> or <NUM> 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. The storage liquid comprises at least <NUM>% by volume of an aqueous alkali metal hydroxide solution.

Thus, it is possible to package, store and transport separation matrices stored in aqueous alkali metal hydroxide solution. This avoids the requirement of storing in alcohol solution and thus avoids the need for using volatile and flammable components in the storage liquid.

The storage receptacle may for example be a bottle, can or drum made from a liquid-impervious material such as plastic or glass. The storage receptacle may also be a pre-packable column, i.e. a separation column that is filled with separation matrix at the production site.

Further objects, advantages and novel features of the present invention will become apparent to one skilled in the art from the following detailed description.

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.

The terms an "Fc-binding polypeptide", "Fc-binding domain" and "Fc-binding protein" mean a polypeptide, domain 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.

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

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

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 (BLASTTM) described in <NPL>. A web-based software for this is freely available from the US National Library of Medicine at http://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. the % identity.

As used herein, the terms "comprises," "comprising," "containing," "having" and the like can have the meaning ascribed to them in U. Patent law and can mean "includes," "including," and the like; "consisting essentially of' or "consists essentially" likewise has the meaning ascribed in U. 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.

For a fuller understanding of the present invention and further objects and advantages of it, the detailed description set out below should be read together with the accompanying figures, and in which:.

The present invention concerns a method of storing a separation matrix comprising multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support.

Throughout this detailed description, two separate numbering conventions may be used. Unless otherwise stated, the amino acid residue position numbering convention of <FIG> is used, and the position numbers are designated as corresponding to those in SEQ ID NO <NUM>-<NUM>. This applies also to multimers, where the position numbers designate the positions in the polypeptide units or monomers according to the convention of <FIG>, unless otherwise stated. However, throughout the claims, summary of invention and on occasion in the detailed description, the position numbers corresponding to those of SEQ ID NO <NUM> and <NUM> are used. Note that position <NUM> of SEQ ID NO <NUM> or SEQ ID NO <NUM> corresponds to position <NUM> of SEQ ID NO <NUM>-<NUM>, and in this manner the different numbering conventions may be interconverted.

The immunoglobulin-binding alkali-stabilized Protein A domains of the invention, also termed herein as "the polypeptide", comprise mutants of a parental Fc-binding domain of Staphylococcus Protein A (SpA), as defined by SEQ ID NO <NUM> or SEQ ID NO <NUM>, wherein the amino acid residues at positions <NUM> and <NUM> of SEQ ID NO <NUM> or <NUM> are asparagines and wherein at least the asparagine residue at position <NUM> of SEQ ID NO <NUM> or <NUM> 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 immunoglobulin-binding alkali-stabilized Protein A domains may comprise mutants of a parental Fc-binding domain of Staphylococcus Protein A (SpA), as defined by, or having at least <NUM>%, at least <NUM>% or at least <NUM>% identity to, SEQ ID NO: <NUM> (E-domain), SEQ ID NO: <NUM> (D-domain), SEQ ID NO:<NUM> (A-domain), SEQ ID NO:<NUM> (variant A-domain), SEQ ID NO: <NUM> (B-domain), SEQ ID NO: <NUM> (C-domain), SEQ ID NO:<NUM> (Protein Z), SEQ ID NO:<NUM> (Zvar), or as defined by SEQ ID NO <NUM> (Zvar without the linker region amino acids <NUM>-<NUM> and <NUM>-<NUM>) or SEQ ID NO <NUM> (C-domain without the linker region amino acids <NUM>-<NUM> and <NUM>-<NUM>) as illustrated in <FIG>, wherein at least the asparagine (or serine, in the case of SEQ ID NO <NUM>) residue at the position corresponding to position <NUM> in SEQ ID NO:<NUM>-<NUM> 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, and wherein the asparagine residues corresponding to positions <NUM> and <NUM> in SEQ ID NO:<NUM>-<NUM> (positions <NUM> and <NUM> of SEQ ID NO <NUM> or <NUM>) are conserved.

A number of the Fc-binding domains listed above are shown aligned in <FIG>. The parental, i.e. non-engineered, Staphylococcus Protein A (SpA) comprises five Fc-dining domains termed domain E (SEQ ID NO <NUM>), D (SEQ ID NO <NUM>), A (SEQ ID NO <NUM>), B (SEQ ID NO <NUM>) and C (SEQ ID NO <NUM>). Protein Z (SEQ ID NO:<NUM>) is a mutated B-domain as disclosed in <CIT>, hereby incorporated by reference in its entirety. SEQ ID NO <NUM> denotes a further mutated variant of Protein Z, here called Zvar, with the mutations N3A,N6D,N23T. SEQ ID NO:<NUM> (not shown in <FIG>) 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>. SEQ ID NO <NUM> is Zvar (SEQ ID NO <NUM>) without the linker region amino acids at positions <NUM>-<NUM> and <NUM>-<NUM>. SEQ ID NO <NUM> is the C-domain of protein A without the linker region amino acids <NUM>-<NUM> and <NUM>-<NUM>) as illustrated in <FIG>.

The mutation of N11 (N3 of SEQ ID NO <NUM>:<NUM>) in these domains, together with the conservation of the asparagine residues N21 and N52 (N13 and N44 of SEQ ID NO <NUM>:<NUM>) 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.

In one embodiment, the immunoglobulin-binding alkali-stabilized Protein A domains may comprise a sequence as defined by SEQ ID NO <NUM>.

Specifically, the amino acid residues in SEQ ID NO <NUM> may individually of each other be:.

In certain embodiments, the amino acid residues in SEQ ID NO <NUM> may be:
X<NUM>=A, X<NUM> = E, X<NUM> = H, X<NUM> = N, X<NUM> = Q, X<NUM> = S, X<NUM> = D, X<NUM> = V, X<NUM> = K, X<NUM> = A, X<NUM> = I, X<NUM> = K, X<NUM> = L. In some embodiments X<NUM> = E, X<NUM> = H, X<NUM> = N, X<NUM>=A, X<NUM> = Q, X<NUM> = S, X<NUM> = D, X<NUM> = I, X<NUM> = K, X<NUM> = L and X<NUM>=D and one or more of X<NUM>, X<NUM>, X<NUM> and X<NUM> is deleted. In further embodiments, X<NUM>=A, X<NUM> = E, X<NUM> = H, X<NUM> = N, X<NUM>= S,Y,Q,T,N,F,L,W,I,M,V,D,E,H,R or K, X<NUM> = Q, X<NUM> = S, X<NUM> = D, X<NUM> = V, X<NUM> = K, X<NUM> = A, X<NUM> = I, X<NUM> = K, X<NUM> = L and X<NUM>=D, or alternatively X<NUM>=A, X<NUM> = E, X<NUM> = H, X<NUM> = N, X<NUM>=A, X<NUM> = Q, X<NUM> = S, X<NUM> = D, X<NUM> = V, X<NUM> = K, X<NUM> = A, X<NUM> = I, X<NUM>= K, X<NUM> = Land X<NUM>= F,Y,W,K or R.

In some embodiments, the amino acid residues may individually of each other be:.

The N11 (X<NUM>) 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 <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> in SEQ ID NO:<NUM>-<NUM>. 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 <NUM>-<NUM> and/or from positions <NUM>-<NUM>.

In some embodiments, the amino acid residue at the position corresponding to position <NUM> in SEQ ID NO:<NUM>-<NUM> (X<NUM>) 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 <NUM> and <NUM> provides particularly good alkali stability, as shown by the examples. In specific embodiments, in SEQ ID NO: <NUM> the amino acid residue at position <NUM> is an alanine and the amino acid residue at position <NUM> is a lysine or glutamic acid, such as a lysine. Mutations at position <NUM> are also discussed in co-pending application <CIT>.

In some embodiments, the amino acid residue at the position corresponding to position <NUM> in SEQ ID NO:<NUM>-<NUM> (X<NUM>) is an arginine or a glutamic acid.

In certain embodiments, the amino acid residue at the position corresponding to position <NUM> in SEQ ID NO:<NUM>-<NUM> is an alanine and/or the amino acid residue at the position corresponding to position <NUM> in SEQ ID NO:<NUM>-<NUM> is an aspartic acid. One of the amino acid residues at positions <NUM> and <NUM> may be an asparagine and in an alternative embodiment both amino acid residues at positions <NUM> and <NUM> may be asparagines.

In some embodiments the amino acid residue at the position corresponding to position <NUM> in SEQ ID NO:<NUM>-<NUM> (X<NUM>) 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 <NUM> and <NUM> in SEQ ID NO: <NUM> are alanine and lysine/glutamic acid respectively, while the amino acid residue at position <NUM> is alanine or glutamic acid.

In certain embodiments the amino acid residue at the position corresponding to position <NUM> in SEQ ID NO:<NUM>-<NUM> (X<NUM>) is an alanine or an asparagine, such as an alanine.

In some embodiments the amino acid residue at the position corresponding to position <NUM> in SEQ ID NO:<NUM>-<NUM> (X<NUM>) is selected from the group consisting of asparagine, alanine, glutamic acid and valine, or from the group consisting of glutamic acid and valine, or valine, or it can be deleted. In specific embodiments, the amino acid residues at positions <NUM> and <NUM> in SEQ ID NO: <NUM> are alanine and glutamic acid respectively, while the amino acid residue at position <NUM> is valine. Optionally, the amino acid residue at position <NUM> may then be alanine or glutamic acid.

In certain embodiments, the amino acid residue at the position corresponding to position <NUM> in SEQ ID NO:<NUM>-<NUM> (X<NUM>) is an alanine, lysine or arginine or it can be deleted.

In some embodiments the amino acid residue at the position corresponding to position <NUM> in SEQ ID NO:<NUM>-<NUM> (X<NUM>) is a lysine or a histidine, such as a lysine.

In certain embodiments the amino acid residue at the position corresponding to position <NUM> in SEQ ID NO:<NUM>-<NUM> (X<NUM>) is a lysine or a serine, such as a lysine.

In some embodiments the amino acid residue at the position corresponding to position <NUM> in SEQ ID NO:<NUM>-<NUM> (X<NUM>) is a glutamic acid or an aspartic acid, such as a glutamic acid.

In certain embodiments the amino acid residue at the position corresponding to position <NUM> in SEQ ID NO:<NUM>-<NUM> (X<NUM>) is a tyrosine or a leucine, such as a tyrosine.

In some embodiments, the amino acid residue at the position corresponding to position <NUM> in SEQ ID NO:<NUM>-<NUM> (X<NUM>) is a leucine or an isoleucine. In specific embodiments, the amino acid residues at positions <NUM> and <NUM> in SEQ ID NO: <NUM> are alanine and lysine/glutamic acid respectively, while the amino acid residue at position <NUM> is isoleucine. Optionally, the amino acid residue at position <NUM> may then be alanine or glutamic acid.

In some variants, the amino acid residues at the positions corresponding to positions <NUM>, <NUM>, <NUM> and <NUM> or to positions <NUM>, <NUM>, <NUM> and <NUM> in SEQ ID NO: <NUM>-<NUM> have been deleted. In specific variants, the parental polypeptide is the C domain of Protein A (SEQ ID NO: <NUM>). The effects of these deletions on the native C domain are described in <CIT> and <CIT>.

In certain embodiments, the mutation in SEQ ID NO <NUM> or <NUM> may correspond to the mutation in SEQ ID NO <NUM>-<NUM>, such as in SEQ ID NO <NUM>, 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,N11E,Q40V,A42K,N43E,L441; Q9A,N11E,Q40V,A42K,N43A,L441; 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,N11E,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 <NUM> or <NUM> may also correspond to the mutation in SEQ ID NO <NUM>-<NUM>, such as in SEQ ID NO <NUM>, 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.

The polypeptide may comprise or consist essentially of a sequence defined by an amino acid sequence selected from the group consisting of: SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM> and SEQ ID NO <NUM>. It may e.g. comprise or consist essentially of a sequence defined an amino acid sequence selected from the group consisting of: SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM> and SEQ ID NO <NUM>. It can also comprise or consist essentially of a sequence defined by an amino acid sequence selected from the group consisting of: SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>; SEQ ID NO <NUM>; SEQ ID NO <NUM>; SEQ NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM>, SEQ ID NO <NUM> and SEQ ID NO <NUM>.

The polypeptide may comprise or consist essentially of a sequence defined by an amino acid sequence selected from the group consisting of SEQ ID NO <NUM>-<NUM>; comprises or consists essentially of a sequence defined by an amino acid sequence selected from the group consisting of SEQ ID NO <NUM>-<NUM>; or it may comprise or consist essentially of a sequence defined by an amino acid sequence selected from the group consisting of SEQ ID NO <NUM>-<NUM>. It may further comprise or consist essentially of a sequence defined by an amino acid sequence selected from the group consisting of SEQ ID NO <NUM>-<NUM>.

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.

The separation matrix comprises multimers of the immunoglobulin-binding alkali-stabilized Protein A domains. Such multimers comprise, consist essentially of, or consist of a plurality of immunoglobulin-binding alkali-stabilized Protein A domains (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. The multimer may 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/conservations 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 <NUM> or <NUM> amino acids, such as <NUM>-<NUM> or <NUM>-<NUM> amino acids. The linkers may e.g. comprise or consist essentially of a peptide sequence defined by, or having at least <NUM>% identity or at least <NUM>% 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 <NUM>% identity or at least <NUM>% 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.

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 <NUM> or less, such as <NUM> or less, such as <NUM> or less or <NUM> or less. As a specific example, the multimer may comprise an AQ, AQGT, VDAKFDKE, AQVDAKFDKE or AQGTVDAKFDKE sequence at the N-terminal end.

The multimer may comprise, or consist essentially, of a sequence selected from the group consisting of: SEQ ID NO <NUM>-<NUM>. These and additional sequences are listed below and named as Parent(Mutations)n, where n is the number of monomer units in a multimer.

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 <NUM>-<NUM> amino acid residues, such as within <NUM>-<NUM> or <NUM>-<NUM> 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 <NUM> amino acids, such as <NUM>-<NUM>, <NUM>-<NUM> or <NUM>-<NUM> 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.

The alkali stability of the polypeptide or multimer can be assessed by coupling it to a surface plasmon resonance (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. <NUM> +/- <NUM>. The incubation can e.g. be performed in <NUM> NaOH for a number of <NUM> cycles, such as <NUM>, <NUM> or <NUM> cycles. The IgG capacity of the matrix after <NUM><NUM> incubation cycles in <NUM> NaOH at <NUM> +/- <NUM> can be at least <NUM>, such as at least <NUM>, at least <NUM> or at least <NUM>% of the IgG capacity before the incubation. Alternatively, the remaining IgG capacity after <NUM> 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 <NUM>%, such as at least <NUM>%, at least <NUM>%, at least <NUM>% or at least <NUM>% of the parental polypeptide/multimer.

The immunoglobulin-binding alkali-stabilized Protein A domains and/or multimers thereof may be encoded by a nucleic acid sequence, such as an RNA sequence or a DNA sequence encoding the polypeptide or multimer. A vector, such as a plasmid, which in addition to the coding sequence comprises the required signal sequences, may be used for expression of the polypeptide or multimer. The vector may comprise nucleic acid encoding a multimer as described above, wherein the separate nucleic acids encoding each unit may have homologous or heterologous DNA sequences.

An expression system, which comprises a nucleic acid or a vector as disclosed above, may be used for expression of the polypeptide or multimer. The expression system may e.g. be a gram-positive or gram-negative prokaryotic host cell system, e.g. E. coli or Bacillus sp. which has been modified to express the present polypeptide or multimer. Alternatively, the expression system may be a eukaryotic host cell system, such as a yeast, e.g. Pichia pastoris or Saccharomyces cerevisiae, or mammalian cells, e.g. CHO cells.

The separation matrix comprises multimers of immunoglobulin-binding alkali-stabilized Protein A domains as described above, covalently coupled to a porous support.

The separation matrix may comprise at least <NUM>, such as <NUM>-<NUM>, <NUM>-<NUM> or <NUM>-<NUM>/ml Fc-binding ligands covalently coupled to a porous support, wherein:.

The alkali-stabilized Protein A domain multimers are highly selective for IgG and the separation matrix can suitably have a dissociation constant for human IgG2 of below <NUM>/ml, such as below <NUM>/ml, in <NUM> phosphate buffer, <NUM> NaCl, pH <NUM>. This can be determined according to the adsorption isotherm method described in<NPL>).

In certain embodiments the separation matrix comprises at least <NUM>, such as <NUM>-<NUM> or <NUM>-<NUM>/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.

In some embodiments the separation matrix comprises <NUM> - <NUM>, such as <NUM>-<NUM>/ml, <NUM>-<NUM>/ml, <NUM>-<NUM>/ml or <NUM> - <NUM>/ml of the polypeptide or multimer coupled to the support. 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.

Such a separation 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. <NUM> +/-<NUM>. The incubation can e.g. be performed in <NUM> or <NUM> NaOH for a number of <NUM> cycles, such as <NUM>, <NUM> or <NUM> cycles, corresponding to a total incubation time of <NUM>, <NUM> or <NUM>. The IgG capacity of the matrix after <NUM>-<NUM><NUM> incubation cycles or a total incubation time of <NUM> or <NUM> in <NUM> NaOH at <NUM> +/- <NUM> can be at least <NUM>, such as at least <NUM>, at least <NUM> or at least <NUM>% of the IgG capacity before the incubation. The capacity of the matrix after a total incubation time of <NUM> in <NUM> NaOH at <NUM> +/- <NUM> can be at least <NUM>, such as at least <NUM> or at least <NUM>% of the IgG capacity before the incubation. The the <NUM>% breakthrough dynamic binding capacity (Qb10%) for IgG at <NUM> or <NUM> residence time may e.g. be reduced by less than <NUM> % after incubation <NUM> in <NUM> aqueous NaOH at <NUM> +/- <NUM>.

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.

The porous support of the separation matrix may be of any suitable well-known kind. 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 (-CONH<NUM>, possibly in N- substituted forms), amino (-NH<NUM>, possibly in substituted form), oligo- or polyethylenoxy groups on their external and, if present, also on internal surfaces. The porosity of the support 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<NPL>. Kav is determined as the ratio (Ve-V<NUM>)/(Vt-V<NUM>), where Ve is the elution volume of a probe molecule (e.g. Dextran <NUM> kD), V<NUM> 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-V<NUM>)/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 <NUM> - <NUM>. The Kav value can advantageously be <NUM> - <NUM>, e.g. <NUM> - <NUM> or <NUM> - <NUM>, as measured with dextran of Mw <NUM> kDa as a probe molecule. The Kd value as measured with dextran of Mw <NUM> kDa can suitably be <NUM>-<NUM>, such as <NUM>-<NUM> or <NUM>-<NUM>. 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.

The polypeptides or multimers may be attached to the porous 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).

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.

In certain embodiments the support comprises a polyhydroxy polymer, such as a polysaccharide. Examples of polysaccharides include e.g. dextran, starch, cellulose, pullulan, agar, agarose etc. Polysaccharides are inherently hydrophilic with low degrees of nonspecific interactions, they provide a high content of reactive (activatable) hydroxyl groups and they are generally stable towards alkaline cleaning solutions used in bioprocessing.

In some embodiments the support comprises agar or agarose. The supports used in the present invention can easily be prepared according to standard methods, such as inverse suspension gelation (<NPL>). 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 <CIT> or <CIT>, which are hereby incorporated by reference in their entireties, and hence renders the matrix more suitable for high flow rates.

In certain embodiments the support, such as a polymer, polysaccharide or 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.

Alternatively, the porous support is based on synthetic polymers, such as polyvinyl alcohol, polyhydroxyalkyl acrylates, polyhydroxyalkyl methacrylates, polyacrylamides, polymethacrylamides etc. In case of hydrophobic polymers, such as matrices based on divinyl and monovinyl-substituted benzenes, the surface of the matrix is often hydrophilised to expose hydrophilic groups as defined above to a surrounding aqueous liquid. Such polymers are easily produced according to standard methods, see e.g. "<NPL>)). Alternatively, a commercially available product, such as SOURCE™ (GE Healthcare) is used. In another alternative, the porous support according to the invention comprises a support of inorganic nature, e.g. silica, zirconium oxide etc..

In yet another embodiment, the solid support is in another form such as a surface, a chip, capillaries, or a filter (e.g. a membrane or a depth filter matrix).

As regards the shape of the matrix according to the invention, in one embodiment the matrix is in the form of a porous monolith. In an alternative embodiment, the matrix is in beaded or particle form that can be porous or non-porous. 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.

The separation matrix as disclosed above has excellent alkali stability and may be stored in an alkaline storage liquid. The method of storing the separation matrix comprises the following steps:.

By using a storage liquid comprising aqueous alkali metal hydroxide solution, a bacteriostatic or bactericidal solution may be obtained without requiring the use of alcohols such as ethanol, isopropanol, or benzyl alcohol. This means that a storage liquid may be used that is cheaper, subject to less regulatory burden, non-flammable and easier to dispose of than the storage solutions presently used for known protein A affinity separation matrices.

The separation matrix is a separation matrix as disclosed above, comprising multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support, wherein the alkali-stabilized Protein A domains comprise mutants of a parental Fc-binding domain of Staphylococcus Protein A (SpA), as defined by SEQ ID NO <NUM> or SEQ ID NO <NUM>, wherein the amino acid residues at positions <NUM> and <NUM> of SEQ ID NO <NUM> or <NUM> are asparagines, and wherein at least the asparagine residue at position <NUM> of SEQ ID NO <NUM> or <NUM> 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. The immunoglobulin-binding alkali-stabilized Protein A domains may comprise further mutations. For example, the glutamine residue at position <NUM> of SEQ ID NO <NUM> or <NUM> may be mutated to an alanine; and/or the asparagine or glutamic acid residue at position <NUM> of SEQ ID NO <NUM> or <NUM> may be mutated to an alanine.

The storage liquid comprises at least <NUM>% by volume of an aqueous alkali metal hydroxide solution. The aqueous alkali metal hydroxide solution may comprise a single alkali metal hydroxide or a mixture of alkali metal hydroxides, such as sodium hydroxide, potassium hydroxide, or a mixture of sodium hydroxide and potassium hydroxide. The aqueous alkali metal hydroxide solution may have a molarity of from <NUM> to <NUM>, such as from <NUM> to <NUM>, expressed as the total combined concentration of alkali metal hydroxides if a mixture of alkali metal hydroxides is used. The storage liquid may essentially consist of, or consist of, the aqueous alkali metal hydroxide solution. However, the storage liquid may in some embodiments also comprise further components. Such further components may include alcohols, such as a C<NUM> - C<NUM> alcohol, such as ethanol, isopropanol or benzyl alcohol. Such further components may include salts, such as sodium chloride. The use of alcohols and/or salts in the storage liquid may increase the efficacy of the storage liquid in inhibiting or inactivating certain microorganisms, such as spore-forming bacteria.

Non-limiting examples of storage liquids include:.

The separation matrix is permeated with the storage liquid prior to and during storage. By permeated with storage liquid, it is meant that the pores and interstices of the separation matrix are to a large extent filled with storage liquid. The separation matrix should be permeated with a quantity of storage liquid sufficient to inhibit growth of microorganisms in the stored separation matrix. The separation matrix may be impregnated, saturated with, or immersed in the storage liquid. Typically, a slurry of separation matrix in storage liquid suitable for storage may comprise about <NUM> % to <NUM>% by weight of separation matrix, relative to the total weight of the slurry.

The separation matrix may be stored in the storage liquid for as extended a period as required. Typically, if the separation matrix is to be stored, this is for at least <NUM> days, often for at least <NUM> days, such as at least <NUM> days, or such as at least <NUM> days, or such as at least <NUM> days. The maximum storage time, or shelf life, of the separation matrix depends on the nature of the storage liquid used, i.e. alkali concentration, as well as the degree of capacity loss acceptable to the user, but may for example be up to <NUM> days or up to <NUM> days.

The mixture of storage liquid and separation matrix may be contained in a suitable storage receptacle, not covered by the appended claims. The storage receptacle may be a bottle, can or drum made from a liquid-impervious material such as plastic, e.g. polyethylene, or glass. The separation matrix may be packaged in such storage receptacles for initial storage and distribution after production, or may be re-filled into such storage receptacles after use in purifying an immunoglobulin. The storage receptacle may alternatively be a pre-packed product for use in development or manufacturing of immunoglobulins. Such pre-packed products include filter plates, such as <NUM>-well filter plates, and pre-packed columns. Such pre-packed columns include columns of all sizes known to the skilled person, from laboratory scale to process scale. Such columns can be shipped prepacked, qualified and sanitized, thus substantially reducing the time required for immunoglobulin purification processes.

The storage receptacle for storing the separation matrix may be open, vented or sealed. Since aqueous alkali metal hydroxide solutions are non-flammable and relatively non-volatile, no special considerations must be given regarding pressure build-up in the storage receptacle and ventilation of the storage room. In order to prevent dry-out of the separation media, it is preferable if the storage media is stored in an airtight receptacle.

The separation matrix may be stored at any temperature known in the art for storage of affinity media, such as from <NUM> to <NUM>, or from <NUM> to <NUM>. However, prolonged storage at elevated temperatures may degrade the capacity of the separation matrix, and therefore it is preferable if the separation matrix can be stored at a temperature of from <NUM> to <NUM>.

If the separation matrix has been previously used for purifying an immunoglobulin prior to storage, e.g. if it has been used in a production campaign that has now concluded, it is preferable that the separation matrix is cleaned and/or sanitized prior to storage. Cleaning liquids comprising at least <NUM>% by volume of an aqueous alkali metal hydroxide solution and having a molarity of <NUM> to <NUM> may suitably be used to clean and/or sanitize the separation matrix.

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 <NUM>% agarose gel. Equal amounts of the pair wise PCR products were hybridized (<NUM> -> <NUM> in <NUM>) 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.

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 <NUM> pore size.

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.

The elution pool was adjusted to a neutral pH (pH <NUM>) 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 <NUM>-<NUM>/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.

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).

The purified monomeric ligands listed in Table <NUM>, further comprising for SEQ ID NO <NUM>-<NUM>, <NUM>-<NUM> and <NUM>-<NUM> 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 <NUM>-<NUM> RU in a Biacore surface plasmon resonance (SPR) instrument (GE Healthcare, Sweden). To follow the IgG binding capacity of the immobilized surface <NUM>/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 <NUM> NaOH for <NUM> minutes at room temperature (<NUM> +/- <NUM>). This was repeated for <NUM>-<NUM> 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 <NUM> and indicate that at least the ligands Zvar(N11K)<NUM>, Zvar(N11E)<NUM>, Zvar(N11Y)<NUM>, Zvar(N11T)<NUM>, Zvar(N11F)<NUM>, Zvar(N11L)<NUM>, Zvar(N11W)<NUM>, ZN11I)<NUM>, Zvar(N11M)<NUM>, Zvar(N11V)<NUM>, Zvar(N11A)<NUM>, Zvar(N11H1), Zvar(N11R)<NUM>, Zvar(N11E,Q32A)<NUM>, Zvar(N11E,Q32E,Q40E)<NUM> and Zvar(N11E,Q32E,K50R)<NUM>, Zvar(Q9A,N11E,N43A)<NUM>, Zvar(Q9A,N11E,N28A,N43A)<NUM>, Zvar(Q9A,N11E,Q40V,A42K,N43E,L44I)<NUM>, Zvar(Q9A,N1E,Q40V,A42K,N43A,L44I)<NUM>, Zvar(Q9A,N11E,N28A,Q40V,A42K,N43A,L44I)<NUM>, Zvar(N11K,H18K,S33K,D37E,A42R,N43A,L44I,K50R,L51Y)<NUM>, Zvar(Q9A,N11K,H18K,S33K,D37E,A42R,N43A,L44I,K50R,L51Y)<NUM>, Zvar(N11K, H18K, D37E, A42R, N43A, L44I)<NUM>, Zvar(Q9A, N11K, H18K, D37E, A42R, N43A, L44I)<NUM> and Zvar(Q9A, N11K, H18K, D37E, A42R, N43A, L44I, K50R)<NUM>, as well as the varieties of Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)<NUM> having G,S,Y,Q,T,N,F,L,W,I,M,V,D,E,H,R or K in position <NUM>, the varieties of Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)<NUM> having F,Y,W,K or R in position <NUM> and the varieties of Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)<NUM> 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)<NUM> and C(Q9A,N11E,E43A)<NUM> have an improved stability compared to the parental B and C domains, used as references.

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 (<NUM><NUM> M-<NUM>s-<NUM>) was <NUM> and the off-rate (<NUM><NUM> s-<NUM>) was <NUM>, giving an affinity (off-rate/on-rate) of <NUM> pM. For Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)<NUM> (SEQ ID NO. <NUM>), the on-rate was <NUM> and the off-rate <NUM>, with affinity <NUM> pM. The IgG affinity was thus somewhat higher for the mutated variant.

The purified dimeric, tetrameric and hexameric ligands listed in Table <NUM> 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 <NUM>-<NUM> RU in a Biacore instrument (GE Healthcare, Sweden). To follow the IgG binding capacity of the immobilized surface <NUM>/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 <NUM> NaOH for <NUM> minutes at room temperature (<NUM> +/- <NUM>). This was repeated for <NUM> 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 <NUM> and in <FIG> and indicate that at least the ligands Zvar(Q9A,N11E,N43A)<NUM>, Zvar(Q9A,N11E,N28A,N43A)<NUM>, Zvar(Q9A,N11E,Q40V,A42K,N43E,L44I)<NUM> and Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)<NUM>, Zvar(Q9A,N11E,D37E,Q40V,A42K,N43A,L44I)<NUM> and Zvar(Q9A,N11E,D37E,Q40V,A42R,N43A,L44I)<NUM> 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)<NUM> 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 V1 in the linker region have improved alkali stability compared to Zvar2.

Example <NUM> was repeated with <NUM> CIP cycles of three ligands using <NUM> NaOH instead of <NUM> as in Example <NUM>. The results are shown in Table <NUM> and show that all three ligands have an improved alkali stability also in <NUM> NaOH, compared to the parental structure Zvar4 which was used as a reference.

The purified tetrameric ligands of Table <NUM> (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 <NUM> and <FIG>.

The base matrix used was rigid cross-linked agarose beads of <NUM> micrometers (volume-weighted, d50V) median diameter, prepared according to the methods of <CIT>, hereby incorporated by reference in its entirety, and with a pore size corresponding to an inverse gel filtration chromatography Kav value of <NUM> for dextran of Mw <NUM> kDa, according to the methods described in <NPL>.

<NUM> (g) of drained base matrix, <NUM> distilled water and <NUM> NaOH (s) was mixed in a <NUM> flask with mechanical stirring for <NUM> at <NUM>. <NUM> of epichlorohydrin was added and the reaction progressed for <NUM> hours. The activated gel was washed with <NUM> gel sediment volumes (GV) of water.

To <NUM> of ligand solution (<NUM>/mL) in a <NUM> Falcon tube, <NUM> NaHCOs, <NUM> Na<NUM>CO<NUM>, <NUM> NaCl and <NUM> EDTA, was added. The Falcon tube was placed on a roller table for <NUM>-<NUM>, and then <NUM> of DTE was added. Reduction proceeded for ><NUM>. The ligand solution was then desalted on a PD10 column packed with Sephadex G-<NUM>. The ligand content in the desalted solution was determined by measuring the <NUM> UV absorption.

The activated gel was washed with <NUM>-<NUM> GV {<NUM> phosphate/<NUM> EDTA pH <NUM>} and the ligand was then coupled according to the method described in <CIT>, hereby incorporated by reference in its entirety. All buffers used in the experiments had been degassed by nitrogen gas for at least <NUM>-<NUM>. The ligand content of the gels could be controlled by varying the amount and concentration of the ligand solution.

After immobilization the gels were washed 3xGV with distilled water. The gels + <NUM> GV {<NUM> phosphate/<NUM> EDTA/<NUM>% thioglycerol pH <NUM>} was mixed and the tubes were left in a shaking table at room temperature overnight. The gels were then washed alternately with 3xGV {<NUM> TRIS/<NUM> NaCl pH <NUM>} and <NUM> HAc and then <NUM>-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.

<NUM> of resin was packed in TRICORN™ <NUM><NUM> columns. The breakthrough capacity was determined with an ÄKTAExplorer <NUM> system at a residence time of <NUM> minutes (<NUM>/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 <NUM>% UV signal was obtained. Then, equilibration buffer was applied again until a stable baseline was obtained.

Sample was loaded onto the column until a UV signal of <NUM>% of maximum absorbance was reached. The column was then washed with <NUM> column volumes (CV) equilibration buffer at flow rate <NUM>/min. The protein was eluted with <NUM> CV elution buffer at a flow rate of <NUM>/min. Then the column was cleaned with <NUM> NaOH at flow rate <NUM>/min and reequilibrated with equilibration buffer.

For calculation of breakthrough capacity at <NUM>%, 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 <NUM>% of the IgG concentration in the feed.

The dynamic binding capacity (DBC) at <NUM>% breakthrough was calculated. The dynamic binding capacity (DBC) was calculated for <NUM> and <NUM>% breakthrough.

The <NUM>% breakthrough DBC (Qb10) was determined both before and after repeated exposures to alkaline cleaning solutions. Each cycle included a CIP step with <NUM> NaOH pumped through the column at a rate of <NUM>/min for <NUM>, after which the column was left standing for <NUM>. The exposure took place at room temperature (<NUM> +/- <NUM>). After this incubation, the column was washed with equilibration buffer for <NUM> at a flow rate of <NUM>/min. Table <NUM> shows the remaining capacity after six <NUM> cycles (i.e. <NUM> cumulative exposure time to <NUM> NaOH), both in absolute numbers and relative to the initial capacity.

Example <NUM> was repeated with the tetrameric ligands shown in Table <NUM>, but with <NUM> NaOH used in the CIP steps instead of <NUM>. The results are shown in Table <NUM> and in <FIG>.

The base matrices used were a set of rigid cross-linked agarose bead samples of <NUM>-<NUM> micrometers (volume-weighted, d50V) median diameter (determined on a Malvern Mastersizer <NUM> laser diffraction instrument), prepared according to the methods of <CIT> and with a pore size corresponding to an inverse gel filtration chromatography Kd value of <NUM>-<NUM> for dextran of Mw <NUM> kDa, according to the methods described above, using HR10/<NUM> columns (GE Healthcare) packed with the prototypes in <NUM> NaCl and with a range of dextran fractions as probe molecules (flow rate <NUM>/min). The dry weight of the bead samples ranged from <NUM> to <NUM>/ml, as determined by drying <NUM> drained filter cake samples at <NUM> overnight and weighing.

<NUM> base matrix was washed with <NUM> gel volumes distilled water on a glass filter. The gel was weighed (<NUM> = <NUM>) and mixed with <NUM> distilled water and <NUM> NaOH (<NUM> mol) in a <NUM> flask with an agitator. The temperature was adjusted to <NUM> +/- <NUM> in a water bath. <NUM> epichlorohydrin (<NUM> mol) was added under vigorous agitation (about <NUM> rpm) during <NUM> +/- <NUM> minutes. The reaction was allowed to continue for another <NUM> +/- <NUM> minutes and the gel was then washed with ><NUM> gel volumes distilled water on a glass filter until neutral pH was reached. This activated gel was used directly for coupling as below.

To <NUM> of ligand solution (<NUM>/mL) in a <NUM> Falcon tube, <NUM> NaHCO<NUM>, <NUM> Na<NUM>CO<NUM>, <NUM> NaCl and <NUM> EDTA, was added. The Falcon tube was placed on a roller table for <NUM>-<NUM>, and then <NUM> of DTE was added. Reduction proceeded for ><NUM>. The ligand solution was then desalted on a PD10 column packed with Sephadex G-<NUM>. The ligand content in the desalted solution was determined by measuring the <NUM> UV absorption.

The activated gel was washed with <NUM>-<NUM> GV {<NUM> phosphate/<NUM> EDTA pH <NUM>} and the ligand was then coupled according to the method described in <CIT>, although with considerably higher ligand amounts (see below). All buffers used in the experiments had been degassed by nitrogen gas for at least <NUM>-<NUM>. The ligand content of the gels was controlled by varying the amount and concentration of the ligand solution, adding <NUM>-<NUM> ligand per ml gel. The ligand was either a tetramer (SEQ ID NO. <NUM>) or a hexamer (SEQ ID NO. <NUM>) of an alkali-stabilized mutant.

The Qb10 % dynamic capacity for polyclonal human IgG at <NUM> and <NUM> residence time was determined as outlined in Example <NUM>.

A series of prototypes, prepared as above, with different ligand content (tetramer, SEQ ID NO:<NUM>) were incubated in <NUM> NaOH for <NUM>, <NUM> and <NUM> hours at <NUM> +/- <NUM> and the dynamic IgG capacity (Qb10%, <NUM> residence time) was measured before and after incubation. The prototypes are shown in Table <NUM> and the results in <FIG> and <FIG>. It can be seen that the stability towards this harsh alkali treatment increases with increasing ligand content.

Two crosslinked agarose bead prototypes, prepared as above, with different ligand content (hexamer, SEQ ID NO:<NUM>), median bead diameter (d50,v) <NUM> and Kd <NUM> for dextran of Mw <NUM> kD, were evaluated with a real mAb feed. The ligand content of prototype A was <NUM>/ml and of prototype B <NUM>/ml. For comparison, the commercial product MabSelect SuRe® LX (GE Healthcare Life Sciences, with ligand SEQ ID NO. <NUM>) was used. The resins were packed in Tricorn columns (GE Healthcare Life Sciences) to bed heights of <NUM>, giving bed volumes of <NUM> and the columns were shown to have peak asymmetry within the <NUM>-<NUM> interval. The sample loaded was a clarified CHO cell supernatant with <NUM>/ml monoclonal IgG1 antibody at physiological pH and the experimental conditions were as listed below in Table <NUM> (CV = column volumes, RT = residence time).

The mAb peak was collected using a UV watch function and the concentration of the mAb was determined by UV measurement at <NUM> (extinction coefficient <NUM>). All absorbance detections were performed using a spectrophotometer, including the measurements for the yield calculations.

Samples for HCP (host cell protein) analyses were prepared by adding <NUM>% Preservation buffer (<NUM> NaH<NUM>PO<NUM>*H<NUM>O (<NUM>%), <NUM> Na<NUM>HPO<NUM>*<NUM><NUM>O (<NUM>%), <NUM>% Tween <NUM>, <NUM>% BSA pH <NUM>) to the samples directly after each run made (e.g. <NUM>µl preservation buffer to <NUM>µl sample). The HCP content was measured using commercial anti-CHO antibodies (Cygnus Technologies) and a Gyrolab (Gyros AB, Sweden) work station.

The results are presented in Table <NUM> below and show that the performance of the prototypes is in the same range as for the commercial product. The HCP content in the feed was <NUM><NUM> ppm.

A crosslinked agarose bead matrix prototype, prepared as above, with <NUM>/ml ligand (hexamer, SEQ ID NO:<NUM>), median bead diameter (d50,v) <NUM>, Kd <NUM> for dextran of Mw <NUM> kD and dry weight <NUM>/ml, was evaluated for elution pH with two real mAb feeds (mAb1 <NUM>/l and mAb2 <NUM>/l) IgG1, physiological pH, and a sample of polyclonal human IgG (Gammanorm, Octapharma). For comparison, the commercial product MabSelect SuRe® LX (GE Healthcare Life Sciences) was used. The resins were packed in Tricorn columns (GE Healthcare Life Sciences) to bed heights of <NUM>, giving bed volumes of <NUM> and the columns were shown to have peak asymmetry within the <NUM>-<NUM> interval. The samples loaded were clarified CHO cell supernatants with IgG1 mAbs at physiological pH and the experimental conditions were as listed below in Table <NUM> (CV = column volumes, RT = residence time).

The results are shown below in Table <NUM> and indicate that the antibodies elute at similar pH levels as on the reference, although with some individual variation depending on the particular antibody-resin combination.

Fractions from the pH-gradient elution of polyclonal IgG were also analysed with respect to content of IgG1, IgG2 and IgG4, using a Biacore SPR instrument (GE Healthcare Life Sciences) with antibodies against the four different IgG classes immobilized on a CM5 Biacore chip.

The chromatograms for polyclonal IgG on the reference and the prototype are shown in <FIG> and the IgG class analyses are shown in <FIG>. The data show that all three classes bind to both resins in a similar way and that the first peak predominantly contains IgG2, while IgG1 and IgG4 elute mainly in the second peak. The anti-IgG3 antibodies cross-reacted with IgG4, so no reliable results for IgG3 were obtained. IgG3 is generally known to show no or only weak binding to Protein A.

A crosslinked agarose bead matrix prototype, prepared as above, with <NUM>/ml ligand (tetramer, SEQ ID NO:<NUM>), <NUM> median bead diameter (d50,v), Kd <NUM> for dextran Mw <NUM> kD and <NUM>/ml dry weight, was evaluated with respect to alkali stability, using the commercial product MabSelect SuRe LX as a reference. Tricorn <NUM> columns packed with the resins to <NUM> bed height were flushed with <NUM> column volumes of <NUM> NaOH. The flow was then stopped for <NUM> minutes (corresponding to <NUM> normal CIP cycles of <NUM>/cycle) before washing out the NaOH solution by <NUM> column volumes of PBS buffer. The dynamic binding capacity for polyclonal IgG (Gammanorm, Octapharma) was then measured and the process was repeated with another injection of <NUM> NaOH. The dynamic capacity was measured after each <NUM> incubation cycle with <NUM> NaOH. In the capacity measurements, the columns were equilibrated with PBS buffer before the <NUM>/ml sample was loaded (residence time <NUM>) until a UV signal of <NUM>% of maximum absorbance was reached. Then the column was washed with PBS buffer, eluted with <NUM> acetic acid pH <NUM> and re-equilibrated. The dynamic binding capacity at <NUM>% and <NUM>% breakthrough was calculated as described above. The results are shown in <FIG> and they show that the prototype was significantly more stable than the commercial product.

The long-term storage stability of a separation matrix according to the invention was assessed. The inventive example (Inv. ) was compared to the commercial product MabSelect SuRe (MSS) (GE Healthcare Life Sciences, with ligand SEQ ID NO. <NUM>) as a reference. The separation matrix (Inv. or MSS) was incubated together with the storage liquid for a predetermined period (two weeks). The storage liquids tested were <NUM>% ethanol solution; <NUM> NaOH solution; <NUM> NaOH solution and <NUM> NaOH solution. After incubation with the storage liquid for the predefined period, the <NUM>% breakthrough dynamic binding capacity at was determined using human polyclonal IgG as described in Example <NUM>, using a residence time of <NUM> minutes. The <NUM>% breakthrough capacity of the separation matrices prior to storage were also determined for comparison. The results are shown in <FIG> and Table <NUM> below.

Claim 1:
A method of storing a separation matrix comprising multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support, wherein the alkali-stabilized Protein A domains comprise mutants of a parental Fc-binding domain of Staphylococcus Protein A (SpA), as defined by SEQ ID NO <NUM> or SEQ ID NO <NUM>, wherein the amino acid residues at positions <NUM> and <NUM> of SEQ ID NO <NUM> or <NUM> are asparagines and wherein at least the asparagine residue at position <NUM> of SEQ ID NO <NUM> or <NUM> 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 to glutamic acid; and wherein the method comprises the steps of:
a) providing a storage liquid comprising at least <NUM>% by volume of an aqueous alkali metal hydroxide solution;
b) permeating the separation matrix with the storage liquid; and
c) storing the storage liquid-permeated separation matrix for a storage time of at least <NUM> days.