NZ574626A

NZ574626A - Process for removing free Fc-moieties from fluids comprising Fc-containing proteins using ion exchange chromatography

Info

Publication number: NZ574626A
Application number: NZ574626A
Authority: NZ
Inventors: Alex Eon-Duval; Alain Lamproye
Original assignee: Ares Trading Sa
Priority date: 2006-08-28
Filing date: 2007-08-27
Publication date: 2011-12-22
Also published as: CN101535335A; BRPI0716382A2; ZA200900837B; CN101535335B; MY157846A; BRPI0716382B8; BRPI0716382B1

Abstract

This patent application discloses a process for reducing the concentration of free Fc- moieties in a fluid comprising an Fc-containing protein, the process comprising a cation exchange chromatography step.

Description

<div class="application article clearfix" id="description"> WO 2008/025748 PCT/EP2007/058887 1 PROCESS FOR THE PURIFICATION OF FC-CONTAINING PROTEINS FIELD OF THE INVENTION The present invention is in the field of protein purification. More specifically, it 5 relates to the purification of an Fc-containing protein via cation exchange chromatography, in particular for reduction of the amount of free Fc-moieties in an Fc-containing protein preparation. BACKGROUND OF THE INVENTION 10 Proteins have become commercially important as drugs that are generally called "biologicals". One of the greatest challenges is the development of cost effective and efficient processes for purification of proteins on a commercial scale. While many methods are now available for large-scale production of proteins, crude products, such as cell culture supernatants, contain not only the desired product but also impurities, 15 which are difficult to separate from the desired product. Although cell culture supernatants of cells expressing recombinant protein products may contain less impurities if the cells are grown in serum-free medium, the host cell proteins (HCPs) still remain to be eliminated during the purification process. Additionally, the health authorities request high standards of purity for proteins intended for human 20 administration. A number of chromatographic systems are known that are widely used for protein purification. Ion exchange chromatography systems are used for separation of proteins primarily on the basis of differences in charge. 25 Anion exchangers can be classified as either weak or strong. The charge group on a weak anion exchanger is a weak base, which becomes de-protonated and, therefore, loses its charge at high pH. DEAE-sepharose is an example of a weak anion exchanger, where the amino group can be positively charged below pH ~ 9 and gradually loses its charge at higher pH values. Diethylaminoethyl (DEAE) or diethyl-(2-30 hydroxy-propyl)aminoethyl (QAE) have chloride as counter ion, for instance. A strong anion exchanger, on the other hand, contains a strong base, which remains positively charged throughout the pH range normally used for ion exchange chromatography (pH WO 2008/025748 PCT/EP2007/058887 2 1-14). Q-sepharose (Q stands for quaternary ammonium) is an example for a strong anion exchanger. Cation exchangers can also be classified as either weak or strong. A strong cation exchanger contains a strong acid (such as a sulfopropyl group) that remains charged from pH 1 - 14; whereas a weak cation exchanger contains a weak acid (such as a carboxymethyl group), which gradually loses its charge as the pH decreases below 4 or 5. Carboxymethyl (CM) and sulphopropyl (SP) have sodium as counter ion, for example. A different chromatography resin is based on an insoluble hydroxylated calcium phosphate matrix called hydroxyapatite. Hydroxyapatite chromatography is a method of purifying proteins that utilizes an insoluble hydroxylated calcium phosphate (Ca5(P04)30H)2, which forms both the matrix and ligand. Functional groups consist of pairs of positively charged calcium ions (C-sites) and clusters of negatively charged phosphate groups (P-sites). The interactions between hydroxyapatite and proteins are complex and multi-mode. In one method of interaction, positively charged amino groups on proteins associate with the negatively charged P-sites and protein carboxyl groups interact by coordination complexation to C-sites (Shepard et al., 2000). Crystalline hydroxyapatite was the first type of hydroxyapatite used in chromatography. Ceramic Hydroxyapatite (CHA) chromatography is a further development in hydroxyapatite chromatography. Ceramic hydroxyapatite has high durability, good protein binding capacity, and can be used at higher flow rates and pressures than crystalline hydroxyapatite. (Vola et al., 1993). Hydroxyapatite has been used in the chromatographic separation of proteins, nucleic acids, as well as antibodies. In hydroxyapatite chromatography, the column is normally equilibrated, and the sample applied, in a low concentration of phosphate buffer and the adsorbed proteins are then eluted in a concentration gradient of phosphate buffer (Giovannini et al., 2000). Yet a further way of purifying proteins is based on the affinity of a protein of interest to another protein that is immobilized to a chromatography resin. Examples for such immobilized ligands are the bacterial cell wall proteins Protein A and Protein G, having specificity to the Fc portion of certain immunoglobulins. Although both Protein A and Protein G have a strong affinity for IgG antibodies, they have varying affinities to other immunoglobulin classes and isotypes as well. WO 2008/025748 PCT/EP2007/058887 Protein A is a 43,000 Dalton protein that is produced by the bacteria Staphylcoccus aureus and contains four binding sites to the Fc regions of IgG. Protein G is produced from group G Streptococci and has two binding sites for the IgG Fc region. Both proteins have been widely characterized for their affinity to various types 5 of immunoglobulins. Another development is Protein A/G, a genetically engineered protein that combines the binding capacities of Protein A and G. Protein L is a further bacterial protein, originating from Peptostreptococcus, binding to Immunoglobulins and fragments thereof containing Ig light chains (Akerstrom and Bjork, 1989). Protein A, Protein G, and Protein L affinity chromatography are widely used for 10 isolation and purification of antibodies. Since the binding sites for Protein A and Protein G reside in the Fc region of an immunoglobulin, Protein A and Protein G (or Protein A/G) affinity chromatography also allows purification of so-called Fc-fusion proteins. Protein L binds to Ig light chains and can thus be used for the purification of light chain containing antibodies. 15 Antibodies, or immunoglobulins (Igs) consist of light chains and heavy chains linked together by disulphide bonds. The first domain located at the amino terminus of each chain is variable in amino acid sequence, providing the vast spectrum of antibody binding specificities. These domains are known as variable heavy (VH) and variable light (VL) regions. The other domains of each chain are relatively invariant in amino 20 acid sequence and are known as constant heavy (CH) and constant light (CL) regions. The major classes of antibodies are IgA, IgD, IgE, IgG and IgM; and these classes may be further divided into subclasses (isotypes). For example, the IgG class has four subclasses, namely, IgG^ lgG2, lgG3, and lgG4. The differences between antibody classes are derived from differences in the 25 heavy chain constant regions, containing between 1 and 4 constant domains (CH1-CH4), depending on the immunoglobulin class. A so-called hinge region is located between the CH1 and CH2 domains. The hinge region is particularly sensitive to proteolytic cleavage; such proteolysis yields two or three fragments depending on the precise site of cleavage. The part of the heavy chain constant region containing the 30 CH2 and CH3 domains is also called the "Fc" part of the immunoglobulin. Antibodies are thus Fc-containing proteins. Another type of Fc-containing proteins are the so-called Fc-fusion proteins. WO 2008/025748 PCT/EP2007/058887 Several antibodies that are used as therapeutic proteins are known. Examples for recombinant antibodies on the market are for instance: Abciximab, Rituximab, Basiliximab, Daclizumab, Palivizumab, Infliximab, Trastuzumab, Alemtuzumab, Adalimumab, Cetuximab, Efalizumab, Ibritumomab, Bevacizumab, or Omalizumab. 5 Fc-fusion proteins are chimeric proteins consisting of the effector region of a protein, such as the Fab region of an antibody or the binding region of a receptor, fused to the Fc region of an immunoglobulin that is frequently an immunoglobulin G (IgG). Fc-fusion proteins are widely used as therapeutics as they offer advantages conferred by the Fc region, such as: 10 - The possibility of purification using protein A or protein G affinity chromatography with affinities varying according to the IgG isotype. Human IgG^ lgG2 and lgG4 bind strongly to Protein A and all human IgGs including lgG3 bind strongly to Protein G; - An increased half-life in the circulatory system, since the Fc region binds to the 15 salvage receptor FcRn which protects from lysosomal degradation; Depending on the medical use of the Fc-fusion protein, the Fc effector functions may be desirable. Such effector functions include antibody-dependent cellular cytotoxicity (ADCC) through interactions with Fc receptors (FcyRs) and complement-dependent cytotoxicity (CDC) by binding to the complement 20 component 1q (C1q). IgG isoforms exert different levels of effector functions. Human IgGi and lgG3 have strong ADCC and CDC effects while human lgG2 exerts weak ADCC and CDC effects. Human lgG4 displays weak ADCC and no CDC effects. Serum half-life and effector functions can be modulated by engineering the Fc 25 region to increase or reduce its binding to FcRn, FcyRs and C1q respectively, depending on the therapeutic use intended for the Fc-fusion protein. In ADCC, the Fc region of an antibody binds to Fc receptors (FcyRs) on the surface of immune effector cells such as natural killers and macrophages, leading to the phagocytosis or lysis of the targeted cells. 30 In CDC, the antibodies kill the targeted cells by triggering the complement cascade at the cell surface. IgG isoforms exert different levels of effector functions increasing in the order of lgG4 < lgG2 < IgGi < lgG3. Human IgGi displays high ADCC WO 2008/025748 PCT/EP2007/058887 and CDC, and is the most suitable for therapeutic use against pathogens and cancer cells. Under certain circumstances, for example when depletion of the target cell is undesirable, abrogating or diminishing effector functions may be required. On the 5 contrary, in the case of antibodies intended for oncology use, increasing effector functions may improve their therapeutic activity (Carter et al., 2006). Modifying effector functions can be achieved by engineering the Fc region to either improve or reduce binding of FcyRs or the complement factors. The binding of IgG to the activating (FcyRI, FcyRlla, FcyRllla and FcyRlllb) and 10 inhibitory (FcyRllb) FcyRs or the first component of complement (C1q) depends on residues located in the hinge region and the CH2 domain. Two regions of the CH2 domain are critical for FcyRs and complement C1q binding, and have unique sequences in lgG2 and lgG4. For instance, substitution of lgG2 residues at positions 233-236 into human IgGi greatly reduced ADCC and CDC (Armour et al., 1999 and 15 Shields et al., 2001). Numerous mutations have been made in the CH2 domain of IgG and their effect on ADCC and CDC was tested in vitro (Shields et al., 2001, Idusogie et al., 2001 and 2000, Steurer et al., 1995). In particular, a mutation to alanine at E333 was reported to increase both ADCC and CDC (Idusogie et al., 2001 and 2000). 20 Increasing the serum half-life of a therapeutic antibody is another way to improve its efficacy, allowing higher circulating levels, less frequent administration and reduced doses. This can be achieved by enhancing the binding of the Fc region to neonatal FcR (FcRn). FcRn, which is expressed on the surface of endothelial cells, binds the IgG in a pH-dependent manner and protects it from degradation. Several 25 mutations located at the interface between the CH2 and CH3 domains have been shown to increase the half-life of IgGi (Hinton et al., 2004 and Vaccaro et al., 2005). The following Table 1 summarizes some known mutations of the IgG Fc-region (taken from Invivogen's website). Engineere d Fc IgG Isotype Mutations Properties Potential Benefits Applications hlgG1e1 human lgG1 T250Q/M428L Increased plasma half-life Improved localization to target; increased efficacy; reduced dose or frequency of administration Vaccination; therapeutic use hlgG1e2 human M252Y/S254T/T256 Increased Improved localization to Vaccination; WO 2008/025748 PCT/EP2007/058887 lgG1 E + H433K/N434F plasma half-life target; increased efficacy; reduced dose or frequency of administration therapeutic us hlgG1e3 human lgG1 E233P/L234V/L235 A/AG236 + A327G/A330S/P331 S Reduced ADCC and CDC Reduced adverse events Therapeutic use without cell depletion hlgG1e4 human lgG1 E333A Increased ADCC and CDC Increased efficacy Therapeutic use with cell depletion hlgG2e1 human lgG2 K322A Reduced CDC Reduced adverse events Vaccination; therapeutic use A class of Fc-fusion proteins having therapeutic utility, Fc-regions have been fused to extracellular domains of certain receptors belonging to the tumor necrosis factor receptor (TNF-R) superfamily (Locksley et al., 2001, Bodmer et al., 2002, Bossen 5 et al., 2006). A hallmark of the members of the TNFR family is the presence of cysteine-rich pseudo-repeats in the extracellular domain, as described e.g. by Naismith and Sprang, 1998. The two TNF receptors, p55 (TNFR1) and p75 TNFR (TNFR2) are examples of such members of the TNFR superfamily. Etanercept is an Fc-fusion protein containing 10 the soluble part of the p75 TNFR (e.g. W091/03553, WO 94/06476). Under the trade name Enbrel®, it is marketed for treatment of Endometriosis, Hepatitis C virus infection, HIV infection, Psoriatic arthritis, Psoriasis, Rheumatoid arthritis, Asthma, Ankylosing spondylitis, Cardiac failure, Graft versus host disease, Pulmonary fibrosis, Crohns disease. Lenercept is a fusion protein containing extracellular components of human 15 p55 TNF receptor and the Fc portion of human IgG, and is intended for the potential treatment of severe sepsis and multiple sclerosis. 0X40 is also a member of the TNFR superfamily. OX40-lgG1 and 0X40-hlG4mut fusion proteins have been prepared for treatment of inflammatory and autoimmune diseases such as Crohn's Disease. 20 An Fc-fusion protein of the BAFF-R, also called BR3, designated BR3-Fc, is a soluble decoy receptor from a series of inhibitors of BAFF (B-cell activating factor of the TNF family), is being developed for the potential treatment of autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). BCMA is a further receptor belonging to the TNFR superfamily. A BCMA-lg 25 fusion protein has been described to inhibit autoimmune disease (Melchers, 2006). Received at IPONZ 09 March 2011 7 Another receptor of the TNF-R superfamily is TACi, the transmembrane activator and CAML-interactor (von Bulow and Bram, 1997; US 5,969,102, Gross et al., 2000), which has an extracellular domain containing two cysteine-rich pseudo-repeats. TACI binds two members of the tumor necrosis factor (TNF) ligand family. One iigand is 5 designated BLyS, BAFF, neutrokine-a, TALL-1, zTNF4, or THANK (Moore et al., 1999). The other ligand has been designated as APRIL, TNRF death ligand-1 or ZTNF2 (Hahne et al., J. Exp.Med. 188: 1185(1998). Fusion proteins containing soluble forms of the TACI receptor fused to an IgG Fc region are known as well and were designated TACI-Fc (WO 00/40716, WO 10 02/094852). TACI-Fc inhibits the binding of BLyS and APRIL to B-celis (Xia et al., 2000). It is being developed for the treatment of autoimmune diseases, including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and hematological malignancies, as well as for treatment of multiple sclerosis (MS). In addition to this, TACI-Fc is being developed in multiple myeloma (MM) (Novak et al., 2004; Moreau et 15 al., 2004) and non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL) and Waldenstrom's macrogiobulemia (WM). Given the therapeutic utility of Fc-containing proteins, particularly antibodies and Fc-fusion proteins, there is a need for significant amounts of highly purified protein that is adequate for human administration. 20 It is an object of the present invention to go someway towards meeting this need and/or to provide the public with a useful choice. SUMMARY OF THE INVENTION One of the problems that may be encountered during production of Fc-containing proteins is the presence of "free Fc-moieties", i.e. polypeptide fragments 25 derived from the Fc-containing protein, which does not contain a substantial portion derived from an antibody variable region or from another specific protein or domain normally present in the Fc-fusion protein. The present invention addresses this problem. It is based on the development of a purification process for a fluid, composition or preparation of an Fc-containing protein, 30 by which the amount of free Fc-moieties that may be present as an impurity can be reduced. Therefore the invention relates to a method for reducing the concentration of free Fc-moieties in a fluid comprising an Fc-containing protein, the method comprising subjecting said fluid to cation exchange chromatography. Received at IPONZ 09 March 2011 8 in a second aspect, the invention relates to the use of cation exchange chromatography for the reduction of free Fc in an Fc-containing protein preparation. In a third aspect, the invention relates to a purified Fc-containing protein, comprising less than 5 % or less than 2 % or less than 1 % or less than 0.5 % or less 5 than 0.2 % or less than 0.1% of free Fc-moieties, produced by the method of the invention. BRIEF DESCRIPTION OF THE DRAWINGS 10 Fig. 1 shows a non-reduced silver stained SDS-PAGE of different fractions stemming from the cation exchange chromatography described in Example 2. Lane 1: Molecular weight markers, Lane 2: purified TACI-Fc, Lane 3: load, Lane 4: wash 2, Lane 5: eluate 2, Lane 6: wash 3, Lane 7: eluate 3, Lane 8: wash 1, Lane 9: eluate 1, Lane 10: purified free Fc; 15 Fig. 2 shows the chromatographic profile of the cation exchange chromatography described in Example 2. BRIEF DESCRIPTION OF THE SEQUENCE LISTING SEQ ID NO: 1 is a Cysteine fingerprint sequence (cysteine rich pseudo repeat) 20 common to members of the TNFR superfamily; SEQ ID NO: 2 is the full length sequence of the human TACI receptor (e.g. described in WO 98/39361); SEQ ID NO: 3 is an example of a human Fc sequence of the invention (e.g. described in WO 02/094852); 25 SEQ ID NO: 4 is a preferred Fc-fusion protein of the invention, comprising sequences derived from the extracellular portion of TACI and a human IgGi Fc portion (e.g. described in WO 02/094852); SEQ ID NO: 5 is a polynucleotide coding for a polypeptide of SEQ ID NO: 2; SEQ ID NO: 6 is a polynucleotide coding for a polypeptide of SEQ ID NO: 3; 30 SEQ ID NO: 7 is a polynucleotide coding for a polypeptide of SEQ ID NO: 4. WO 2008/025748 PCT/EP2007/058887 DETAILED DESCRIPTION OF THE INVENTION The present invention is based on the finding that cation exchange chromatography can reduce the amount or extent of free Fc-moieties that may be present in a fluid or composition of an Fc-containing protein. 5 The invention therefore relates to a method for reducing the concentration of free Fc-moieties in a fluid comprising an Fc-containing protein, the method comprising subjecting said fluid to cation exchange chromatography. The fluid comprising the Fc-containing protein may be any composition or preparation, such as e.g. a body fluid derived from a human or animal, or a fluid 10 derived from a cell culture, such as e.g. a cell culture supernatant. It may also be a fluid derived from another purification step, such as e.g. the eluate or flow-through from a capture step or any other suitable purification step such as the ones explained in more detail below. The term "Fc-containing protein", as used herein, refers to any protein having at 15 least one immunoglobulin constant domain selected from the CH1, hinge, CH2, CH3, CH4 domain, or any combination thereof, and preferably a hinge, CH2 and CH3 domain. The immunoglobulin constant domain may be derived from any of IgG, IgA, IgE, IgM, or combination or isotype thereof. Preferably, it is IgG, such as e.g. IgG^ lgG2, lgG3 or lgG4. More preferably, it is IgGv 20 An Fc-containing protein, in accordance with the present invention, may thus be e.g. an antibody or an Fc-fusion protein, or variants thereof, such as fragments, muteins or functional derivatives of antibodies or Fc-fusion proteins. The Fc-containing protein of the invention may be a monomer, dimer or multimer. The Fc-containing protein may also be a "pseudo-dimer" (sometimes called 25 "monomer"), containing a dimeric Fc-moiety (e.g. a dimer of two disulfide-bridged hinge-CH2-CH3 constructs), of which only one is fused to a further moiety such as an immunoglobulin variable domain, a ligand binding and optionally inhibiting fragment of a receptor, or any other protein. An example for such a pseudo-dimer is an Fc-fusion protein having lnterferon-p fused to one of the two IgG hinge-CH2-CH3 constructs such 30 as e.g. the one described in WO 2005/001025. The Fc-containing protein may also be a heterodimer, containing two different non-immunoglobulin portions or immunoglobulin variable domains, or a homodimer, WO 2008/025748 PCT/EP2007/058887 containing two copies of a single non-immunoglobulin portion or immunoglobulin variable domain. Preferably, the Fc-containing protein is a dimer. It is also preferred that the Fc-containing protein of the invention is a homo-dimer. 5 In accordance with the present invention, the Fc-moiety of the Fc-containing protein may also be modified in order to modulate effector functions. For instance, the following Fc mutations, according to EU index positions (Kabat et al., 1991), can be introduced if the Fc-moiety is derived from IgGi: T250Q/M428L 10 M252Y/S254T/T256E + H433K/N434F E233P/L234V/L235A/AG236 + A327G/A330S/P331S E333A; K322A. Further Fc mutations may e.g. be the substitutions at EU index positions selected from 330, 331 234, or 235, or combinations thereof. An amino acid 15 substitution at EU index position 297 located in the CH2 domain may also be introduced into the Fc-moiety in the context of the present invention, eliminating a potential site of N-linked carbohydrate attachment. Furthermore, the cysteine residue at EU index position 220 may also be replaced with a serine residue, eliminating the cysteine residue that normally forms disulfide bonds with the immunoglobulin light chain 20 constant region. In accordance with the present invention, it is preferred that the Fc-moiety comprises or consists of SEQ ID NO: 3 or is encoded by a polynucleotide comprising or consisting of SEQ ID NO: 6. In a preferred embodiment, the Fc-containing protein comprises an 25 immunoglobulin variable region, e.g. one or more heavy chain variable domains and/or one or more light chain variable domains. Preferably, the antibody contains one or two heavy chain variable domains. More preferably, the antibody additionally contains one or two light chain constant and/or variable domains. It is preferred that the Fc-containing protein is an antibody. 30 The term "antibody" refers to an immunoglobulin or fragment thereof, and encompasses any polypeptide comprising an antigen-binding site. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, polyspecific, non-specific, humanized, human, chimeric, single-chain, synthetic, recombinant, hybrid, mutated, WO 2008/025748 PCT/EP2007/058887 grafted, or in vitro generated antibodies. The antibody may be selected from any of the known antibody isotypes, for example, IgA, IgG, IgD, IgE, IgM. The antibody may be a monomer, dimer, or multimer such as a trimer, or pentamer. Examples of antibodies that can be purified in accordance with the present 5 invention are Abciximab, Rituximab, Basiliximab, Daclizumab, Palivizumab, Infliximab, Trastuzumab, Alemtuzumab, Adalimumab, Cetuximab, Efalizumab, Ibritumomab, Bevacizumab, or Omalizumab. Further examples of antibodies that can be subjected to cation exchange chromatography in accordance with the present invention are antibodies directed against: 10 CD2, CD3, CD4, CD8, CD11a, CD11b, CD14, CD18, CD20, CD22, CD23, CD25, CD33, CD40, CD44, CD52, CD80, CD86, CD147, CD164, IL-2 receptor, IL-4 receptor, IL-6 receptor, IL-12 receptor, IL-18 receptor subunits (IL-18R-alpha, IL-18R-beta), TACI, BCMA, BAFF-R, EGF receptor, VEGF receptor, integrin a4|37, the integrin VLA4, B2 integrins, TRAIL receptors 1, 2, 3, and 4, RANK, RANK ligand, epithelial cell 15 adhesion molecule (EpCAM), intercellular adhesion molecule-3 (ICAM-3), CTLA4, Fc-gamma-l, II or III receptor, HLA-DR 10 beta, HLA-DR antigen, or L-selectin. Antibodies directed against TNF, Blys, or Interferon^ are further examples of therapeutically interesting antibodies. Fc-fusion proteins are also Fc-containing proteins that are preferably subjected 20 to the method of the invention. The term "Fc-fusion protein", as used herein, is meant to encompass proteins, in particular therapeutic proteins, comprising an immunoglobulin-derived moiety, which will be called herein the "Fc-moiety", and a moiety derived from a second, non-immunoglobulin protein, which will be called herein the "therapeutic moiety", 25 irrespective of whether or not treatment of disease is intended. Therapeutic Fc-fusion proteins, i.e. Fc-fusion proteins intended for treatment or prevention of disease of an animal or preferably for human treatment or administration, are especially suitable to be purified in accordance with the invention. Any Fc-fusion protein may be purified in accordance with the present invention, 30 such as e.g. an lnterferon-p-containing fusion protein. Preferably, the method of the invention is for purifying an Fc-fusion protein comprising a ligand binding fragment, such as all or part of an extracellular domain, of a member of the tumor necrosis factor receptor (TNFR) superfamily. WO 2008/025748 PCT/EP2007/058887 The therapeutic moiety of an Fc-fusion protein may e.g. be or be derived from EPO, TPO, Growth Hormone, Interferon-alpha, Interferon-beta, Interferon-gamma, PDGF-beta, VEGF, IL-1 alpha, IL-1 beta, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12, IL-18, IL-18 binding protein, TGF-beta, TNF-alpha, orTNF-beta. 5 The therapeutic moiety of an Fc-fusion protein may also be derived from a receptor, e.g a transmembrane receptor, preferably be or be derived from the extracellular domain of a receptor, and in particular a ligand binding fragment of the extracellular part or domain of a given receptor. Examples for therapeutically interesting receptors are CD2, CD3, CD4, CD8, CD11a, CD11b, CD14, CD18, CD20, CD22, 10 CD23, CD25, CD33, CD40, CD44, CD52, CD80, CD86, CD147, CD164, IL-2 receptor, IL-4 receptor, IL-6 receptor, IL-12 receptor, IL-18 receptor subunits (IL-18R-alpha, IL-18R-beta), EGF receptor, VEGF receptor, integrin alpha 4 10 beta 7, the integrin VLA4, B2 integrins, TRAIL receptors 1, 2, 3, and 4, RANK, RANK ligand, epithelial cell adhesion molecule (EpCAM), intercellular adhesion molecule-3 (ICAM-3), CTLA4 15 (which is a cytotoxic T lymphocyte- associated antigen), Fc-gamma-l, II or III receptor, HLA-DR 10 beta, HLA-DR antigen, L-selectin. It is highly preferred that the therapeutic moiety is derived from a receptor belonging to the TNFR superfamily. The therapeutic moiety may e.g. be or be derived from the extracellular domain of TNFR1 (p55), TNFR2 (p75), 0X40, Osteoprotegerin, 20 CD27, CD30, CD40, RANK, DR3, Fas ligand, TRAIL-R1, TRAIL-R2, TRAIL-R3, TAIL-R4, NGFR, AITR, BAFFR, BCMA, TACI. In accordance with the present invention, the therapeutic moiety derived from a member of the TNFR superfamily preferably comprises or consists of all or part of the extracellular domain of the member of the TNFR, and more preferably comprises a 25 ligand binding fragment of such a member of the TNFR. The following Table 5 lists members of the TNFR superfamily from which a therapeutic moiety in accordance with the present invention may be derived, and their respective ligands. A "ligand binding fragment" of a member of the TNFR family can easily be determined by the person skilled in the art, e.g. in a simple in vitro assay 30 measuring binding between protein fragment of a given receptor and the respective ligand. Such an assay can e.g. be a simple in vitro RIA- or ELISA-type sandwich assay wherein one of the proteins, e.g. the receptor fragment, is immobilized to a carrier (e.g. an ELISA plate) and is incubated, following appropriate blocking of the protein binding WO 2008/025748 PCT/EP2007/058887 13 sites on the carrier, with the second protein, e.g. the ligand. After incubation, ligand binding is detected e.g. by way of radioactive labeling of the ligand and determination of the bound radioactivity, after appropriate washing, in a scintillation counter. Binding of the ligand can also be determined with a labeled antibody, or a first ligand-specific 5 antibody and a second, labeled antibody directed against the constant part of the first antibody. Ligand binding can thus be easily determined, depending of the label used, e.g. in a color reaction. Preferably, the method of the present invention is for purifying an Fc-fusion protein comprising a therapeutic moiety derived from a member of the TNFR 10 superfamily selected from those listed in Table 5. Table 5: The TNFR superfamily (according to Locksley et al., 2001 and Bossen et al., 2006) Member of TNFR superfamily Ligand NGFR NGF EDAR EDA-A1 XEDAR EDA-A2 CD40 CD40L Fas FasL 0x40 OX40L AITR AITRL GITR GITRL CD30 CD30L CD40 CD40L HveA LIGHT, LT-alpha 4-1BB 4-1BBL TNFR2 TNF-alpha, LT-alpha, LT-alpha-beta LT-betaR LIGHT, LT-alpha, LT-alpha-beta DR3 TL1A CD27 CD27L TNFR1 TNF-alpha, LT-alpha, LT-alpha-beta LTBR LT-beta RANK RANKL WO 2008/025748 PCT/EP2007/058887 14 Member of TNFR superfamily Ligand TACI BlyS, APRIL BCMA BlyS, APRIL BAFF-R BAFF (= BlyS) TRAILR1 TRAIL TRAILR2 TRAIL TRAILR3 TRAIL TRAILR4 TRAIL Fn14 TWEAK OPG RANKL, TRAIL DR4 TRAIL DR5 TRAIL DcR1 TRAIL DcR2 TRAIL DcR3 FasL, LIGHT, TL1A In a preferred embodiment, the Fc-fusion protein comprises a therapeutic moiety selected from an extracellular domain of TNFR1, TNFR2, or a TNF binding fragment thereof. 5 In a further preferred embodiment, the Fc-fusion protein comprises a therapeutic moiety selected from an extracellular domain of BAFF-R, BCMA, or TACI, or a fragment thereof binding at least one of Blys or APRIL. An assay for testing the capability of binding to Blys or APRIL is described e.g. in Hymowitz et al., 2006. 10 In yet a further preferred embodiment, the therapeutic moiety of an Fc-fusion protein comprises the Cysteine rich pseudo-repeat of SEQ ID NO: 1. If is further preferred that the therapeutic moiety is derived from TACI. TACI is preferably human TACI. SEQ ID NO: 2, which corresponds to the amino acid sequence of human full-length TACI receptor (also SwissProt entry 014836). More preferably, the 15 therapeutic moiety comprises a soluble portion of TACI, preferably derived from the extracellular domain of TACI. Preferably, the TACI-derived therapeutic moiety comprises at least amino acids 33 to 67 of SEQ ID NO: 2 and/or amino acids 70 to 104 of SEQ ID NO: 2. In a preferred embodiment, the TACI extracellular domain included in WO 2008/025748 PCT/EP2007/058887 15 the therapeutic moiety according to the invention comprises or consist of amino acids 1 to 166 of SEQ ID NO: 2 or amino acids 30 to 166 of SEQ ID NO: 2, or amino acids 30 to 119 of SEQ ID NO: 2, or amino acids 30 to 110 of SEQ ID NO: 2. All of those therapeutic moieties are preferred for the preparation of the Fc-fusion protein to be 5 purified by the method of the invention and are combined with the Fc-moieties described in detail above, and in particular with an Fc-moiety comprising or consisting of SEQ ID NO: 3. A highly preferred Fc-fusion protein to be purified in accordance with the present invention comprises or consists of SEQ ID NO: 4 or encoded by the polynucleotide of SEQ ID NO: 7. 10 Hence, it is highly preferred that the Fc-fusion protein comprises a polypeptide selected from a. amino acids 34 to 66 of SEQ ID NO: 2; b. amino acids 71 to 104 of SEQ ID NO: 2; c. amino acids 34 to 104 of SEQ ID NO: 2; 15 d. amino acids 30 to 110 of SEQ ID NO: 2; e. SEQ ID NO: 3; f. SEQ ID NO: 4; g. a polypeptide encoded by a polynucleotide hybridizing to the complement of SEQ ID NO: 5 or 6 or 7 under highly stringent 20 conditions; and h. a mutein of any of (c), (d), (e), or (f) having at least 80 % or 85 % or 90 % or 95 % sequence identity to the polypeptide of (c), (d), (e) or (f); wherein the polypeptide binds to at least one of Blys or APRIL. In accordance with the present invention, the Fc-containing protein is subjected 25 to cation exchange chromatography in order to reduce, decrease, or eliminate free Fc-moieties, preferably at least by 50, 40, 30, 20 or 10 % of the total protein concentration, or more preferably less than 10 %. The term "free Fc moieties", "free Fc moiety", or simply "free Fc", as used herein, is meant to encompass any part of the Fc-containing protein to be purified in 30 accordance with the present invention, which is derived from the immunoglobulin constant domain or domains without comprising complete further domains. Thus, if the Fc-containing protein comprises immunoglobulin variable domains, free Fc does not contain significant portions of the variable domains. If the Fc-containing protein is an WO 2008/025748 PCT/EP2007/058887 Fc-fusion protein, free Fc does not contain significant portions of the therapeutic moiety of the Fc-fusion protein. Free Fc may e.g. contain dimers of the IgG hinge, CH2 and CH3 domains, which are not linked or bound to significant portions of a therapeutic moiety or immunoglobulin variable domains, such as e.g. the Fc part that is generated 5 by papain cleavage. A "significant portion" may e.g. be no less than 80, 85, 90, 95, 98 or 99 % of the full-length variable domain or therapeutic moiety present in the Fc-containing protein. Monomers derived from the Fc-moiety may also be contained in the free Fc fraction. It is understood that free Fc may still contain a number of amino acid residues 10 from the therapeutic moiety or the Ig variable domains, such as e.g. one to fifty or one to twenty, or one to ten, or one to five amino acids, or one or two single amino acids, belonging to the therapeutic moiety or variable domain, still fused to the Fc-moiety. The cation exchange chromatography may be carried out on any suitable cation exchange resin, such as e.g. weak or strong cation exchangers as explained above in 15 the Background of the Invention. Preferably, the cation exchange chromatography is carried out on a strong cation exchange resin. More preferably, the cation exchange material comprises a cross-linked methacrylate modified with S03~ groups. A column commercially available under the name Fractogel EMD S03~ (from Merck) is an example of a cation exchange 20 resin that is particularly suitable in the context of the present method. Preferably, the fluid or composition comprising the Fc-containing protein is loaded to a cation exchange resin at a pH of at least one unit below the isoelectric point (pi) of said Fc-fusion protein. In a preferred embodiment, the cation exchange resin is washed with a buffer 25 having a conductivity of 6 to 10 mS/cm and at a pH of 5.5 to 7.5. The buffer may e.g. have a conductivity at 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, or 9.9 mS/cm. More preferably, the conductivity ranges from 7.6 to 9.2, i.e. 8.4 + 0.8 mS/cm. 30 The washing step is preferably carried out at a pH ranging from 5.5 to 7.5, preferably from 6.0 to 7.0. The pH of the buffer may e.g. be at 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5. WO 2008/025748 PCT/EP2007/058887 In a further preferred embodiment, the washing step is carried out in a buffer comprising 60 to 140, preferably 70 to 130, more preferably 75 to 125 mM sodium phosphate. The buffer may e.g. comprise 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, or 140 mM sodium phosphate. 5 In a further preferred embodiment, the cation exchange column is eluted at a pH ranging from 7.0 to 8.5, preferably 7.25 or 7.3 or 7.35 or 7.4 or 7.45 or 7.5 or 7.55 or 7.6 or 7.65 or 7.7 or 7.75 or 7.8 or 7.85 or 7.9 or 7.95 or 8.0 or 8.05 or 8.1 or 8.15 or 8.2 or 8.25 or 8.3 or 8.35 or 8.4 or 8.45 or 8.5. Elution may preferably be carried out at a conductivity ranging from 15 to 22 10 mS/cm. For instance, the conductivity may be selected from 15, 16, 17, 18, 19, 20, 21, or 22 mS/cm. A preferred buffer for elution is a phosphate buffer. In accordance with the present invention, cation exchange chromatography can preferably be used for elimination or reduction of free Fc in the range of 5 to 15 fold. 15 Thus, a reduction of the concentration of free Fc in the Fc-containing protein comprising fluid, preparation or composition to less than 20 % or less than 15 % or less than 10 % or less than 5 % or less than 2 % or less than 1 % or less than 0.8 % or less than 0.5 % or less than 0.3 % or less than 0.2 % or less than 0.1 % of the total protein concentration can be achieved. 20 In a preferred embodiment, the cation exchange chromatography may be used in a purification method having one or more additional steps, preferably selected from affinity chromatography, anion exchange chromatography and hydroxyapatite chromatography. In a highly preferred embodiment, the method of the invention is used as a 25 second step of a purification scheme of an Fc-containing protein comprising the following steps: a. Subjecting a fluid comprising said Fc-containing protein to Protein A or Protein G or Protein L affinity chromatography; b. Subjecting the eluate of step (a) to Cation exchange chromatography; 30 c. Subjecting the eluate of step (b) to Anion exchange chromatography; d. Subjecting the flow-through of step (c) to Hydroxyapatite chromatography and collecting the eluate to obtain purified Fc-containing protein. WO 2008/025748 PCT/EP2007/058887 In accordance with the present invention, a fluid comprising an Fc-containing protein is first subjected to Protein A or Protein G or Protein L or Protein A/G affinity chromatography. The fluid may preferably be cell culture material, e.g. solubilized cells, more preferably cell culture supernatant. The term "cell culture supernatant", as used 5 herein, refers to a medium in which cells are cultured and into which proteins are secreted provided they contain appropriate cellular signals, so-called signal peptides. It is preferred that the Fc-containing protein expressing cells are cultured under serum-free culture conditions. Thus, preferably, the cell culture supernatant is devoid of animal-serum derived components. Most preferably, the cell culture medium is a 10 chemically defined medium. The Protein A, G, A/G or L used for the affinity chromatography may e.g. be recombinant. It may also be modified in order to improve its properties (such as e.g. in the resin called MabSelect SuRe, commercially available from GE Healthcare). In a preferred embodiment, step (a) is carried out on a resin comprising cross-linked 15 agarose modified with recombinant Protein A. A column commercially available under the name Mabselect Xtra (from GE Healthcare) is an example of an affinity resin that is particularly suitable for step (a) of the present method. The Protein A or G or L affinity chromatography is preferably used as a capture step, and thus serves for purification of the Fc-containing protein, in particular 20 elimination of host cell proteins and Fc-containing protein aggregates, and for concentration of the Fc-containing protein preparation. The term "aggregates", as used herein, is meant to refer to protein aggregates, and encompasses multimers (such as dimers, tetramers or higher order aggregates) of the Fc-containing protein to be purified and may result e.g. in high molecular weight 25 aggregates. The affinity chromatography has the further advantage of reducing aggregate levels by 2 to 4 fold. In using the Protein A or G or A/G or L affinity chromatography, host cell protein levels may be reduced by 100 to 300 fold. 30 In a preferred embodiment of the invention, the elution in step (a) is carried out at a pH ranging from 2.8 to 4.5, preferably from 3.0 to 4.2, more preferably at 3.5, 3.55, 3.6, 3.65, 3.7, 3.75, 3.8, 3.85, 3.9, 3.95, 4.0, 4.05, 4.1, or 4,15. The elution in step (a) may also be carried out with a pH gradient, preferably a gradient from pH 4.5 to 2.8. WO 2008/025748 PCT/EP2007/058887 In a further preferred embodiment, the elution in step (a) is carried out in a buffer selected from sodium acetate or sodium citrate. Suitable buffer concentrations are e.g. selected from 50 mM or 100 mM or 150 mM or 200 mM or 250 mM. In accordance with the present invention, the eluate from the Protein A or 5 Protein G or Protein A/G or Protein L chromatography is subjected to cation exchange chromatography, as explained in detail above. Preferably, the eluate of the cation exchange chromatography, i.e. of step (b), is diluted or dialysed into an appropriate loading buffer before loading it on the anion exchange column. The anion exchange column is also preferably equilibrated with the 10 loading buffer. A preferred pH for the loading buffer is one unit below the pi. Suitable pH values range from 6.0 to 8.5, preferably from 7.0 to 8.0, e.g. 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, 7.4, 7.45, 7.5, 7.55, 7,6, 7.65, 7.7, 7.75, 7.8, 7.85, 7.9, 7.95, or 8.0. A preferred conductivity for the loading buffer is in the range of 3.0 to 4.6 mS/cm. 15 An appropriate equilibration/loading buffer may e.g. be sodium phosphate at a concentration ranging from 5 to 35, preferably from 20 to 30 mM. The buffer concentration may e.g. be at 10, 15, 20, 25, 30 mM. In the frame of the present invention, the flow-through (also called break-through) of the anion exchange chromatography, comprising the Fc-containing protein of interest, is being collected. 20 Step (c) of the method of the invention further reduces aggregates 3 to 5 fold and host cell proteins 30 to 70 fold. In accordance with the present invention, the flow-through of the anion exchange chromatography of step (c) is then used for further purification by hydroxyapatite chromatography. Any hydroxyapatite resin may be used to carry out 25 step (d) of the method according to the invention. In a preferred embodiment, step (d) is carried out on a ceramic hydroxyapatite resin, such as a type I or type II hydroxyapatite resin. The hydroxyapatite resin may have particles of any size such as 20, 40 or 80 [im. In a highly preferred embodiment, the ceramic hydroxyapatite resin comprises particles having a size of 40 [im. A hydroxyapatite resin that is particularly suitable for step (d) of 30 the present method is a column commercially available under the name CHT Ceramic Hydroxyapatite Type I, 40 prn. In a preferred embodiment, the flow-through from step (c) is directly loaded on the hydroxyapatite resin, i.e. without previous dilution or dialysis into an appropriate WO 2008/025748 PCT/EP2007/058887 loading buffer. Loading is preferably carried out at a pH of 6.5 to 7.5, such as 6.6, 6.7, 6.8, 6.9, 7.1, 7.2, 7.3, or 7.4, and preferably 7.0. In a further preferred embodiment, the elution in step (d) is carried out in the presence of sodium phosphate ranging from 2 to 10 mM, preferably ranging from 2.75 5 to 5.25 mM, such as e.g. at 3, 3.25, 3.5, 3.75, 4, 4.25, 4,5, 4.75, 5. In yet a further preferred embodiment, the elution in step (d) is carried out at a pH ranging from 6.0 to 7.0, e.g. at 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9. In another preferred embodiment, elution in step (d) is carried out in the presence of potassium chloride ranging from 0.4 to 1 M, preferably at 0.45, 0.5, 0.55, 10 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95 M, most preferably at 0.6 M. In accordance with the present invention, the eluate of the hydroxyapatite chromatography is collected, containing the finally purified Fc-containing protein preparation. Suitable matrix materials, i.e. carrier materials for the chromatographic resins 15 used in steps (a) to (c), that may be used in connection with the present invention may e.g. be agarose (sepharose, superose) dextran (sephadex), polypropylene, methacrylate cellulose, polystyrene/divinyl benzene, or the like. The resin materials may be present in different cross-linked forms, depending on the specific use. The volume of the resin, the length and diameter of the column to be used, as 20 well as the dynamic capacity and flow-rate depend on several parameters such as the volume of fluid to be treated, concentration of protein in the fluid to be subjected to the process of the invention, etc. Determination of these parameters for each step is well within the average skills of the person skilled in the art. In a preferred embodiment of the present purification process, one or more 25 ultrafiltration steps are performed. Ultrafiltration is useful for removal of small organic molecules and salts in the eluates resulting from previous chromatrographic steps, to equilibrate the Fc-containing protein in the bulk buffer, or to concentrate the Fc-containing protein to the desired concentration. Such ultrafiltration may e.g. be performed on ultrafiltration membranes, with pore sizes allowing the removal of 30 components having molecular weights below 5, 10, 15, 20, 25, 30 or more kDa. Preferably, ultrafiltration is carried out between steps (b) and (c), and/or after step (d). More preferably, two ultrafiltration steps are carried out, one between steps (b) and (c) and one after step (d). WO 2008/025748 PCT/EP2007/058887 If the protein purified according to the process of the invention is intended for administration to humans, it is advantageous to include one or more steps of virus removal in the process. Preferably, a virus removal filtration step is carried out after step (d). More preferably, the virus removal filtration step is a nanofiltration step where 5 the filter has a nominal pore size of 20nm. The method of the present invention, and in particular steps (a), (c), (d) in combination with nanofiltration efficiently eliminates virus load to a combined LRV (log reduction value) of up to about 15 to 25. In order to facilitate storage or transport, for instance, the material may be frozen and thawed before and/or after any purification step of the invention. 10 In accordance with the present invention, the recombinant Fc-containing protein may be produced in eukaryotic expression systems, such as yeast, insect, or mammalian cells, resulting in glycosylated Fc-containing proteins. In accordance with the present invention, it is most preferred to express the Fc-containing protein in mammalian cells such as animal cell lines, or in human cell lines. 15 Chinese hamster ovary cells (CHO) or the murine myeloma cell line NSO are examples of cell lines that are particularly suitable for expression of the Fc-containing protein to be purified. The Fc-containing protein can also preferably be produced in human cell lines, such as e.g. the human fibrosarcoma HT1080 cell line, the human retinoblastoma cell line PERC6, or the human embryonic kidney cell line 293, or a permanent 20 amniocyte cell line as described e.g. in EP 1 230 354 . If the Fc-containing protein to be purified is expressed by mammalian cells secreting it, the starting material of the purification process of the invention is cell culture supernatant, also called harvest or crude harvest. If the cells are cultured in a medium containing animal serum, the cell culture supernatant also contains serum 25 proteins as impurities. Preferably, the Fc-containing protein expressing and secreting cells are cultured under serum-free conditions. The Fc-containing protein may also be produced in a chemically defined medium. In this case, the starting material of the purification process of the invention is serum-free cell culture supernatant that mainly contains host cell 30 proteins as impurities. If growth factors are added to the cell culture medium, such as insulin, for example, these proteins will be eliminated during the purification process as well. WO 2008/025748 PCT/EP2007/058887 In order to create soluble, secreted Fc-containing protein, that are released into the cell culture supernatant, either the natural signal peptide of the therapeutic moiety of the Fc-containing protein is used, or preferably a heterologous signal peptide, i.e. a signal peptide derived from another secreted protein being efficient in the particular 5 expression system used, such as e.g. the bovine or human Growth Hormone signal peptide, or the immunoglobulin signal peptide. As mentioned above, a preferred Fc-containing protein to be purified in accordance with the present invention is a fusion protein having a therapeutic moiety derived from human TACI (SEQ ID NO: 2), and in particular a fragment derived from its 10 extracellular domain (amino acids 1 to 165 of SEQ ID NO: 2). A preferred fragment comprises amino acids 30 to 110 of SEQ ID NO: 2. In the following, therapeutic moieties derived from the extracellular domain of TACI will be called "soluble TACI" or "sTACI". A preferred Fc-moiety comprises SEQ ID NO: 3, resulting in an Fc-fusion protein according to SEQ ID NO: 4, in the following called "TACI-Fc". The term TACI-Fc, 15 as used herein, also encompasses muteins of TACI-Fc. The term "muteins", as used herein, refers to analogs of sTACI or TACI-Fc, in which one or more of the amino acid residues of sTACI or TACI-Fc are replaced by different amino acid residues, or are deleted, or one or more amino acid residues are added to the original sequence of sTACI or TACI-Fc without changing considerably the 20 activity of the resulting products as compared with the original sTACI or TACI-Fc. These muteins are prepared by known synthesis and/or by site-directed mutagenesis techniques, or any other known technique suitable therefor. Muteins in accordance with the present invention include proteins encoded by a nucleic acid, such as DNA or RNA, which hybridizes to the complement of a DNA or 25 RNA, which encodes a sTACI or TACI-Fc according to any of SEQ ID NOs: 2 or 4 under stringent conditions. An example for a DNA sequence encoding a TACI-Fc is SEQ ID NO: 7. The term "stringent conditions" refers to hybridization and subsequent washing conditions, which those of ordinary skill in the art conventionally refer to as "stringent". 30 See Ausubel et al., Current Protocols in Molecular Biology, supra, Interscience, N.Y., §§6.3 and 6.4 (1987, 1992). Without limitation, examples of stringent conditions include washing conditions 12-20°C below the calculated Tm of the hybrid under study in, e.g., 2 x SSC and 0.5% SDS for 5 minutes, 2 x SSC and 0.1% SDS for 15 minutes; 0.1 x WO 2008/025748 PCT/EP2007/058887 SSC and 0.5% SDS at 37°C for 30-60 minutes and then, a 0.1 x SSC and 0.5% SDS at 68°C for 30-60 minutes. Those of ordinary skill in this art understand that stringency conditions also depend on the length of the DNA sequences, oligonucleotide probes (such as 10-40 bases) or mixed oligonucleotide probes. If mixed probes are used, it is 5 preferable to use tetramethyl ammonium chloride (TMAC) instead of SSC. See Ausubel, supra. In another embodiment, any such mutein has at least 50 %, at least 60 %, at least 70 %, at least 75 %, at least 80%, at least 85 %, at least 90%, or at least 95 % identity or homology thereto. 10 Identity reflects a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by comparing the sequences. In general, identity refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of the two polynucleotides or two polypeptide sequences, respectively, over the length of the sequences being compared. 15 For sequences where there is not an exact correspondence, a "% identity" may be determined. In general, the two sequences to be compared are aligned to give a maximum correlation between the sequences. This may include inserting "gaps" in either one or both sequences, to enhance the degree of alignment. A % identity may be determined over the whole length of each of the sequences being compared (so-called 20 global alignment), that is particularly suitable for sequences of the same or very similar length, or over shorter, defined lengths (so-called local alignment), that is more suitable for sequences of unequal length. Methods for comparing the identity and homology of two or more sequences are well known in the art. Thus for instance, programs available in the Wisconsin Sequence 25 Analysis Package, version 9.1 (Devereux J et al., 1984), for example the programs BESTFIT and GAP, may be used to determine the % identity between two polynucleotides and the % identity and the % homology between two polypeptide sequences. BESTFIT uses the "local homology" algorithm of Smith and Waterman (1981) and finds the best single region of similarity between two sequences. Other 30 programs for determining identity and/or similarity between sequences are also known in the art, for instance the BLAST family of programs (Altschul S F et al, 1990, Altschul S F et al, 1997, accessible through the home page of the NCBI at www.ncbi.nlm.nih.gov) and FASTA (Pearson W R, 1990). WO 2008/025748 PCT/EP2007/058887 24 Any such mutein preferably has a sequence of amino acids sufficiently duplicative of that of sTACI or TACI-Fc, such as to have substantially similar ligand binding activity as a protein of SEQ ID NO: 2 or 4. For instance, one activity of TACI is its capability of binding to Blys or APRIL (Hymowitz et al., 2006). As long as the mutein 5 has substantial APRIL or Blys binding activity, it can be considered to have substantially similar activity to TACI. Thus, it can be easily determined by the person skilled in the art whether any given mutein has substantially the same activity as a protein of SEQ ID NO: 2 or 4 by means of routine experimentation. 10 what are known as "conservative" substitutions. Conservative amino acid substitutions of sTACI or TACI-Fc, may include synonymous amino acids within a group which have sufficiently similar physicochemical properties that substitution between members of the group will preserve the biological function of the molecule (Grantham, 1974). It is clear that insertions and deletions of amino acids may also be made in the above-defined 15 sequences without altering their function, particularly if the insertions or deletions only involve a few amino acids, e.g., under thirty, under twenty, or preferably under ten, and do not remove or displace amino acids which are critical to a functional conformation, e.g., cysteine residues. Proteins and muteins produced by such deletions and/or insertions come within the purview of the present invention. 20 Preferably, the conservative amino acid groups are those defined in Table 2. More preferably, the synonymous amino acid groups are those defined in Table 3; and most preferably the synonymous amino acid groups are those defined in Table 4. Preferred changes for muteins in accordance with the present invention are 25 TABLE 2 Preferred Groups of Synonymous Amino Acids 35 30 Amino Acid Ser Arg Leu Pro Thr Ala Val Gly lie Phe Tyr Cys Synonymous Group Ser, Thr, Gly, Asn Arg, Gin, Lys, Glu, His lie, Phe, Tyr, Met, Val, Leu Gly, Ala, Thr, Pro Pro, Ser, Ala, Gly, His, Gin, Thr Gly, Thr, Pro, Ala Met, Tyr, Phe, lie, Leu, Val Ala, Thr, Pro, Ser, Gly Met, Tyr, Phe, Val, Leu, lie Trp, Met, Tyr, lie, Val, Leu, Phe Trp, Met, Phe, lie, Val, Leu, Tyr Ser, Thr, Cys WO 2008/025748 PCT/EP2007/058887 25 His Gin Asn Lys Asp Glu Met Trp Glu, Lys, Gin, Thr, Arg, His Glu, Lys, Asn, His, Thr, Arg, Gin Gin, Asp, Ser, Asn Glu, Gin, His, Arg, Lys Glu, Asn, Asp Asp, Lys, Asn, Gin, His, Arg, Glu Phe, lie, Val, Leu, Met Trp 10 TABLE 3 More Preferred Groups of Synonymous Amino Acids Amino Acid Synonymous Group Ser Ser Arg His, Lys, Arg 15 Leu Leu, lie, Phe, Met Pro Ala, Pro Thr Thr Ala Pro, Ala Val Val, Met, lie 20 Gly Gly lie lie, Met, Phe, Val, Leu Phe Met, Tyr, lie, Leu, Phe Tyr Phe, Tyr Cys Cys, Ser 25 His His, Gin, Arg Gin Glu, Gin, His Asn Asp, Asn Lys Lys, Arg Asp Asp, Asn 30 Glu Glu, Gin Met Met, Phe, lie, Val, Leu Trp Trp 35 TABLE 4 Most Preferred Groups of Synonymous Amino Acids Amino Acid Synonymous Group Ser Ser Arg Arg Leu Leu, lie, Met Pro Pro Thr Thr Ala Ala Val Val Gly Gly lie lie, Met, Leu Phe Phe Tyr Tyr WO 2008/025748 PCT/EP2007/058887 26 Cys His Gin Asn Lys Asp Glu Met Trp His Gin Asn Lys Asp Glu Cys, Ser Met, lie, Leu Met 10 15 20 25 30 A functional derivative may be prepared from an Fc-fusion protein purified in accordance with the present invention. "Functional derivatives" as used herein cover derivatives of the Fc-containing protein to be purified in accordance with the present invention, which may be prepared from the functional groups which occur as side chains on the residues or the N- or C-terminal groups, by means known in the art, and are included in the invention as long as they remain pharmaceutical^ acceptable, i.e. they do not destroy the activity of the protein which is substantially similar to the activity of the unmodified Fc-containing protein as defined above, and do not confer toxic properties on compositions containing it. Functional derivatives of an Fc-containing protein can e.g. be conjugated to polymers in order to improve the properties of the protein, such as the stability, half-life, bioavailability, tolerance by the human body, or immunogenicity. To achieve this goal, the Fc-containing protein may be linked e.g. to polyethylene glycol (PEG). PEGylation may be carried out by known methods, described in WO 92/13095, for example. Functional derivatives may also, for example, include aliphatic esters of the carboxyl groups, amides of the carboxyl groups by reaction with ammonia or with primary or secondary amines, N-acyl derivatives of free amino groups of the amino acid residues formed with acyl moieties (e.g. alkanoyl or carbocyclic aroyl groups) or O-acyl derivatives of free hydroxyl groups (for example that of seryl or threonyl residues) formed with acyl moieties. In a third aspect, the invention relates to a protein purified by the process of purification according to the invention. In the following, such protein is also called "purified Fc-containing protein". Such purified Fc-containing protein is preferably highly purified Fc-containing protein. Highly purified Fc-fusion protein is determined e.g. by the presence of a single band in a silver-stained, non-reduced SDS-PAGE-gel after loading of protein in the Received at IPONZ 09 March 2011 27 amount of 2 meg per lane. Purified Fc-fusion protein may also be defined as eluting as a single peak in HPLC. The Fc-containing protein preparation obtained from the purification process of the invention may contain less than 20 % of impurities, preferably less than 10%, 5%, 5 3%, 2% or 1% of impurities, or it may be purified to homogeneity, i.e. being free from any detectable proteinaceous contaminants contaminants as determined e.g. by silver stained SDS-PAGE or HPLC, as explained above. Purified Fc-containing protein may be intended for therapeutic use, in particular for administration to human patients. If purified Fc-containing protein is administered to 10 patients, it is preferably administered systemically, and preferably subcutaneously or intramuscularly, or topically, i.e. locally. Rectal or intrathecal administration may also be suitable, depending on the specific medical use of purified Fc-containing protein. For this purpose, in a preferred embodiment of the present invention, the purified Fc-containing protein may be formulated into pharmaceutical composition, i.e. 15 together with a pharmaceutical^ acceptable carrier, excipients or the like. The term "comprising" as used in this specification and claims means "consisting at least in part of". When interpreting statements in this specification and claims which include the "comprising", other features besides the features prefaced by this term in each statement can also be present. Related terms such as "comprise" and 20 "comprised" are to be interpreted in similar manner. The definition of "pharmaceutical^ acceptable" is meant to encompass any carrier, which does not interfere with effectiveness of the biological activity of the active ingredient and that is not toxic to the host to which it is administered. For example, for parenteral administration, the active protein(s) may be formulated in a unit dosage form 25 for injection in vehicles such as saline, dextrose solution, serum albumin and Ringer's solution. The active ingredients of the pharmaceutical composition according to the invention can be administered to an individual in a variety of ways. The routes of administration include intradermal, transdermal (e.g. in slow release formulations), 30 intramuscular, intraperitoneal, intravenous, subcutaneous, oral, intracranial, epidural, topical, recta!, and intranasal routes. Any other therapeutically efficacious route of administration can be used, for example absorption through epithelial or endothelial tissues or by gene therapy wherein a DNA molecule encoding the active agent is Received at IPONZ 09 March 2011 27a administered to the patient (e.g. via a vector), which causes the active agent to be expressed and secreted in vivo. In addition, the protein(s) according to the invention can be administered together with other components of biologically active agents such as pharmaceutically acceptable surfactants, excipients, carriers, diluents and vehicles. WO 2008/025748 PCT/EP2007/058887 For parenteral (e.g. intravenous, subcutaneous, intramuscular) administration, the active protein(s) can be formulated as a solution, suspension, emulsion or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle (e.g. water, saline, dextrose solution) and additives that maintain isotonicity (e.g. 5 mannitol) or chemical stability (e.g. preservatives and buffers). The formulation is sterilized by commonly used techniques. The therapeutically effective amounts of the active protein(s) will be a function of many variables, including the type of Fc-containing protein, the affinity of the Fc-containing protein for its ligand, the route of administration, the clinical condition of the 10 patient. A "therapeutically effective amount" is such that when administered, the Fc-containing protein results in inhibition of its ligand of the therapeutic moiety of the Fc-fusion protein, as explained above and referring particularly to Table 5 above. The dosage administered, as single or multiple doses, to an individual will vary 15 depending upon a variety of factors, including pharmacokinetic properties of the Fc-fusion protein, the route of administration, patient conditions and characteristics (sex, age, body weight, health, size), extent of symptoms, concurrent treatments, frequency of treatment and the effect desired. Adjustment and manipulation of established dosage ranges are well within the ability of those skilled in the art, as well as in vitro 20 and in vivo methods of determining the inhibition of the natural ligand of the therapeutic moiety in an individual. Purified Fc-containing protein may be used in an amount of 0.001 to 100 mg/kg or 0.01 to 10 mg/kg or body weight, or 0. 1 to 5 mg/kg of body weight or 1 to 3 mg/kg of body weight or 2 mg/kg of body weight. 25 In further preferred embodiments, the purified Fc-containing protein is administered daily or every other day or three times per week or once per week. The daily doses are usually given in divided doses or in sustained release form effective to obtain the desired results. Second or subsequent administrations can be performed at a dosage which is the same, less than or greater than the initial or 30 previous dose administered to the individual. A second or subsequent administration can be administered during or prior to onset of the disease. WO 2008/025748 PCT/EP2007/058887 The present invention further relates to the use of cation exchange chromatography for the reduction of the concentration of free Fc-moieties in a composition comprising an Fc-containing protein. In a preferred embodiment, the concentration of free Fc is reduced to less than 5 20 % or less than 15 % or less than 10 % or less than 5 % or less than 2 % or less than 1 % or less than 0.8 % or less than 0.5 % or less than 0.2 % or less than 0.1 % of the total protein concentration of said composition. Having now fully described this invention, it will be appreciated by those skilled in the 10 art that the same can be performed within a wide range of equivalent parameters, concentrations and conditions without departing from the spirit and scope of the invention and without undue experimentation. While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is 15 intended to cover any variations, uses or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims. 20 All references cited herein, including journal articles or abstracts, published or unpublished U.S. or foreign patent application, issued U.S. or foreign patents or any other references, are entirely incorporated by reference herein, including all data, tables, figures and text presented in the cited references. Additionally, the entire contents of the references cited within the references cited herein are also entirely incorporated by reference. 25 Reference to known method steps, conventional methods steps, known methods or conventional methods is not in any way an admission that any aspect, description or embodiment of the present invention is disclosed, taught or suggested in the relevant art. The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art 30 (including the contents of the references cited herein), readily modify and/or adapt for various application such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning a range of equivalents of the WO 2008/025748 PCT/EP2007/058887 30 disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art. EXAMPLES: PURIFICATION OF RECOMBINANT, HUMAN TACI-Fc FROM SERUM-FREE CHO CELL SUPERNATANT Glossary used in all Examples BV: bed volume CHO: Chinese Hamster Ovary DSP: Downstream Process EDTA: Ethylene Diamine Tetraacetic Acid ELISA: Enzyme-Linked Immunosorbent Assay HAC: Hydroxyapatite Chromatography HCP: Host Cell Protein HPLC: High Performance Liquid Chromatography id: internal diameter K: potassium kD: kilo Dalton MES: 2-Morpholinoethanesulfonic acid Na: sodium NaAc: Sodium Acetate n/d: not determined PA-SE-HPLC: Protein A Size-Exclusion High Performance Liquid SE-HPLC: SDS-PAGE: PPm: RO: RT: Chromatography parts per million Reverse Osmosis Room Temperature Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis Size-Exclusion High Performance Liquid Chromatography WO 2008/025748 PCT/EP2007/058887 31 T°C: TMAC: Temperature Tetra-Methyl Ammonium Chloride Ultra-Violet UV: WFI: Water For Injection Water Reverse Osmosis 5 WRO Example 1: Capture Step: Affinity purification on Protein A Starting material was clarified harvest of a TACI-Fc expressing CHO cell clone 10 cultured under serum-free conditions and stored frozen until use. The Capture Step on a MabSelect Xtra™ column (GE Healthcare 17-5269-03) was carried out according to the following protocol, on a column having a bed height of 17cm. All operations were performed at room temperature, except for the load solution, 15 which was kept at a temperature below 15°C. The UV signal at 280 nm was recorded. Sanitization The column was sanitised with at least 3BV of 0.1 M acetic acid + 20% ethanol in reverse flow at 250cm/h. The flow was stopped for 1 hour. 20 Wash step The column was washed with at least 2BV of RO water in reverse flow at 250cm/h. Equilibration The column was equilibrated with at least 5BV of 25mM sodium phosphate + 25 150mM NaCI pH7.0 (until conductivity and pH parameters are within specified range: pH 7.0 ±0.1, conductivity 18 ± 2 mS/cm) in down flow at 450cm/h. 30 Loading The column was loaded with clarified harvest kept at a temperature below 15°C to a capacity of up to 15mg total TACI-Fc as determined by Biacore assay per ml of packed resin at a flow rate of 350cm/h. WO 2008/025748 PCT/EP2007/058887 32 Wash step Wash the column with at least 2BV of equilibration buffer at 350cm/h then with at least 4BV of equilibration buffer (until the UV signal is back to baseline) at 450cm/h. Elution 5 The material was eluted with different elution buffers as shown in Table I at a flow rate of 350 cm/h. The eluate fraction was collected from start of UV signal increase to 6.0±0.5BV of elution. The eluate was incubated for 1 hour at room temperature at a pH below 4.1 (adjused by addition of citric acid solution, if necessary) and then the pH was adjusted to 5.0±0.1 by addition of 32% NaOH solution. 10 Regeneration The column was regenerated with at least 3BV of 50mM NaOH + 1M NaCI in reverse flow at 450cm/h, stop the flow for 15min then re-start the flow at 450cm/h for at least 3BV (until the UV signal is back to baseline). From this step, the column was operated in reverse flow mode. 15 Wash step The column was washed with at least 2BV of RO water at 450cm/h. Sanitisation The column was santitised with at least 3BV of sanitisation buffer at 250cm/h, the flow stopped and the column incubated for 60min. 20 Final Wash steps The column was washed with at least 1BV of RO water at 250cm/h, then with at least 3BV of equilibration buffer at 250cm/h and finally with at least 2BV of RO water at 250cm/h. Finally, the column was stored after flushing with at least 3BV of 20% ethanol at 25 250cm/h. 30 Results Table I: Results using different elution buffers WO 2008/025748 PCT/EP2007/058887 33 Run # Elution buffer TACI-Fc yield (%) Aggregates (%) HCPs (ppm) 1 50mM NaAc pH3.7 47.7 30.3 5558 2 100mM NaAc pH3.8 55.7 25.2 n/d 3 200mM NaAc pH3.8 58.0 28.2 n/d 4 100mM NaAc pH3.7 68 30.0 n/d 5 0.2M NaAc+150mM NaCI pH4 75.1 3.8 n/d 6 100mM NaAc pH3.7 84.6 22 3491 7 250mM NaAc pH3.7 82.8 18.7 3318 8 100mM Na citrate pH3.7 79.2 8.8 4710 9 250mM Na citrate pH3.7 71.9 23 2347 10 100mM Na citrate pH3.75 82.8 8.5 1576 11 100mM Na citrate pH3.75 66.6 9.0 664 12 100mM NaAc pH3.85 83.3 15.0 n/d 13 100mM Na citrate pH3.75 81.0 9.1 3490 14 100mM Na citrate pH3.65 75.1 14.6 2580 14 100mM Na citrate pH3.75 44.7 18.4 3783 16 100mM Na citrate pH3.75 47.1 15.8 3217 17 100mM Na citrate pH3.75 50.7 9.4 2349 18 100mM Na citrate pH3.75 58.0 10.4 2550 19 100mM Na citrate pH3.75 67.1 28.7 2372 20 100mM Na citrate pH3.75 65.6 17.5 2353 21 100mM Na citrate pH3.75 75.6 19.4 1807 22 100mM Na citrate pH3.75 57.1 20.7 2465 23 100mM Na citrate pH3.75 51.9 18.4 2030 24 100mM Na citrate pH3.75 58 11.5 1746 25 100mM Na citrate pH3.75 41.8 22.9 3029 26 100mM Na citrate pH3.9 39.4 6.0 2424 27 100mM Na citrate pH3.9 31.0 8.8 2936 28 100mM Na Ac pH4.1 28.3 25.0 3311 29 100mM Na citrate pH3.9 46.4 9.1 n/d 30 100mM NaAc pH4.1 42.8 13.4 n/d 31 100mM Na citrate pH3.75 57.5 26.5 n/d 32 100mM NaAc pH4.2 38.1 10.1 n/d 33 100mM Na citrate pH3.9 43.3 8.3 2011 WO 2008/025748 PCT/EP2007/058887 34 Run # Elution buffer TACI-Fc yield (%) Aggregates (%) HCPs (ppm) 34 100mM Na citrate pH3.9 63.6 6.6 1749 35 100mM Na citrate pH3.9 65.7 7.3 1689 36 100mM Na citrate pH3.9 62.7 7.4 1609 37 100mM Na citrate pH3.9 61.6 7.4 1479 38 100mM Na citrate pH3.9 60.6 7.4 1623 39 100mM Na citrate pH3.9 64.6 8.0 1497 Conclusions TACI-Fc5 in clarified harvest was captured directly on a MabSelect Xtra column at a dynamic capacity of 15g total TACI-Fc5 per L of packed resin at a flow rate of 350 5 cm/h. Elution conditions, especially pH, were optimized to maximize recovery of product while providing significant reduction in aggregate levels. An elution buffer of 0.1 M sodium citrate pH 3.9 was selected giving about 5-10% aggregate levels starting from about 25-40% in clarified harvest and with no turbidity observed. HCP levels were typically 1500-2000ppm. The HCP levels were measured by ELISA using polyclonal 10 antibodies. The antibod mixture w generated against host cell proteins derived from clarified and concentrated cell culture supernatant of non-transfected CHO cells. Example 2: Cation Exchange Chromatography The eluate from the capture step on Protein A, dialysed into suitable loading 15 buffer, was used as a starting material for the cation exchange chromatography. A Fractogel EMD S03~ column (Merck 1.16882.0010) having a bed height of 10 cm was used in this step. A Fractogel S03~ column with a bed height of 15 cm may be used as well. In the latter case, the dynamic capacity and flow rate may need adaptation, which is well within routine knowledge of the person skilled in the art. 20 All the operations were performed at room temperature and the flow rate was kept constant at 150 cm/h. The UV signal at 280 nm was recorded at all time. Wash step The column was washed with at least 1BV of WRO (water reverse osmosis). 25 Sanitisation WO 2008/025748 PCT/EP2007/058887 35 Then, the column was sanitised with at least 3BV of 0.5M NaOH + 1.5M NaCI in up-flow mode. Rinsing The column was rinsed with at least 4BV of WRO in down-flow mode. 5 Equilibration The column was equilibrated with at least 4BV of 100mM sodium citrate pH5.0 (or until the target conductivity of 12±1 mS/cm and pH 5.0 ± 0.1 are reached). Loading The column was loaded with post capture material at pH 5.0 (pH at 5.0±0.1, 10 conductivity at 12±1 mS/cm) and at a capacity of no more than 50mg TACI-Fc, as determined by SE-HPLC assay per ml of packed resin. Wash step The column was then washed with at least 5BV of 100mM sodium phosphate pH6.5. 15 Elution The column was eluted with different buffers and under different conditions as reported in tables II - IV below. Regeneration and sanitisation The column was regenerated and sanitised with 4BV of 0.5M NaOH + 1.5M 20 NaCI in up-flow mode. Then, the flow was stopped for 30min. Rinsing The column was rinsed with at least 4BV of WRO. Storing The column was stored in at least 3BV of 20% ethanol. 25 Results Table II: Effect of elution pH and conductivity HCP levels in the load: 189ppm PH Conductivity (mS/cm) TACI-Fc recovery HCPs (ppm) HCP clearance (x) 6.5 15.0 25% 118 1.6 7.3 22.5 100% 50 3.8 8.0 15.0 95% 34 5.5 7.3 22.5 100% 56 3.4 WO 2008/025748 PCT/EP2007/058887 36 PH Conductivity (mS/cm) TACI-Fc recovery HCPs (ppm) HCP clearance (x) 7.3 33.0 98% 133 1.4 7.3 22.5 96% 45 4.2 7.3 22.5 97% 53 3.6 7.3 12.0 54% 79 2.4 6.3 22.5 83% 47 4.1 8.0 30.0 96% 108 1.8 8.2 22.5 97% 46 4.2 6.5 30.0 91% 116 1.6 7.3 22.5 93% 48 3.9 7.3 22.5 95% 40 4.8 Table III shows the TACI-Fc recovery and HCP clearance when loading at a capacity of 10 and 32 mg TACI-Fc per ml of resin and eluting in a phosphate buffer at a conductivity of between 12 to 33 mS/cm. Collection of the peak was done from the 5 beginning of the UV increase for 10 + 0.5 BV. Table III: Effect of optimal elution pH and conductivity when loading at capacity HCP levels in load: 201 ppm Loading capacity (mg/ml) PH Conductivity (mS/cm) TACI-Fc recovery HCPs (ppm) HCP clearance (x) 10 8.0 15.0 91% 67 3.0 20.7 93% 61 3.3 32 8.0 20.7 88% 54 3.7 10 Table IV shows the effect of a wash step with 50 or 100 or 150mM sodium phosphate pH 6.5 on TACI-Fc recovery and HCP clearance. 15 Table IV: Effect of wash step conditions on column performance WO 2008/025748 PCT/EP2007/058887 37 HCP levels in the load: 190ppm and aggregate levels: 2.0% Sodium Phosphate TACI-Fc TACI-Fc Aggregates HCPs in concentration in wash yield in yield in in eluate eluate (mM] wash eluate (ppm) wash 1 50 0.7% 99% 2.8% 62 wash 2 100 2.1% 98% 2.9% 59 wash 3 150 9.1% 90% 2.7% 49 The buffer used in wash 2, containing 100 mM sodium phosphate pH 6.5, had a conductivity of 8.4 mS/cm. 5 Fig. 1 shows a silver stained, non-reduced SDS-PAGE gel of samples derived from experiments using the three wash step conditions shown in Table IV on the free Fc clearance. Fig. 2 shows overlapping chromatograms of the wash step experiments with sodium phosphate at different concentrations. 10 The wash step was optimized at pH 6.5 with increasing concentrations of sodium phosphate (50 to 150mM). As can be seen in Fig. 1, a wash buffer concentration of 150mM (wash 3, lane 6) resulted in losses of TACI-Fc. A wash buffer concentration of 50 mM (wash 1, lane 8) resulted in a peak of pure TACI-Fc, however, the eluate contained traces of free Fc. A wash step with 100mM sodium phosphate pH 6.5 15 resulted in 98% recovery in the main peak of elution and only 2% losses in the wash (Fig. 2). HCP clearance was 3.2 fold. Analysis of wash and eluate fractions by SDS-PAGE show that the wash step contained Free Fc with some intact TACI-Fc at buffer concentrations of 100 mM or above (Fig. 1, lanes 4 and 6). A concentration of 100 mM or more is necessary to completely remove Free Fc from the eluate fraction (Fig. 1, 20 lanes 5 and 7). Conclusions A cation-exchange step was developed as a second purification step, after the capture step. The capture eluate was at low pH (5.0) and low conductivity and could be directly 25 loaded onto the cation-exchanger. A Fractogel EMD S03" resin was selected with a loading capacity of 50 mg/ml. The non-bioactive degradation product free Fc could be efficiently removed in a wash step with 0.1 M sodium phosphate pH 6.5. Elution WO 2008/025748 PCT/EP2007/058887 conditions were optimised for best clearance of HCPs and high TACI-Fc recovery (179 mM sodium phosphate pH 8.0, conductivity 20.7 mS/cm). Alternatively, elution can be carried out in 10 BV of 20 mM sodium phosphate and 180 mM NaCI pH8.0 from the start of the rise in absorbance at 280 nm. 5 Example 3: Anion Exchange Chromatography The starting material used for this purification step was the eluate from the cation exchange step on Fractogel SO ~3 (see Example 2), dialysed or diluted into suitable loading buffer. 10 This anion-exchange chromatography step was carried out on a SOURCE 30Q column (GE Healthcare 17-1275-01) with a bed height of 10 cm. A SOURCE 30Q column with a bed height of 15 cm may be used as well in this step. In the latter case, the dynamic capacity and flow rate may need adaptation, which is well within routine knowledge of the person skilled in the art. 15 All operations were carried out at room temperature and the UV signal at 280 nm was recorded. The steps were carried out at a flow rate of either 150 or 200cm/h. Rinsing First, the column was rinsed with at least 1BV of RO water at a flow rate of 150cm/h. 20 Sanitisation Then, the column was sanitised with at least 3BV of 0.5M NaOH + 1,5M NaCI. Wash step The column was washed with at least 3BV, preferably 4 to 10BV, of 0.5M Na phosphate pH 7.5 at a flow rate of 200cm/h. 25 Equilibration The column was equilibrated with at least 5 BV of 10, 15, 20, 25, or 30 mM sodium phosphate pH 7.5. Optionally, the column can be pre-equilibrated with 3 BV of 0.5M sodium phosphate pH7.5. Loading, washing and concomitant collection of TACI-Fc in the flow-through 30 The column was loaded with post-cation exchange material diluted to obtain a phosphate concentration of 10 to 30 mM, pH 7.5, at a capacity of no more than 50 mg TACI-Fc as determined by SE-HPLC assay per ml of packed resin, collecting the flow- WO 2008/025748 PCT/EP2007/058887 39 through from start of UV increase until the end of the wash step, which is carried out in 4±0.5 BV of equilibration buffer. Regeneration/sanitisation The column was regenerated and sanitised with at least 3BV of 0.5M NaOH + 5 1.5M NaCI in reverse flow mode (until UV signal is back to the baseline) at a flow rate of 150cm/h. At the end of the regeneration, the pump is stopped for 30min. Wash step The column was washed with at least 3BV of RO water at a flow rate of 200cm/h. 10 Storing The column is stored in at least 3BV of 20% ethanol (v/v) at a flow rate of 150cm/h. Results 15 The following Table V summarizes the results obtained with the purification process described above. Table V: Effect of loading phosphate concentation Load PH Load phosphate conc (mM) Load TACI-Fc conc (mg/L) Load capacity (mg/ml) TACI-Fc recovery Aggregates HCPs (ppm) 7.5 30 773 39 94% 10.4% 82.8 7.5 25 639 39 90% 6.9% 50.4 7.5 20 651 49 90% 5.6% 43.9 7.5 15 437 46 88% 3.4% 45.0 7.5 10 283 n./d. 82% 2.8% 26.3 Conclusions 20 The anion-exchange step on a Source 30Q column in flow-through mode was optimised to maximise clearance of HCPs and aggregates. Loading cation-exchange eluate either diluted or diafiltered in 20mM sodium phosphate buffer at pH7.5 gave the best compromise between product recovery (90%) and clearance of HCPs (from about 2000ppm to 44ppm) and aggregates (from about 25% to 5.6%). Dynamic capacity of 25 50mg TACI-Fc per ml of packed resin at a flow rate of 150-200cm/h was used. WO 2008/025748 PCT/EP2007/058887 Example 4: Hydroxyapatite chromatography The starting material used for this purification step was anion-exchange chromatography flow-through (see Example 3). 5 A CHT Ceramic Hydroxyapatite Type I, 40 [im column (Biorad 157-0040) with a bed height of 10cm was used. All operations were carried out at room temperature. The flow rate was kept constant at 175 cm/h and the UV signal at 280 nm was recorded. All solutions were sterile filtered and the equipment sanitised with sodium hydroxide before use. The 10 column was stored in 0.5M NaOH solution when not in use. Initial wash steps (Rinsing and pre-equilibration) The column was washed with at least 1BV of 20mM sodium phosphate pH7.5 buffer, and then with at least 3BV of 0.5M sodium phosphate buffer pH7.5 to lower the pH. 15 Equilibration The column was equilibrated with at least 5BV of 20mM sodium phosphate pH7.5 (or until the target conductivity of 3.0±0.3 mS/cm and pH 7.5 ± 0.1 were reached). Loading 20 The column was loaded with the SOURCE 30Q flow-through with calcium chloride added to 0.1 mM final concentration from a stock solution at 0.5M and pH adjusted to 7.0 by addition of 85% ortho-phosphoric acid, at a capacity of NMT 50mg TACI-Fc as determined by SE-HPLC assay per ml of packed resin. It is also possible to load the SOURCE 30Q flow-through without calcium chloride, adjusted to pH 7.0, on 25 the hydroxyapatite column. Wash steps The column was washed with at least 4BV of 3, 4 or 5 mM sodium phosphate, 10mM MES, 0.1mM CaCI2 pH6.5. it is also possible to use the same buffer without calcium chloride. 30 Elution The column was eluted with 5, 4, 3 or 2 mM sodium phosphate (see Table VI), 10mM MES, 0.1mM CaCI2, and 0.6, 0.7, 0.8 or 0.9 M KCI pH 6.5 buffer (see Table VII) WO 2008/025748 PCT/EP2007/058887 41 from the beginning of the UV increase for different BV (see Tables VI and VII). It is also possible to use the same buffer without calcium chloride for the elution. Rinsing The column was rinsed with: 5 - at least 1BV of 20mM sodium phosphate pH7.5 buffer; at least 3BV of 0.5M sodium phosphate pH7.5 buffer; and - with at least 1BV of 20mM sodium phosphate pH7.5 buffer. Storing The column was stored in at least 3BV of 0.5M NaOH. 10 Results Table VI shows the effect of phosphate concentration (from 2 to 5mM) in the elution buffer on the clearance of aggregates and product recovery. Elution peak fractions were pooled and analysed by SE-HPLC for TACI-Fc concentration and 15 aggregate levels. Table VI: Effect of phosphate concentration in the elution buffer Phosphate conc (mM) BV of elution TACI-Fc yield Aggregates 5 12 73% 0.49% 13 74% 0.52% 14 68% 0.65% 15 77% 0.67% 16 77% 0.70% 17 70% 0.73% 18 76% 0.85% 4 12 68% 0.34% 13 67% 0.29% 14 66% 0.36% 15 67% 0.39% 16 66% 0.38% 17 66% 0.32% 18 66% 0.40% WO 2008/025748 PCT/EP2007/058887 42 3 12 70% 0.46% 13 76% 0.42% 14 73% 0.51% 15 71% 0.52% 16 69% 0.55% 17 69% 0.50% 18 70% 0.53% 2 12 65% 0.19% 13 66% 0.00% 14 66% 0.18% 15 68% 0.14% 16 66% 0.17% 17 71% 0.19% 18 65% 0.16% Table VII shows the effect of KCI concentration in the elution buffer on the clearance of aggregates and product recovery. Two sodium phosphate concentrations were investigated: 2 and 3mM. Elution peak fractions were pooled and analysed by SE-5 HPLC for TACI-Fc concentration and aggregate levels. Table VII: Effect of potassium chloride concentration in the elution buffer Phosphate conc (mM) KCI conc (M) BV of elution TACI-Fc yield aggregates 3 0.6 10 102% 0.48% 11 109% 0.46% 12 106% 0.43% 13 105% 0.42% 14 103% 0.43% 3 0.7 10 96% 0.42% 11 97% 0.40% 12 98% 0.41% 13 96% 0.40% 14 96% 0.43% WO 2008/025748 PCT/EP2007/058887 43 3 0.8 10 106% 0.58% 11 110% 0.55% 12 112% 0.57% 13 101% 0.59% 14 110% 0.57% 2 0.6 10 71% 0.29% 11 79% 0.28% 12 80% 0.29% 13 80% 0.29% 14 81% 0.26% 2 0.9 10 64% 0.27% 11 72% 0.25% 12 73% 0.29% 13 70% 0.33% 14 66% 0.24% Conclusions: Hydroxyapatite chromatography provides a reliable, efficient way of reducing TACI-Fc aggregate levels. Starting from anion-exchange chromatography purified material (see Example 3) with aggregate levels of about 5-8%, hydroxyapatite chromatography can reduce these levels to below 0.8% with a recovery of TACI-Fc of 85-90%. WO 2008/025748 PCT/EP2007/058887 44 REFERENCES 1. Akerstrom and Bjork, J Biol Chem. 1989 Nov 25;264(33): 19740-6. 2. Altschul S F et al, J Mol Biol, 215, 403-410, 1990 5 3. Altschul S F et al, Nucleic Acids Res., 25:389-3402, 1997 4. Armour KL. et al., 1999. Recombinant human IgG molecules lacking Fcgamma receptor I binding and monocyte triggering activities. Eur J Immunol. 29(8):2613-24 5. Bodmer et al., TIBS 27(1), 19-24 6. Boschetti, E., Jungbauer, Sep. Sci. & Tech. 2 No. 15, Acad. Press (2000) 53 10 7. Boschetti et al., Genetic Engineering Vol. 20, No. 13, July, 2000 8. Bossen et al., JBC 281(2), 13964-13971 9. Bram and von Bulow, US 5,969,102 (1999) 10. Bram et al., WO 98/39361 11. Carter PJ., 2006. Potent antibody therapeutics by design. Nature Reviews 15 Immunology. Advance online publication 12. Devereux J etal, Nucleic Acids Res, 12, 387-395, 1984 13. Feng et al., Bioprocessing 2005, 1-7 14. Giovannini, Biotechnology and Bioengineering 73:522-529 (2000) 15. Grantham et al., Science, Vol. 185, pp. 862-864 (1974) 20 16. Gross et al., Nature 404(27), 995-999 17. Gross et al., WO 00/40716 18. Hinton PR. et al., 2004. Engineered human IgG antibodies with longer serum half-lives in primates. J Biol Chem. 279(8):6213-6 19. Hymowitz et al., JBC 280(8), 7218-7227 25 20. Idusogie EE. et al., 2000. Mapping of the C1q binding site on rituxan, a chimeric antibody with a human lgG1 Fc. J Immunol. 164(8):4178-84 21. Idusogie EE. et al., 2001. Engineered antibodies with increased activity to recruit complement. J Immunol. 166(4):2571-5 22. Kabat, E. A., Wu, T. T., Perry, H. M., Gottesman, K. S., and Foeller, C. (1991), 30 Sequences of Proteins of Immunological Interest, 5th Ed., National Institutes of Health, Bethesda, MD 23. Kochanek et al., EP 1 230 354 24. Locksley et al., Cell 104, 487-501, 2001 WO 2008/025748 PCT/EP2007/058887 45 25. Melchers, Ann. Rheum. Dis 2003, 62, 25-27. 26. Moore et al., Science 285: 260-3 27. Moreau et al., Blood 103(8), 3148-3157 28. Naismith and Sprang, TIBS 23, 74-79, 1998 5 29. Novak et al., Blood 103(2), 689-694 30. Parker et al., WO 02/094852 31. Pearson, Methods Enzymol. 1990;183:63-98 32. Porath, J. Carlsson, I. Olsson, and G. Belfrage, Nature (London) 258, 598-599 (1975) 10 33. Porath and B. Olin, Biochemistry 22, 1621-1630 (1983) 34. Puren et al., Proc Natl Acad Sci USA. 1999 Mar 2;96(5):2256-61 35. Shepard, J. of Chromatography 891:93- 98 (2000) 36. Shields RL. et al., 2001. High resolution mapping of the binding site on human lgG1 for Fc gamma Rl, Fc gamma Rll, Fc gamma Rill, and FcRn and design of lgG1 15 variants with improved binding to the Fc gamma R. J Biol Chem. 276(9):6591-604. 37. Smith et al., WO 91/03553 38. Smith et al., WO 94/06476 39. Stanker, J. Immunological Methods 76:157-169 (1985) (10 mM to 30 mM sodium phosphate elusion gradient) 20 40. Steurer W. et al., 1995. Ex vivo coating of islet cell allografts with murine CTLA4/Fc promotes graft tolerance. J Immunol. 155(3): 1165-74 41. Sun et al., WO 2005/044856 42. Tarditi, J. Chromatography 599:13-20 (1992) 43. Vaccaro C. et al., 2005. Engineering the Fc region of immunoglobulin G to modulate 25 in vivo antibody levels. Nat Biotechnol. 23(10): 1283-8 44. Vigers et al., Nature. 1997 Mar 13;386(6621): 190-4 45. Vedantham et al., WO 03/59935 46. Vola et al., BioTechniques 14:650-655 (1993) 47. von Bulow and Bram, Science 228 : 138 (1997) 30 48. Xia et al., J. Exp. Med. 2000, 137-143. Received at IPONZ: 02-November-2011 45a In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be 5 construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art. WO 2008/025748 PCT/EP2007/058887 46 </div>

Claims

<div class="application article clearfix printTableText" id="claims"> CLAIMS

1. Method for reducing the concentration of free Fc-moieties in a fluid comprising an Fc-containing protein, the method comprising subjecting said fluid to cation exchange chromatography.

2. The method according to claim 1, comprising the step of: Loading said fluid on a cation exchange resin at a pH of at least one unit below the isoelectric point (pi) of said Fc-containing protein.

3. The method according to claims 1 or 2, comprising the step of: Washing the cation exchange resin with a buffer having a conductivity of 6 to 10 mS/cm and at a pH of 5.5 to 7.5.

4. The method according to claim 3, wherein the washing step is carried out at a conductivity of 8.2 to 8.6 mS/cm.

5. The method according to claims 3 or 4, wherein the washing step is carried out at a pH of 6.0 to 7.0.

6. The method according to any of claims 3 to 5, wherein the washing step is carried out in a buffer comprising 75 to 125 mM sodium phosphate.

7. The method according to any of the preceding claims, comprising the step of: Eluting the Fc-containing protein at a pH ranging from 7.0 to 8.5.

8. The method according to claim 7, wherein the eluting step is carried out at a conductivity of 15 to 22 mS/cm.

9. The method according to any of the preceding claims, wherein the cation exchange chromatography is carried out on a strong cation exchange resin. WO 2008/025748 PCT/EP2007/058887 47

10. The method according to claim 9, wherein the cation exchange resin comprises S03" groups.

11. The method according to claim 9 or 10, wherein the resin comprises a cross-5 linked methacrylate matrix.

12. The method according to any of the preceding claims, further comprising a purification step selected from affinity chromatography, anion exchange chromatography and hydroxyapatite chromatography. 10

13. The method according to any of the preceding claims, wherein the Fc-containing protein comprises an Immunoglobulin (Ig) constant region.

14. The method according to claim 13, wherein the constant region is a human 15 constant region.

15. The method according to claim 13 or 14, wherein the immunoglobulin is an IgGi.

16. The method according to any of claims 13 to 15, wherein the constant region 20 comprises a CH2 and a CH3 domain.

17. The method according to any of the preceding claims, wherein the Fc-containing protein comprises an immunoglobulin variable region. 25

18. The method according to claim 17, wherein the Fc-containing protein is an antibody.

19. The method according to any of claims 1 to 16, wherein the Fc-containing protein is an Fc-fusion protein. 30

20. The method according to claim 19, wherein the Fc-fusion protein comprises a ligand binding portion of a member of the tumor necrosis factor receptor (TNFR) superfamily. Received at IPONZ 09 March 2011 48

21. The method according to cfaim 20, wherein the ligand binding portion is selected from an extracellular domain of TNFR1, TNFR2, or a TNF binding fragment thereof.

22. The method according to claim 20, wherein the ligand binding portion is selected from an extracellular domain of BAFF-R, BCMA, TACI, or a fragment thereof binding at least one of Blys or APRIL.

23. The method according claim 22, wherein the Fc-fusion protein comprises a polypeptide selected from a. amino acids 34 to 66 of SEQ ID NO: 2; b. amino acids 71 to 104 of SEQ ID NO: 2; c. amino acids 34 to 104 of SEQ ID NO: 2; d. amino acids 30 to 110 of SEQ ID NO: 2; e. SEQ ID NO: 3; f. SEQ ID NO: 4; g. a polypeptide encoded by a polynucleotide hybridizing to the complement of SEQ ID NO: 5 or 6 or 7 under highly stringent conditions; and h. a mutein of any of (c), <d), (e), or (f) having at least 80 % or 85 % or 90 % or 95 % sequence identity to the polypeptide of (c), (d), <e) or (f); wherein the polypeptide binds to at least one of Blys or APRIL.

24. Use of cation exchange chromatography for the reduction of the concentration of free Fc-moieties in a composition comprising an Fc-containing protein.

25. Use according to claim 24, wherein the concentration of free Fc is reduced to less than 5 % or less than 2 % or less than 1 % or less than 0.5 % or less than 0.2 % or less than 0.1 % of the total protein concentration of said composition.

26. A purified Fc-containing protein having reduced free Fc-moieties, produced by the method of any one of claims 1-22. Received at IPONZ: 02-November-2011 49

27. A method as claimed in claim 1 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings.

28. A use as claimed in claim 24 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings.

29. A purified Fc-containing protein as claimed in claim 26 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings. Reference has been directed, in pursuance of Section 16(1) of the Patents Act 1953, to patent application no. 574623. </div>