HK1131399A - Process for the purification of fc-fusion proteins - Google Patents

Description

Method for purifying Fc fusion protein

Technical Field

The present invention is in the field of protein purification. More specifically, the present invention relates to the purification of Fc-fusion proteins by protein a or protein G affinity chromatography, cation exchange chromatography, anion exchange chromatography and hydroxyapatite chromatography.

Background

Proteins are becoming commercially important as drugs commonly referred to as "biologicals". One of its greatest challenges is to develop cost-effective and efficient protein purification processes on a commercial scale. Although many large scale methods for purifying proteins are available, crude products, such as cell culture supernatants, contain not only the desired product but also impurities that are difficult to separate from the desired product. If the cells are cultured in serum-free medium, the cell culture supernatant of the cells expressing the recombinant protein product may contain less impurities, but the Host Cell Proteins (HCPs) still need to be removed during the purification process. In addition, the health authorities have high standards for the purity of proteins for human use.

Many purification methods involve the need to employ low or high pH, high salt concentrations, or other extreme conditions that may compromise the biological activity of a given protein. Thus, for any protein, it is a challenge to establish a purification process that can adequately purify while retaining the biological activity of the protein.

Several known chromatography systems have been widely used for protein purification.

Ion exchange chromatography systems for separating proteins are based primarily on differences in charge. In ion exchange chromatography, charged regions on the surface of the solute are adsorbed to the chromatography matrix by being attracted to opposite charges, provided that the ionic strength provided by the surrounding buffer is low. Elution is typically achieved by increasing the ionic strength (i.e., conductivity) of the buffer in competition with the solute for the charged sites of the ion exchange matrix. Changing the pH and thus the charge of the solute is another way to achieve elution of the solute. This change in conductivity or pH can be incremental (gradient elution) or stepwise (step elution).

Anion exchangers can be classified as weak or strong exchangers. The charged groups on weak anion exchangers are weakly basic groups that lose their charge by deprotonation when subjected to high pH. DEAE-Sepharose is an example of a weak anion exchanger, the amino group of which is positively charged below about pH9 and gradually loses this charge at higher pH. For example, methylphenylethylamine bromide (DEAE) or diethyl- (2-hydroxy-propyl) aminoethyl (diethyl- (2-hydroxy-propyl) aminoethyl, QAE) contains the counter ion chloride. On the other hand, strong anion exchangers contain strong base groups and are positively charged over the entire pH range (pH1-14) normally used for ion exchange chromatography. Q-Sepharose (Q stands for quaternary amine) is an example of a strong anion exchanger.

Cation exchangers can be classified as weak or strong exchangers. Strong cation exchangers contain strong acid groups (e.g., sulfopropyl groups) and remain charged at pH1-14, while weak cation exchangers contain weak acid groups (e.g., carboxymethyl groups) and gradually lose charge as pH decreases below 4 or 5. For example, Carboxymethyl (CM) and Sulfopropyl (SP) contain the counterion sodium ion.

A different chromatography resin is based on an insoluble calcium hydroxy phosphate matrix, known as hydroxyapatite. Hydroxyapatite chromatography is a method of using insoluble hydroxylated calcium phosphate (Ca) that can form matrices and ligands₅(PO₄)₃OH)₂A method for purifying a protein. The functional groups includePositively charged calcium ions (C-site) and clustered negatively charged phosphate groups (P-site). The interaction between hydroxyapatite and proteins has a complex, multiple pattern. In one interaction, positively charged amino groups on proteins bind to negatively charged P-sites on the (resin) and carboxyl groups of proteins interact by coordination complexation with C-sites on the (resin) (Shepard et al, 2000).

Crystalline hydroxyapatite was the type of hydroxyapatite first used for chromatography. Ceramic Hydroxyapatite (CHA) chromatography was further developed in hydroxyapatite chromatography. Ceramic hydroxyapatite has high durability and good protein binding capacity compared to crystalline hydroxyapatite and can be used at high flow rates and high pressures (Vola et al, 1993).

Hydroxyapatite has been used in the chromatography to separate proteins, nucleic acids, and antibodies. In hydroxyapatite chromatography, the column is usually equilibrated with a low concentration of phosphate buffer and the sample is loaded, and then the adsorbed protein is eluted with a stepwise concentration of phosphate buffer (Giovannini et al, 2000).

Yet another method for purifying a protein is based on the affinity of the protein of interest for another protein immobilized on a chromatography resin. Examples of such immobilized ligands (proteins) are cell wall proteins of bacteria specific for the Fc part of certain immunoglobulins: protein a and protein G. While both protein a and protein G have strong affinity for IgG antibodies, they differ in their affinity for other types of immunoglobulins and their isotypes.

Protein a is a 43,000 dalton protein produced by staphylococcus aureus and contains 4 binding sites for the Fc region of IgG. Protein G is produced by Streptococcus G and contains 2 binding sites for the Fc region of IgG. The properties of these two proteins have been widely characterized and they have affinity for various types of immunoglobulins. Protein L is another bacterial protein from the genus Peptostreptococcus, which binds immunoglobulins and fragments thereof containing Ig light chains (Akerstrom and Bjork, 1989).

Protein a, protein G and protein L affinity chromatography have been widely used for the isolation and purification of immunoglobulins.

Since the binding sites for protein A and protein G are located in the Fc region of immunoglobulins, affinity chromatography of protein A and protein G also enables the purification of so-called Fc-fusion proteins.

An Fc-fusion protein is a chimeric protein comprising an effector portion of a protein (e.g., a receptor binding region) fused to the Fc region of an immunoglobulin (often immunoglobulin G, IgG). Fc-fusion proteins are widely used as therapeutic agents because they have the advantages conferred by the Fc region, for example:

-ability to purify with protein a or protein G affinity chromatography, the affinity varying depending on the IgG isotype. Human IgG₁、IgG₂And IgG₄Can strongly bind to protein A, and all human IgG includes IgG₃Can strongly bind to protein G;

an extended half-life in the circulation, since its Fc region binds to the salvage receptor FcRn protecting it from degradation by lysosomal enzymes;

depending on the medical application of this Fc fusion protein, effector functions of the Fc region may be required. Such effector functions include Antibody Dependent Cellular Cytotoxicity (ADCC) through interaction with Fc receptors (Fc γ R), and Complement Dependent Cytotoxicity (CDC) in combination with complement component 1q (C1 q). IgG subtypes exert different levels of effector function. Human IgG₁And IgG₃Has potent ADCC and CDC effects, and human IgG₂Has weak ADCC and CDC effects. Human IgG₄Shows weak ADCC effect without CDC effect.

Depending on the therapeutic application for which the Fc-fusion protein is intended, its serum half-life and effector function can be improved by engineering the Fc region to increase or decrease its ability to bind to FcR, fcyr, and C1q, respectively.

In ADCC, the Fc region of an antibody binds to Fc receptors (fcyr) on the surface of immune effector cells (e.g., natural killer cells and macrophages) resulting in phagocytosis or lysis of the target cell.

In CDC, antibodies kill target cells by triggering the complement cascade at the cell surface. IgG subtypes exert different levels of effector function, as IgG₄＜IgG₂＜IgG₁≤IgG₃Sequentially increasing. Human IgG₁Shows high ADCC and CDC effects, and is most suitable for the treatment application of pathogens and cancer cells.

In some cases, it may be desirable to remove or reduce effector function, for example, when eliminating target cells that are detrimental to the body. Conversely, when the antibody is used for tumor therapy, increasing effector function may enhance its therapeutic activity (Carter et al, 2006).

Modification of effector function can be achieved by engineering the Fc region to increase or decrease its binding to Fc γ R or complement factors.

The binding of IgG to activating Fc γ R (Fc γ RI, Fc γ RIIa, Fc γ RIIIa and Fc γ RIIIb) and inhibitory Fc γ R (Fc γ RIIb) or complement 1 component (C1q) depends on the residues located in the hinge region and CH2 domain. Two regions of the CH2 domain are critical for Fc γ R binding to complement C1q, in IgG₂And IgG₄Contains unique sequence. For example, with IgG₂Substitution of human IgG with residue 233-236₁Will greatly reduce IgG₁ADCC and CDC functions (Armour et al, 1999; and Shield et al, 2001).

A number of mutations have been made in the CH2 domain of IgG and their effects on ADCC and CDC have been studied in vitro (Shieid et al, 2001; Idusogene et al, 2001 and 2002; Steurer et al, 1995). Specifically, it has been reported that mutation of the E333 position to alanine improves both ADCC and CDC functions (Idusogene et al, 2001 and 2002).

Increasing the serum half-life of a therapeutic antibody is another method of improving its efficacy, allowing higher circulating levels, reducing the frequency of administration, and reducing the dosage. This may be achieved by increasing the binding of the Fc region to neonatal fcr (fcrn). FcRn is expressed on the surface of endothelial cells and binds IgG in a pH-dependent manner, protecting it from degradation. Several mutations at the interface between the CH2 and CH3 domains have been shown to prolong the half-life of IgG (Hinton et al, 2004 and vaccarao et al, 2005).

Table 1 below summarizes some of the known mutations in the Fc region (taken from Invivogen's website).

TABLE 1

Engineered Fc	IgG isotypes	Mutations	Performance of	Potential benefits	Applications of
						hIgG1e1	Human IgG1	T250Q/M428L	Increase the half-life of plasma	Improving target positioning; the efficacy is improved; reducing the dosage or frequency of administration	A vaccine; therapeutic applications
hIgG1e2	Human IgG1	M 252Y/S254T/T256E+H433K/N434F	Increase the half-life of plasma	Improving target positioning; the efficacy is improved; reducing the dosage or frequency of administration	A vaccine; therapeutic applications
						hIgG1e3	Human gG1	E233P/L234V/L235A/ΔG236+A327G/A330S/P331S	Reduced ADCC and CDC function	Reduce side effects	Therapeutic applications without the need to eliminate cells
hIgG1e4	Human gG1	E333A	ADCC and CDC function enhancement	Increase the efficacy	Therapeutic applications requiring elimination of cells
						hIgG2e1	Human gG2	K322A	CDC function reduction	Reduce side effects	A vaccine; therapeutic applications

In certain known Fc fusion proteins having therapeutic utility, the Fc region is fused to the extracellular domain of certain receptors belonging to the tumor necrosis factor receptor (TNF-R) superfamily (Locksley et al, 2001, Bodmer et al, 2002, Bossen et al, 2006). Members of the TNFR family are marked by the presence of cysteine-rich pseudo-repeats in their extracellular domains, as described by Naismith and Sprang 1998.

Two TNF receptors: p55(TNFR1) and p75TNFR (TNFR2) are examples of such members of the TNF superfamily. Etanercept (an antagonist of TNF-alpha) is an Fc fusion protein containing a soluble portion of p75TNFR (e.g., WO91/03553, WO94/06476), which is under the trade name EtanerceptMarketed for the treatment of endometriosis, hepatitis c virus infection, HIV infection, psoriatic arthritis, psoriasis, rheumatoid arthritis, asthma, ankylosing spondylitis, heart failure, graft-versus-host disease, pulmonary fibrosis, crohn's disease. Lenercept (Lenercept) is a fusion protein containing the extracellular component of the human p55TNF receptor and the Fc portion of human IgG and is expected to be useful in the treatment of severe sepsis and multiple sclerosis.

OX40 is also a member of the TNFR superfamily. OX40-IgG1 and OX40-HIG4mut fusion proteins have been prepared for the treatment of inflammatory and autoimmune diseases, such as Crohn's disease.

The Fc-fusion protein of BAFF-R (also known as BR3), also known as BR3-Fc, is a soluble decoy receptor belonging to the family of inhibitors of BAFF (B cell activator of the TNF family) and is under development for potential treatment of autoimmune diseases such as Rheumatoid Arthritis (RA) and Systemic Lupus Erythematosus (SLE).

BCMA is another receptor belonging to the TNFR superfamily. BCMA-Ig fusion proteins have been reported to inhibit autoimmune diseases (Melchers, 2006).

Another receptor of the TNF-R superfamily is TACI, a transmembrane activator and CAML-interacting factor (von Bulow and Bram, 1997; US 5,969,102, Gross et al, 2000), whose extracellular domain contains two cysteine-rich pseudo-repeats. TACI binds to two members of the Tumor Necrosis Factor (TNF) ligand family, one class of ligands being designated BLyS, BAFF, neutrokine-alpha, TALL-1, zTNF4, or THANK (Moore et al, 1999), and the other class of ligands being designated APRIL, TNFR death ligand-1, or ZTNF2(Hahne et al, J Exp Med.188: 1185, 1998).

Fusion proteins containing a soluble form of the TACI receptor fused to the Fc region of IgG are well known and are designated TACI-Fc (WO 00/40716, WO 02/094852). TACI-Fc inhibits the binding of BLyS and APRIL to B-cells (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 the treatment of Multiple Sclerosis (MS). In addition, TACI-Fc is being developed for the treatment of Multiple Myeloma (MM) (Novak et al, 2004; Moreau et al, 2004) and non-Hodgkin's lymphoma (NHL), Chronic Lymphocytic Leukemia (CLL) and Waldenstrom's Macroglobulinemia (WM).

For the therapeutic application of Fc-fusion proteins, particularly those containing the extracellular portion of the TNFR superfamily, a sufficient amount of highly pure protein suitable for human administration is needed.

WO 02/094852 describes a method for partial purification of TACI-Fc, involving protein A chromatography followed by S-200 molecular size exclusion chromatography.

WO 03/059935 discloses a method for purifying p75TNFR: Fc-fusion proteins by combining hydroxyapatite chromatography with protein A affinity chromatography. However, in the method described in WO 03/059935, the Fc-fusion protein does not bind to hydroxyapatite but is contained in the effluent of a hydroxyapatite column. In addition, no mention is made of ion exchange chromatography for the purification of p75TNFR: Fc-fusion proteins.

WO 2005/044856 discloses a method for removing high molecular weight aggregates in antibodies prepared by hydroxyapatite chromatography and also discloses a purification method using protein a, anion exchange chromatography and hydroxyapatite chromatography. However, firstly, the method described is only for antibodies and secondly, there is no disclosure of using a cation exchange chromatography step between a protein a affinity chromatography and an anion exchange chromatography step.

The proposed protocol for the purification of recombinant soluble TNF receptors proposed in WO94/06476 is based on TNF or lectin affinity chromatography, anion or cation exchange chromatography and reverse phase high performance liquid chromatography (RP-HPLC). Hydroxyapatite chromatography is not mentioned in this document as a suitable purification step for soluble TNF receptors.

US 2002/0115175 describes a method for purifying metalloproteinases such as TNF α convertase. The theoretical isoelectric point of TACE is about 5.4, as calculated using the "isoelectric point service EMBL WWW portal" on the internet. TACE is a protease that cleaves the N-terminal 8 amino acids of membrane-bound (pro-) TNF α, thus releasing and activating the cytokine TNF α from the cell membrane. The TACE purification method disclosed in US 2002/0115175 comprises a wheat germ agglutinin agarose step. This document also describes Fc fusion proteins of TACE, but not purified.

EP 1561756 discloses that protein a or G based chromatography alone may not be sufficient to separate contaminating DNA from the protein, and that other steps such as anion or cation exchange chromatography, hydroxyapatite chromatography or combinations thereof may be employed for the purification of the protein, but no specific sequence of these chromatography steps is suggested. Furthermore, the proteins involved in EP 1561756 are hematopoeitic factors, cytokines and antibodies. EP 1561756 does not mention Fc-fusion proteins.

EP 1614693 describes a method for purifying antibodies according to protein a affinity chromatography, anion exchange chromatography and cation exchange layer. In this document, the purification of antibodies by anion exchange and cation exchange chromatography, or by cation exchange chromatography followed by hydrophobic chromatography, is specifically mentioned. This hydrophobic chromatography may be replaced by any other type of chromatography, including hydroxyapatite chromatography. In EP 1614693, no Fc-fusion proteins are mentioned.

The antibody purification method described by Feng et al, 2005 is based on an initial capture step with protein A followed by a polishing step which may be hydrophobic interaction chromatography, anion exchange chromatography, cation exchange chromatography, or hydroxyapatite chromatography. However, Feng et al only describe antibody purification and not Fc-fusion proteins. Furthermore, no specific sequence was proposed to systematically remove all undesirable impurities such as Host Cell Proteins (HCP), aggregates, DNA, contaminating viruses and abscisin a, except the initial protein a affinity step.

Thus, there is still no method that satisfies the need for sufficient purification for the production of Fc fusion proteins of purity suitable for human administration.

Summary of The Invention

The present invention is based on the development of a method for the purification of Fc-fusion proteins.

Thus, in a first aspect, the present invention relates to a method for the purification of an Fc-fusion protein comprising the steps of:

a. subjecting the fluid containing the Fc-fusion protein to protein a or protein G affinity chromatography;

b. subjecting the eluate of step (a) to cation exchange chromatography;

c. subjecting the eluate of step (b) to anion exchange chromatography; and

d. subjecting the effluent of step (c) to hydroxyapatite chromatography and collecting the eluate to obtain a purified Fc-fusion protein.

This method can be used to purify Fc-fusion proteins having an isoelectric point (pI) in the range of 7.0-9.5. This method is preferably used for the purification of therapeutic Fc-fusion proteins, i.e. Fc-fusion proteins intended for human administration. More preferred are Fc-fusion proteins for use comprising the extracellular portion (particularly the ligand binding portion and optionally the inhibitory extracellular portion) of a member of the Tumor Necrosis Factor Receptor (TNFR) superfamily.

It has surprisingly been shown that step (b) is suitable for removing so-called free Fc, i.e. immunoglobulin heavy chain domains that are not fused to the entire therapeutic molecule, e.g. the ligand-binding extracellular portion of a TNF family member.

In a second aspect, the present invention relates to a purified Fc-fusion protein, preferably a therapeutic Fc-fusion protein, more preferably an Fc-fusion protein comprising an extracellular portion, in particular a ligand-binding extracellular portion, of a member of the Tumor Necrosis Factor Receptor (TNFR) superfamily, which purified Fc-fusion protein contains less than 1% or 0.5% or 0.2% or 0.1% free Fc protein.

It was also shown that the combination of steps (a), (c) and (d) significantly removed the therapeutically inactive Fc-fusion protein aggregates that are not required for administration to humans.

Thus, in a third aspect, the present invention relates to a purified Fc-fusion protein composition, preferably a therapeutic Fc-fusion protein, more preferably an Fc-fusion protein comprising an extracellular portion, particularly a ligand-binding extracellular portion, of a member of the Tumor Necrosis Factor Receptor (TNFR) superfamily. The purified Fc-fusion protein comprises less than 1% or less than 0.5% Fc-fusion protein aggregates and/or comprises less than 0.5% or less than 0.2% or less than 0.1% free Fc protein.

Another aspect of the invention relates to the removal of free Fc from a preparation of Fc-fusion proteins, preferably a therapeutic preparation of Fc-fusion proteins, more preferably an Fc-fusion protein comprising an extracellular portion or a ligand binding portion of a member of the tumor necrosis factor receptor family, and optionally an inhibitory fragment thereof, using cation exchange chromatography.

In a further aspect the invention relates to the use of hydroxyapatite chromatography for removing aggregates from a preparation of Fc-fusion protein, preferably a therapeutic preparation of Fc-fusion protein, more preferably an Fc-fusion protein comprising an extracellular portion of a member of the Tumor Necrosis Factor Receptor (TNFR) superfamily, or a ligand-binding fragment thereof.

Brief description of the drawings

FIG. 1 shows a non-reducing silver stained SDS-PAGE of the different components of the countercurrent flow from the cation exchange chromatography described in example 2. Lane 1: labeling with molecular weight; lane 2: purified TACE-Fc; lane 3: loading a cargo; lane 4: washing liquid 2; lane 5: eluate 2; lane 6: washing solution 3; lane 7: eluate 3; lane 8: washing solution 1; lane 9: eluate 1; lane 10: purified free Fc.

FIG. 2 shows the 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) common to members of the TNFR superfamily;

SEQ ID NO: 2 is the full-length sequence of the human TACI receptor (as described in WO 98/39361);

SEQ ID NO: 3 is an example of the Fc sequence of the present inventors (as described in WO 02/094852);

SEQ ID NO: 4 is a preferred Fc-fusion protein of the invention, which contains an extracellular part derived from TACI and human IgG₁The sequence of the Fc portion (as described in WO 02/094852).

SEQ ID NO: 5 is a polypeptide encoding SEQ ID NO: 2 (as described in WO 02/094852).

SEQ ID NO: 6 is a polypeptide encoding SEQ ID NO: 3 (as described in WO 02/094852).

SEQ ID NO: 7 is a polypeptide encoding SEQ ID NO: 4 (as described in WO 02/094852).

Detailed Description

The present invention is based on the development of an exemplary therapeutic Fc-fusion protein purification process, referred to as TACI-Fc fusion protein, that produces a high purity TACI-Fc preparation suitable for human administration.

The present invention therefore relates to a method for the purification of an Fc-fusion protein comprising the steps of:

a. subjecting a liquid comprising the Fc-fusion protein to protein a or protein G affinity chromatography;

b. subjecting the eluate of step (a) to cation exchange chromatography;

c. subjecting the eluate of step (b) to anion exchange chromatography;

d. subjecting the effluent of step (c) to hydroxyapatite chromatography and collecting the eluate to obtain a purified Fc-fusion protein.

In one embodiment of the invention, the purification method does not comprise a lectin affinity chromatography step, in particular a wheat germ agglutinin agarose step.

The method of the invention can be used for purifying Fc-fusion proteins with isoelectric points ranging from 6.9 to 9.5. The "isoelectric point" or "pI" of a protein is the pH at which the net total charge of the protein is zero, i.e., the pH at which the number of positive and negative charges is equal. The pI of any given protein can be determined according to well established techniques, such as isoelectric focusing.

The pI of the Fc-fusion protein to be purified according to the invention can be, for example, 6.9, 6.95, 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, 8.0, 8.05, 8.1, 8.15, 8.2, 8.25, 8.3, 8.35, 8.4, 8.45, 8.5, 8.55, 8.6, 8.65, 8.7, 8.75, 8.8, 8.85, 8.9, 8.95, 9.0, 9.05, 9.1, 9.15, 9.2, 9.25, 9.3, 9.35, 9.4, 9.5.

Preferably, the pI of the Fc-fusion protein to be purified according to the invention is 8-9 or 8.0-9.0, more preferably 8.3-8.6.

The method of the invention is preferably used for the purification of therapeutic Fc-fusion proteins, i.e. Fc-fusion proteins intended for the treatment or prevention of animal diseases or preferably for the treatment of humans. The method of the present invention is more preferably used for purifying an Fc-fusion protein containing an extracellular portion of a member of the Tumor Necrosis Factor Receptor (TNFR) superfamily. The extracellular portion is preferably a ligand-binding fragment of the extracellular portion or domain of the respective receptor. Preferred Fc-fusion proteins that can be purified according to the invention are capable of binding to a ligand, inhibiting or blocking the function of the ligand, e.g., receptor activation function.

The term "Fc-fusion protein" as used herein is meant to include proteins, particularly proteins containing a moiety derived from an immunoglobulin (referred to herein as an "Fc-moiety") and a moiety derived from a second, non-immunoglobulin (referred to herein as a "therapeutic moiety"), whether or not intended for use in the treatment of disease.

The term "free Fc" as used in the present description means any part of an Fc-fusion protein to be purified according to the method of the invention which is derived from the immunoglobulin part of the Fc-fusion protein but does not contain a significant therapeutic part of the Fc-fusion protein. Thus, free Fc may include dimers consisting of IgG hinge, CH2, and CH3 regions, but not linked or bound to a significant therapeutic moiety, which corresponds to an Fc-moiety generated, for example, by cleavage with papain. Monomers derived from an Fc-moiety may also be contained in the free Fc component. It is known that such free Fc may also contain some amino acid residues from the therapeutic moiety, for example 1-10 (e.g. 2, 3, 4,5, 6, 7, 8 or 9) amino acids belonging to the therapeutic moiety fused to the Fc moiety.

This Fc-moiety can be derived from human or animal immunoglobulins (Ig), preferably IgG. The IgG may be IgG₁、IgG₂、IgG₃Or IgG₄. It is also preferred that this Fc-moiety is derived from the heavy chain of an immunoglobulin, preferably IgG. More preferably, the Fc-portion comprises, for example, an immunoglobulin heavy chain constant region portion. Such an Ig constant region preferably comprises at least one Ig constant region selected from the group consisting of a hinge region, a CH2 region, a CH3 region, or any combination thereof. Preferably, this Fc-moiety comprises at least the CH2 and CH3 regions. More preferably, this Fc-moiety comprises the IgG hinge region, the CH2 and the CH3 regions.

The Fc-fusion protein of the present invention may be a monomer or a dimer. The Fc-fusion protein may also be a "pseudo-dimer" comprising a dimeric Fc-moiety (e.g., a dimer consisting of two disulfide-linked hinge-CH 2-CH3 structures), one of which is fused to a therapeutic moiety.

The Fc-fusion protein may be a heterodimer comprising two different therapeutic moieties, or a homodimer comprising two copies of one therapeutic moiety.

According to the invention, this Fc-moiety can also be modified to modulate its effector function. For example, if the Fc-moiety is derived from IgG₁The following Fc mutations can be introduced at EU index positions (Kabat et al, 1991):

T250Q/M428L

M252Y/S254T/T256E+H433K/N434F

E233P/L234V/L235A/ΔG236+A327G/A330S/P331S

E333A；K322A

the further Fc mutation may be, for example, a substitution at the EU index position selected from 330, 331, 234 or 235, or a combination thereof. Any amino acid substitution at EU index position 297 in the CH2 region can also be introduced into the Fc-portion of the invention to remove potential N-linked glycosyl attachment sites. The cysteine residue at EU index position 220 can also be substituted with a serine residue to remove cysteine residues that typically can form disulfide bonds with immunoglobulin light chain constant regions.

According to the invention, preferably this Fc-portion comprises the amino acid sequence of SEQ ID NO: 3 or consists thereof, or consists of a polypeptide comprising SEQ ID NO: 6.

Therapeutic moieties of the invention can be derived, for example, from EPO, TPO, growth hormone, interferon- α, interferon- β, interferon- γ, PDGF- β, VEGF, IL-1 α, IL-1 β, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12, IL-18 binding protein, TGF- β, TNF- α, or TNF- β.

The therapeutic moieties of the invention may also be derived from a receptor, e.g., a transmembrane receptor, preferably or derived from an extracellular domain of a receptor, particularly a ligand binding fragment and optionally an inhibitory fragment of an extracellular portion or domain of a given receptor. Examples of receptors of therapeutic interest are CD2, CD3, CD4, CD8, CD11a, 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 subunit (IL-18R-alpha, IL-18R-beta), EGF receptor, VEGF receptor, integrin-alpha 410 beta 7, integrin VLA4, B2 integrin, TRAIL receptor 1, 2, 3 and 4, RANK ligand, epithelial cell adhesion molecule (EpCAM), intercellular adhesion molecule-3 (ICAM-3), CTLA4 (a cytotoxic T lymphocyte-associated antigen), Fc-gamma-1 receptor, HLA-10 beta, DR-antigen, DR-selectin.

It is highly preferred that the therapeutic moieties of the invention are derived from receptors belonging to the TNFR superfamily. The therapeutic moiety may be or be derived from the extracellular domain of TNFR1(p55), TNFR2(p75), OX40, Osteoprotegerin (Osteoprotegerin), CD27, CD30, CD40, RANK, DR3, Fas ligand, TRAIL-R1, TRAIL-R2, TRAIL-R3, TAIL-R4, NGFR, AITR, BAFFR, BCMA, TACI.

According to the present invention, the therapeutic moiety derived from a member of the TNFR superfamily comprises or consists of all or part of the extracellular domain of that TNFR member, more preferably a ligand binding fragment and optionally an inhibitory fragment of that TNFR member.

Table 5 below lists the TNFR superfamily members from which the therapeutic moieties of the invention can be derived and their respective ligands. One skilled in the art can readily determine the "ligand-binding fragment" of a TNFR family member by, for example, simple in vitro assays to detect binding between a protein fragment of a given receptor and the respective ligand. Such assays may be, for example, simple in vitro RIA-or ELISA sandwich assays in which one protein, such as a receptor fragment, is immobilized on a carrier (e.g., an ELISA plate) and incubated, and the protein binding site on the carrier is then suitably blocked with a second protein, such as a ligand. After incubation, binding of the ligand is detected, for example, using a radiolabelled ligand, and the bound radioactivity is measured in a scintillation counter after appropriate washing. Binding of the ligand may also be measured using a labeled antibody, or using a ligand-specific primary antibody and a labeled secondary antibody directed against the constant region of the primary antibody. Depending on the label used, e.g.a colour reaction, the binding of the ligand can easily be determined. The blocking or inhibition of ligand binding function can be detected using an appropriate cellular assay.

The methods of the invention are preferably used to purify an Fc-fusion protein comprising a therapeutic moiety derived from a member selected from the TNFR superfamilies listed in table 5.

Table 5: TNFR superfamily (according to Locksley et al, 2001 and Bossen et al, 2006)

Members of the TNFR superfamily	Ligands
		NGFR	NGF
EDAR	EDA-AI
		XEDAR	EDA-A2
CD40	CD40L
		Fas	FasL
Ox40	OX40L
		AITR	AITRL
GITR	GITRL
		CD30	CD30L
CD40	CD40L
		HveA	LIGHT，LT-α
4-1BB	4-1BBL
		TNFR2	TNF-α、LT-α、LT-α-β
LT-βR	LIGHT、LT-α、LT-α-β
		DR3	TL1A
CD27	CD27L
		TNFR1	TNF-α、LT-α、LT-α-β
LTBR	LT-β
		RANK	RANKL
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 TNFR1, TNFR2 extracellular domain, or a TNF binding fragment and optionally an inhibitory fragment thereof.

In another preferred embodiment, the Fc-fusion protein comprises a therapeutic moiety selected from the extracellular domain of BAFF-R, BCMA or TACI, or a fragment thereof that binds to at least one of Blys or APRIL.

Assays for detecting the ability to bind to Blys or APRIL are described, for example, in Hymowitz et al, 2006.

TACI is preferably human TACI. SEQ ID NO: 2 corresponds to the amino acid sequence of the full-length human TACI receptor (see also SwissProt entry O14836). More preferably, the therapeutic moiety comprises a soluble portion of TACI, preferably derived from the extracellular domain of TACI. This TACI-derived therapeutic moiety preferably comprises SEQ ID NO: 2, and/or at least amino acids 33-67 of SEQ ID NO: 2, amino acids 70-104. In a preferred embodiment, the TACI extracellular domain comprised by the therapeutic moiety according to the present invention comprises the amino acid sequence of SEQ ID NO: 2, or amino acids 1-166 of SEQ ID NO: 2, or seq id NO: 2, or amino acids 30-119 of SEQ ID NO: 2, or consists of amino acids 30-110. For the Fc-fusion protein preparation to be purified by the method of the invention, preferably all these therapeutic moieties are combined with the Fc-moieties detailed above, in particular with a Fc-fusion protein preparation comprising the amino acid sequence of SEQ ID NO: 3 or an Fc-moiety combination consisting thereof. The Fc-fusion protein to be purified by the method of the invention highly preferably comprises SEQ id no: 4 or consists thereof, or consists of SEQ ID NO: 7 is encoded by the polynucleotide of seq id no.

Thus, this Fc-fusion protein highly preferably comprises a polypeptide selected from the group consisting of:

(a) SEQ ID NO: 2 amino acids 34-66;

(b) SEQ ID NO: 2 amino acids 71-104;

(c) SEQ ID NO: 2 amino acids 34-104;

(d) SEQ ID NO: 2 amino acids 30-110;

(e)SEQ ID NO：3：

(f)SEQ ID NO：4：

(g) can be combined with the sequence shown in SEQ ID NO: 5 or 6 or 7;

(h) a mutein of (c), (d), (e) or (f) having at least 80% or 85% or 90% or 95% sequence identity to a polypeptide of (c), (d), (e) or (f); wherein the polypeptide binds to at least one of Blys or APRIL.

In another preferred embodiment, the Fc-fusion protein comprises the heavy chain constant region of a globulin, more preferably the constant region of a human. In one embodiment of the invention, the immunoglobulin is an IgG₁. It is also preferred that the constant region comprises the hinge, CH2 and CH3 regions.

In another embodiment, the therapeutic moiety SEQ ID NO: 1, a cysteine-rich pseudo-repeat sequence.

According to the invention, the liquid containing the Fc-fusion protein is first passed through a protein A or protein G affinity chromatography column. The liquid is preferably a cell culture, such as lysed cells, more preferably a cell culture supernatant. The term "cell culture supernatant" as used herein refers to a culture solution for culturing cells, which contains a protein secreted from the cells, the protein containing an appropriate cell signal, a so-called signal peptide. The Fc-fusion protein-expressing cells are preferably cultured under serum-free culture conditions. Thus, the cell culture supernatant is preferably free of animal serum components. Such a cell culture solution is most preferably a chemically defined culture solution.

The protein a used for affinity chromatography may be recombinant. May also be modified to improve its properties (e.g., a resin known as MabSelect SuRe, commercially available from GE Healthcare). In a preferred embodiment, step (a) is performed on a cross-linked agarose resin containing a modification with recombinant protein a. Chromatography columns available under the trade name MabSelect Xtra (from GE Healthcare) are examples of affinity resins particularly suitable for step (a) of the process.

Protein A or protein G affinity chromatography is preferably used as a capture step for purification of Fc-fusion proteins, in particular for removal of aggregates of host proteins and Fc-fusion proteins, and for concentration of Fc-fusion protein preparations.

The term "coacervate" as used in the present specification refers to protein coacervates, including multimers (e.g. dimers, tetramers or larger coacervates) in the Fc-fusion protein to be purified, which may result in, for example, high molecular weight coacervates.

Another advantage of this affinity chromatography is that the level of aggregates can be reduced by a factor of 2-4.

Using protein A or protein G affinity chromatography, host cell protein levels can be reduced by 100-fold.

In a preferred embodiment of the invention, the elution of step (a) is performed at a pH in the range of 2.8 to 4.5, preferably 3.0 to 4.2, more preferably 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 of step (a) may also be carried out with a gradient pH, preferably a pH of 4.5-2.8.

In another preferred embodiment, the elution of step (a) is performed with a buffer selected from sodium acetate or sodium citrate. Suitable buffer concentrations are selected, for example, from 50mM or 100mM or 150mM or 200mM or 250 mM.

According to the invention, the eluate after protein A or protein G chromatography is subjected to cation exchange chromatography. Cation exchange chromatography may be performed on any suitable cation exchange resin (e.g., a weak or strong cation exchanger as described in the background of the invention above).

Preferably step (b) is carried out on a strong cation exchange resin. More preferably, the cation exchange material contains SO₃ ^-Group-modified crosslinked methacrylates. Under the trade name Fractogel EMD SO₃ ^-Chromatography columns available commercially (from Merck) are examples of cation exchange resins that are particularly suitable for step (b) of the process.

Preferably, the eluate of protein a is loaded directly onto the cation exchange column. This loading is preferably carried out at a pH at least one unit lower than the pI of the Fc-fusion protein to be purified.

It is also preferred that after loading, the sample is washed with a buffer having a conductivity of 6-10mS/cm, e.g., 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, this conductivity is in the range of 7.6-9.2, i.e. 8.4. + -. 0.8 mS/cm. The washing step is preferably carried out at pH5.5-7.5, preferably 6.0-7.0.

In another preferred embodiment, the cation exchange chromatography column is eluted at a pH in the range of 7.0 to 8.5, preferably at a pH of 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 is preferably carried out with a buffer having a conductivity in the range from 15 to 22 mS/cm. For example, the conductivity may be selected from 16, 17, 18, 19, 20, 21, or 22 mS/cm. The preferred elution buffer is a phosphate buffer.

In a highly preferred embodiment, step (b) further comprises the steps of:

b1. washing the cation exchange resin with a buffer solution with pH of 6.0-7.0 and conductivity of 6-10 mS/cm; and

b2. eluting the column with a buffer solution having a pH of 7.0-8.5 and a conductivity of 15-22 mS/cm.

The preferred buffer for step (b1) is 75-125mM sodium phosphate buffer.

In the framework of the present invention it was surprisingly found that step (b) is effective in removing free Fc. Thus, cation exchange chromatography may preferably be used to remove or reduce free Fc about 5-15 fold in accordance with the present invention.

The advantage of step (b) of the method of the invention is that it also allows a reduction of the concentration of host cell protein, e.g.1-2 fold, in the Fc-fusion protein preparation, thus contributing significantly to the elimination of Host Cell Protein (HCP).

According to the invention, the eluate after the cation exchange step is passed through an anion exchange chromatography column. Anion exchange chromatography may be performed on any suitable anion exchange resin (e.g., a weak or strong anion exchanger as described in the background of the invention above). Preferably step (c) is carried out on a strong anion exchange resin. More preferably, the anion exchange resin contains a charge N⁺(CH₃)₃Group-modified polystyrene/divinylbenzene. A chromatography column available under the trade name Source 30Q (from GE Healthcare) is an example of an anion exchange resin that is particularly suitable for step (C) of the process.

The eluate of step (b) is preferably diluted or dialyzed with a suitable loading buffer prior to loading onto the anion exchange column. The anion exchange column is also preferably first equilibrated with the loading buffer.

The pH of the loading buffer is preferably less than one unit pI. Suitable pH ranges are 6.0 to 8.5, preferably 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. The preferred conductivity range for the loading buffer is 3.0-4.6 mS/cm.

A suitable equilibration/loading buffer may be, for example, a sodium phosphate solution in a concentration range of 5-35mM, preferably 20-30 mM. The concentration of the buffer may be, for example, 10, 15, 20, 25, 30 mM. In the framework of the present invention, the anion exchange chromatography effluent (also called flow-through) containing the Fc-fusion protein of interest is collected.

Step (c) of the method of the invention may further reduce the aggregates by a factor of 3-5 and the host cell protein by a factor of 30-70.

According to the present invention, the effluent of the anion exchange chromatography of step (c) is further purified using a hydroxyapatite chromatography column. Step (d) of the process of the invention may be carried out using any hydroxyapatite resin. In a preferred embodiment, step (d) is performed on a ceramic hydroxyapatite resin, such as a type I or type II hydroxyapatite resin. The hydroxyapatite resin may be in the form of particles of any size, for example 20, 40 or 80 μm. In a more preferred embodiment, the ceramic hydroxyapatite resin is in the form of particles having a size of 40 μm. A hydroxyapatite resin particularly suitable for step (d) of the process is a chromatographic column available under the trade name CHT ceramic Hydroxyapatite type I (40 μm).

In a preferred embodiment, the effluent of step (c) is loaded directly onto the hydroxyapatite resin, i.e. without prior dilution or dialysis with a suitable loading buffer. The loading is preferably carried out at a pH of 6.5 to 7.5, for example 6.6, 6.7, 6.8, 6.9, 7.1, 7.2, 7.3 or 7.4, preferably 7.0.

In another preferred embodiment, the elution of step (d) is performed in the presence of 2-10mM sodium phosphate, preferably 1.75-5.25mM, such as 2,25, 2.5, 2,75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4,5, 4.75, 5 mM.

In a further preferred embodiment, the elution of step (d) is carried out at a pH in the range of 6.0 to 7.0, e.g.6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9.

In another preferred embodiment, the elution of step (d) is carried out in the presence of 0.4-1M potassium chloride, preferably 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95M, most preferably 0.6M.

According to the invention, the eluate of step (d) is collected, which contains the final purified Fc-fusion protein preparation.

Suitable matrix materials, i.e.support materials, for the chromatography resins used in steps (a) to (c) which can be used in connection with the present invention may be, for example, agarose (superse), dextran (sephadex), polypropylene, cellulose methacrylate, polystyrene/divinylbenzene, etc. depending on the specific use, the resin materials may be present in various cross-linked forms.

The volume of resin in the chromatography column used, the length and diameter of the column, as well as the dynamic capacity and flow rate, depend on several parameters, such as the volume of liquid to be treated, the concentration of protein in the liquid in which the method of the invention is to be carried out, etc. Methods for determining these parameters in each step are well known to those of ordinary skill in the art.

In a preferred embodiment of the purification process according to the invention, one or more ultrafiltration steps are carried out. Ultrafiltration is used to remove small organic molecules and salts from the eluate from the previous chromatography step, to equilibrate the Fc-fusion protein with a large amount of buffer, or to concentrate the Fc-fusion protein to the desired concentration. Such ultrafiltration may be carried out on an ultrafiltration membrane having a pore size that allows removal of components having a molecular weight of less than 5, 10, 15, 20, 25, 30kDa or greater.

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).

If the protein purified according to the method of the invention is intended for human administration, the method preferably includes one or more viral removal steps. Preferably, a virus filtration removal step is performed after step (d). More preferably, the virus filtration removal step is a nanofiltration step, wherein the filter membrane used has a nominal pore size of 20 nm. The method of the invention and the specific steps (a), (c), (d) in combination with nanofiltration enable efficient removal of viral load to a total LRV (log reduction value) of not higher than about 15-25.

To facilitate storage or transport, for example, such materials may be freeze-thawed before and/or after the purification steps of the invention.

According to the present invention, eukaryotic expression systems, such as yeast, insect or mammalian cells, can be used to produce recombinant Fc-fusion proteins to produce glycosylated Fc-fusion proteins.

According to the present invention, it is most preferred to express the Fc-fusion protein in mammalian cells, such as animal cell lines or human cell lines. Chinese Hamster Ovary (CHO) or mouse myeloma cell line NS0 are examples of cell lines particularly suitable for expressing the Fc-fusion protein to be purified. It is also preferred to produce Fc-fusion proteins using human cell lines, such as the human fibrosarcoma HT1080 cell line, the human retinoblastoma cell line PERC6, or the human embryonic kidney cell line 293, or the constant amniotic fluid cell line described in EP 1230354.

If the Fc-fusion protein to be purified is expressed secretionally by mammalian cells, the starting material for the purification process according to the invention is a cell culture supernatant, also referred to as harvest or crude harvest. If the cells are cultured in a culture medium containing animal serum, the cell culture supernatant still contains serum protein impurities.

Cells expressing and secreting Fc-fusion proteins are preferably cultured under serum-free conditions. The Fc-fusion proteins can also be produced in chemically defined media. In this case, the starting material for the purification process of the invention is a serum-free cell culture supernatant containing mainly host cell protein impurities. For example, if growth factors such as insulin are added to the cell culture broth, these proteins will also need to be removed during the purification process.

In order to produce a soluble secreted Fc-fusion protein for release into the cell culture supernatant, the native signal peptide of the Fc-fusion protein therapeutic moiety, or preferably a heterologous signal peptide, i.e. a signal peptide derived from another secreted protein effective in the particular expression system used, such as a bovine or human growth hormone signal peptide, or an immunoglobulin signal peptide, may be utilized.

As mentioned above, preferred Fc-fusion proteins to be purified by the method of the invention are fusion proteins having a therapeutic moiety derived from human TACI (SEQ ID NO: 2), in particular having a fragment derived from the extracellular domain thereof (amino acids 1-165 of SEQ ID NO: 2). A preferred fragment comprises SEQ ID NO: 2, amino acids 30-110. Hereinafter, the therapeutic moiety derived from the extracellular domain of TACI will be referred to as "soluble TACI" or "sttaci". A preferred Fc-portion comprises SEQ ID NO: 3, which can produce seq id NO: 4, hereinafter referred to as "TACI-Fc".

The term TACI-Fc as used in the present specification also includes muteins of TACI-Fc.

The term "mutein" as used herein refers to analogs of sTACI or TACI-Fc in which one or more amino acid residues of sTACI or TACI-Fc are replaced with, or deleted from, different amino acid residues, or one or more amino acid residues are added to the original sequence of sTACI or TACI-Fc, without significantly altering the activity of the resulting product as compared to the original sTACI or TACI-Fc. Such muteins can be prepared by known synthetic techniques and/or site-directed mutagenesis techniques, or any other known suitable technique.

The muteins of the present invention comprise a polypeptide consisting of a sequence that hybridizes under stringent conditions to the nucleic acid sequence encoding SEQ ID NO: 2 or 4 or TACI-Fc, or a complement of DNA or RNA of TACI-Fc. An example of a DNA sequence encoding TACI-Fc is SEQ ID NO: 7.

the term "stringent conditions" refers to hybridization and subsequent washing conditions, and is conventionally referred to as "stringent conditions" by those of ordinary skill in the art. See Ausubel et al, Current Protocols in Molecular Biology, supra, Interscience, N.Y. § 6.3 and 6.4(1987, 1992). Non-limiting examples of stringent conditions include wash conditions at 12-20 ℃ below the calculated Tm for the hybridization, e.g., wash with 2XSSC and 0.5% SDS for 5 minutes, wash with 2XSSC and 0.1% SDS for 15 minutes; washing with 0.1XSSC and 0.5% SDS at 37 ℃ for 30-60 minutes, and then washing with 0.1XSSC and 0.5% SDS at 68 ℃ for 30-60 minutes. One of ordinary skill in the art will recognize that stringency conditions will also depend on the DNA sequence, the length of the oligonucleotide probe (e.g., 10-40 bases), or the length of the mixed oligonucleotide probe. If a mixed probe is used, tetramethylammonium chloride is preferably used in place of SSC. See Ausubel, supra.

In another embodiment, any such mutant protein is at least 50%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical or homologous.

Identity reflects the relationship between two or more polypeptide sequences or between two or more polynucleotide sequences as determined by sequence comparison. In general, identity refers to the exact correspondence of nucleotides to nucleotides, or amino acids to amino acids, of two polynucleotide or polypeptide sequences over the length of the sequences being compared.

For sequences that do not correspond exactly,% identity can be determined. In general, two sequences to be compared are aligned to give the maximum correlation between the two sequences. This may include inserting "spaces" in one or both sequences to improve the degree of alignment. The percent identity over the entire length of the sequences to be compared can be determined (so-called global alignment), which is particularly suitable for sequences of the same or very similar length, or shorter and well-defined length (so-called local alignment), more particularly for sequences of unequal length.

Methods for comparing the identity and homology of two or more sequences are well known in the art. For example, the% identity between two polynucleotides and the% identity and% homology between two polypeptide sequences can be determined using the programs in Wisconsin sequence analysis package version 9.1 (Devereux J et al, 1984), such as the BESTFIT and GAP programs. BESTFIT uses the "local homology" algorithm of Smith and Waterman (1981) to find a region of optimal similarity between two sequences. Other procedures for determining identity and/or similarity between sequences are known in the art, such as the BLAST (Altschul S F et al, 1990, Altschul S F et al, 1997) and FASTA (Pearson W R, 1990) family of programs accessed through the NCBI homepage of www.ncbi.nlm.nih.gov.

Any such mutein preferably has an amino acid sequence that is sufficiently duplicated in the sTACI or TACI-Fc sequence to have a sequence identical to the sequence of SEQ ID NO: 2 or 4 substantially similar ligand binding activity. For example, one activity of TACI is the ability to bind Blys or APRIL (Hymowitz et al, 2006). Such muteins are considered to have substantially similar activity to TACI, as long as they have substantial APRIL or Blys binding activity. Thus, one skilled in the art can readily determine by routine experimentation whether any given mutein has an amino acid sequence that is identical to SEQ ID NO: 2 or 4 proteins with substantially the same activity.

Preferred alterations of the muteins of the present invention are those known as "conservative" substitutions. Conservative amino acid substitutions of sTACI or TACI-Fc can include synonymous amino acid substitutions in a group of amino acid members with sufficiently similar physicochemical properties that the biological function of the molecule will be retained (Grantham, 1974). It is clear that amino acid insertions and deletions can be made in the above sequences without altering their function, in particular if such insertions or deletions involve only a few amino acids, such as less than 30, less than 20, or preferably less than 10, and do not remove or replace amino acids which are critical to the functional configuration, such as cysteine residues. Proteins and muteins produced by such deletions and/or insertions fall within the scope of the present invention.

The conservative amino acid grouping is preferably those listed in Table 2. The synonymous amino acid groupings are more preferably those listed in Table 3, and the synonymous amino acid groupings are most preferably those listed in Table 4.

TABLE 2Preferred group of synonymous amino acids

Synonymous group of amino acids

Ser Ser，Thr，Gly，Asn

Arg Arg，Gln，Lys，Glu，His

Leu Ile，Phe，Tyr，Met，Val，Leu

Pro Gly，Ala，Thr，Pro

Thr Pro，Ser，Ala，Gly，His，Gln，Thr

Ala Gly，Thr，Pro，Ala

Val Met，Tyr，Phe，Ile，Leu，Val

Gly Ala，Thr，Pro，Ser，Gly

Ile Met，Tyr，Phe，Val，Leu，Ile

Phe Trp，Met，Tyr，Ile，Val，Leu，Phe

Tyr Trp，Met，Phe，Ile，Val，Leu，Tyr

Cys Ser，Thr，Cys

His Glu，Lys，Gln，Thr，Arg，His

Gln Glu，Lys，Asn，His，Thr，Arg，Gln

Asn Gln，Asp，Ser，Asn

Lys Glu，Gln，His，Arg，Lys

Asp Glu，Asn，Asp

Glu Asp，Lys，Asn，Gln，His，Arg，Glu

Met Phe，Ile，Val，Leu，Met

Trp Trp

TABLE 3More preferred group of synonymous amino acids

Synonymous group of amino acids

Ser Ser

Arg His，Lys，Arg

Leu Leu，Ile，Phe，Met

Pro Ala，Pro

Thr Thr

Ala Pro，Ala

Val Val，Met，Ile

Gly Gly

Ile Ile，Met，Phe，Val，Leu

Phe Met，Tyr，Ile，Leu，Phe

Tyr Phe，Tyr

Cys Cys，Ser

His His，Gln，Arg

Gln Glu，Gln，His

Asn Asp，Asn

Lys Lys，Arg

Asp Asp，Asn

Glu Glu，Gln

Met Met，Phe，Ile，Val，Leu

Trp Trp

TABLE 4Most preferred group of synonymous amino acids

Synonymous group of amino acids

Ser Ser

Arg Arg

Leu Leu，Ile，Met

Pro Pro

Thr Thr

Ala Ala

Val Val

Gly Gly

Ile Ile，Met，Leu

Phe Phe

Tyr Tyr

Cys Cys，Ser

His His

Gln Gln

Asn Asn

Lys Lys

Asp Asp

Glu Glu

Met Met，Ile，Leu

Trp Met

From the Fc-fusion protein purified according to the present invention, functional derivatives thereof can be prepared. "functional derivatives" as used in the present specification covers derivatives of the Fc-fusion proteins to be purified according to the process of the invention, which derivatives can be prepared from the side chain functional groups or the N-or C-terminal groups of the residues by methods known in the art, and are included in the invention as long as they are still pharmaceutically acceptable, i.e.their protein activity is not impaired, they are still substantially similar to the activity of the above-described unmodified Fc-fusion proteins, and they do not poison the compositions in which they are contained.

Functional derivatives of Fc-fusion proteins can be coupled to polymers, for example, to improve their protein properties, such as stability, half-life, bioavailability, tolerance or immunogenicity in humans. To achieve this, TACI-Fc can be linked to, for example, polyethylene glycol (PEG). PEGylation may be carried out by known methods, for example, as described in WO 92/13095.

For example, such functional derivatives may also include aliphatic esters of carboxyl groups, amides of carboxyl groups by reaction with ammonia or primary or secondary amines, N-acyl derivatives of free amino groups of amino acid residues with acyl groups (e.g.alkanoyl or cycloaroyl groups), or O-acyl derivatives of free hydroxyl groups (e.g.of seryl or threonyl residues) with acyl groups.

In a third aspect, the invention relates to a protein purified by the purification method of the invention. Hereinafter, such proteins are also referred to as "purified Fc-fusion proteins".

Such a purified Fc-fusion protein is preferably a highly purified Fc-fusion protein. For example, 2mcg of protein was loaded in each lane for non-reducing SDS-PAGE gel silver staining to confirm high purity of the Fc-fusion protein by the presence of only one band. It is also possible to determine the purified Fc-fusion protein by using only one peak in the HPLC eluate.

The Fc-fusion protein preparation obtained from the purification method of the present invention may contain less than 20% impurities, preferably less than 10%, 5%, 3%, 2% or 1% impurities, or may be purified to homogeneity, i.e., free of any detectable contaminating proteins as detected by silver-stained SDS-PAGE or HPLC as described above.

The purified Fc-fusion protein is expected to be useful in therapy, particularly for administration to patients. If the purified Fc-fusion protein is to be administered to a patient, it is preferably administered systemically, preferably subcutaneously or intramuscularly, or locally. Depending on the specific medical use of the purified Fc-fusion protein, intrarectal or intrathecal administration may also be suitable.

For this purpose, in a preferred embodiment of the present invention, the purified Fc-fusion protein may be formulated into a pharmaceutical composition, i.e. together with pharmaceutically acceptable carriers, excipients, etc.

The definition of "pharmaceutically acceptable" is intended to include any carrier that does not interfere with the effectiveness of the biological activity of the active ingredient and is not toxic to the host to which it is administered. For example, for parenteral administration, the active protein may be formulated in unit dosage forms for injection using carriers such as saline, dextran solution, serum albumin, and ringer's solution.

The active ingredients of the pharmaceutical compositions of the present invention may be administered to an individual by a variety of routes. The administration routes include: intradermal, transdermal (e.g., sustained release formulations) intramuscular, intraperitoneal, intravenous, subcutaneous routes, oral, intracranial, epidural, topical, rectal, and intranasal routes. Any other therapeutically effective route of administration may be used, for example absorption through epithelial or endothelial tissue, or gene therapy, wherein administration of a DNA molecule encoding the active agent to a patient (e.g., via a vector) results in expression and secretion of the active agent in vivo. In addition, the proteins of the present invention may be administered with other biologically active pharmaceutical ingredients, such as pharmaceutically acceptable surfactants, adjuvants, carriers, diluents, and the like.

For parenteral (e.g., intravenous, subcutaneous, intramuscular) administration, the active protein may be formulated as a solution, suspension, emulsion or lyophilized powder in combination with a pharmaceutically acceptable parenteral carrier (e.g., water, saline, dextrose solution) and additives that maintain isotonicity (e.g., mannitol) or chemical stability (e.g., preservatives and buffers). The formulation is sterilized by conventional techniques.

The therapeutically effective amount of active protein may be a function of a number of variables including the type of Fc-fusion protein, the affinity of the Fc-fusion protein for its ligand, the route of administration, the clinical condition of the patient.

As described above and with particular reference to Table 5 above, a "therapeutically effective amount" is an amount that results in inhibition of the ligand of the therapeutic portion of the Fc-fusion protein after administration of the Fc-fusion protein.

The dose administered to an individual in a single dose or multiple doses will vary depending on various factors, including the pharmacokinetic properties of the Fc-fusion protein, the route of administration, the condition and characteristics of the patient (sex, age, body weight, health condition, body size), the degree of symptoms, concurrent treatments, the frequency of treatment, and the desired effect. It is within the ability of those skilled in the art to adjust and manipulate established dosage ranges, as are in vitro and in vivo methods for determining the inhibition of therapeutic moieties of natural ligands on an individual.

The amount of purified Fc-fusion protein is 0.001-100mg, or 0.01-10mg, or 0.1-5mg, or 1-3mg, or 2mg per kg body weight.

In other preferred embodiments, the purified Fc-fusion protein is administered daily or every other day, or three times per week or once per week.

The daily dose is usually administered in divided doses or in sustained release form effective to achieve the desired result. The second or subsequent administration may be at the same, lesser or greater dose as the dose originally or previously administered to the individual. A second or subsequent administration may be made during or before the onset of the disease.

The present invention also relates to a purified Fc-fusion protein composition comprising an extracellular portion of a member of the Tumor Necrosis Factor Receptor (TNFR) superfamily obtained with the method of the present invention as detailed above, wherein the composition comprises less than 2%, or less than 1.5%, or less than 1%, or less than 0.7%, or less than 0.6%, or preferably less than 0.5% of protein aggregates. The compositions of the invention preferably comprise full-length, intact Fc-fusion proteins with no more than 1 or 2 amino acids deleted from the N-or C-terminus, more preferably without any amino acids deleted from the N-or C-terminus.

The present invention also relates to a purified Fc-fusion protein composition comprising the extracellular portion of a member of the Tumor Necrosis Factor Receptor (TNFR) superfamily obtained using the method of the present invention, wherein the composition comprises less than 1%, or less than 0.8%, or less than 0.5% of the above-mentioned free Fc.

Such Fc-fusion proteins may, for example, be derived from the TNFR superfamily member OX 40. Such OX 40-functional proteins, e.g. OX40-IgG₁And OX40-hIG₄mut, which can be preferably used for the treatment and/or prevention of inflammatory and autoimmune diseases, such as Crohn's disease.

Such an Fc-fusion protein comprising a therapeutic moiety is preferably an extracellular domain selected from TNFR1, TNFR2, or a TNF binding fragment thereof.

In a preferred embodiment, the Fc-fusion protein is Etanercept (Etanercept), an Fc-fusion protein containing a soluble portion of p75TNFR (e.g., WO9l/03553, WO 94/06476). Etanercept purified according to the invention may be used, for example, for the treatment and/or prevention of endometriosis, hepatitis c virus infection, HIV infection, psoriatic arthritis, psoriasis, rheumatoid arthritis, asthma, ankylosing spondylitis, heart failure, graft-versus-host disease, pulmonary fibrosis, crohn's disease. Lenercept (Lenercept) is a fusion protein containing the extracellular component of the human p55TNF receptor and the Fc portion of human IgG and is expected to be useful in the treatment of severe sepsis and multiple sclerosis.

In another preferred embodiment, the Fc-fusion protein comprises a therapeutic portion selected from the group consisting of BAFF-R, BCMA or TACI extracellular domain, or a fragment thereof that binds to at least one of Blys or APRIL.

The BAFF-R-derived Fc-fusion protein purified according to the method of the invention is preferably used for the treatment and/or prevention of autoimmune diseases, such as Rheumatoid Arthritis (RA) and Systemic Lupus Erythematosus (SLE).

The BCMA-Ig fusion protein purified according to the method of the invention is preferably used for the treatment and/or prevention of autoimmune diseases.

The Fc-fusion protein derived from TACI (TACI-Fc) preferably comprises a polypeptide selected from the group consisting of:

seq ID NO: 2 amino acids 34-66;

seq ID NO: 2 amino acids 71-104;

seq ID NO: 2 amino acids 34-104;

d.SEQ ID NO: 2 amino acids 30-110;

e.SEQ ID NO：3：

f.SEQ ID NO：4：

g. can be combined with the sequence shown in SEQ ID NO: 5 or 6 or 7;

h. a mutein of one of (c), (d), (e) or (f) having at least 80% or 85% or 90% or 95% sequence identity to a polypeptide of (c), (d), (e) or (f); wherein the polypeptide binds to at least one of Blys or APRIL.

The purified TACI-Fc can be preferably used for the preparation of a medicament for the treatment and/or prevention of various diseases or conditions. Such diseases or conditions are preferably selected from autoimmune diseases such as Systemic Lupus Erythematosus (SLE), Rheumatoid Arthritis (RA), and the treatment of Multiple Sclerosis (MS). Purified TACI-Fc can also be used to treat cancers, e.g., hematologic malignancies such as Multiple Myeloma (MM) and/or non-hodgkin's lymphoma (NHL), Chronic Lymphocytic Leukemia (CLL), and Waldenstrom's Macroglobulinemia (WM).

Having now fully described this invention, it will be appreciated by those skilled in the art that the method of the present invention can be carried out 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 the invention has been described in conjunction with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is 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.

All references cited in this specification, including articles or abstracts in journals, published or unpublished U.S. or foreign patent applications, issued U.S. or foreign patents, or any other references, are incorporated herein by reference in their entirety, including all data, tables, figures, and text in the references. In addition, the bibliography of the references cited in this specification is also incorporated herein by reference in its entirety.

Reference to known method steps, conventional method steps, known methods or conventional methods does not in any way constitute 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 (including the contents of the references cited herein), readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the general concept of the present invention. Therefore, such adaptations and modifications as come within the meaning and range of equivalents of the disclosed embodiments, as taught and guided by the present specification, are intended to be within the scope of equivalents of the disclosed embodiments. It is also to be understood that the phraseology or terminology employed 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 supernatants

Glossary

BV: volume of the column bed

CHO: chinese hamster ovary

And (4) DSP: downstream process

EDTA: ethylenediaminetetraacetic acid

ELISA: enzyme linked immunosorbent assay

HAC: chromatography on hydroxyapatite

HCP: host proteins

HPLC: high performance liquid chromatography

id: inner diameter

K: potassium salt

kD: kilodalton (kilodalton)

MES: 2-morpholinylethanesulfonic acid

Na: sodium salt

NaAc: sodium acetate

n/d: not determined

PA-SE-HPLC: protein A molecular size exclusion high performance liquid chromatography

ppm: parts per million

And (3) RO: reverse osmosis

RT: at room temperature

SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis

SE-HPLC: molecular size exclusion high performance liquid chromatography

T ℃ is as follows: temperature of

TMAC: tetramethylammonium chloride

UV: ultraviolet ray

WFI: water for injection

WRO reverse osmosis water

Example 1: a capturing step: protein a affinity purification

Starting material was a clarified harvest of clones of TACI-Fc expressing CHO cells cultured under serum-free conditions and stored frozen until use.

The following scheme is adopted in MabSelect Xtra^TMThis capture step was carried out on a column (GE Healthcare 17-5269-03) having a bed height of 17 cm. All manipulations were performed at room temperature, except that the loading solution was kept below 15 ℃. UV signal at 280nm was recorded.

Cleaning of

The column was cleaned with at least 3BV of 0.1M acetic acid + 20% ethanol at a countercurrent flow rate of 250 cm/h. The flow was stopped for 1 hour.

Washing step

The column was washed with at least 2BV of RO water at a countercurrent flow rate of 250 cm/h.

Balancing

The column was equilibrated with a downward flow rate of 450cm/h using at least 5BV of 25mM sodium phosphate +150mM NaCl solution pH7.0 (unit conductivity and pH parameters within specified ranges: pH 7.0. + -. 0.1, conductivity 18. + -.2 mS/cm).

Loading

The collected supernatant, kept at 15 ℃ or lower, was loaded onto this column at a flow rate of 350cm/h, and the loading capacity per ml of packed resin, as determined by the Biacore test, was not higher than 15mg of total TACI-Fc protein.

Washing step

The column was washed with at least 2BV of equilibration buffer at a flow rate of 350cm/h, then with at least 4BV of equilibration buffer at a flow rate of 450cm/h (until the UV signal returned to baseline).

Elution is carried out

The material was eluted at a flow rate of 350cm/h with different elution buffers as indicated in Table 1. When the UV signal started to rise to elute to 6.0. + -. 0.5BV, the eluted fractions were collected. The eluate was incubated at pH4.1 or less (adjusted by adding citric acid solution if necessary) at room temperature for 1 hour, and then adjusted to pH 5.0. + -. 0.1 by adding 32% NaOH solution.

Regeneration

The column was regenerated with at least 3BV of 50mM NaOH +1M NaCl at a reverse flow rate of 450cm/h, stopping the flow for 15 minutes, and then restarted with at least 3BV of liquid at a flow rate of 450cm/h (until the UV signal returned to baseline).

From this step, the column was operated in countercurrent.

Washing step

The column was washed with at least 2BV of RO water at a flow rate of 450 cm/h.

Cleaning of

The column was cleaned with at least 3BV of cleaning buffer at a flow rate of 250cm/h and the flow incubation was stopped for 60 minutes.

Final washing step

The column was washed with at least 1BV of RO water at a flow rate of 250cm/h, then with at least 3BV of equilibration buffer at a flow rate of 250cm/h, and finally with at least 2BV of RO water at a flow rate of 250 cm/h.

Finally, the column was stored after washing with 3BV of 20% ethanol at a flow rate of 250 cm/h.

Results

TABLE I: results with different elution buffers

Operation #	Elution buffer	TACL-Fc production (%)	Aggregate (%)	HCPs(ppm)
					1234	50mM NaAc pH3.7100mM NaAc pH3.8200mM NaAc pH3.8100mM NaAc pH3.7	47.755.758.068	30.325.228.230.0	5558n/dn/dn/d
567891011121314	0.2M NaAc +150mM NaClpH4100mM NaAc pH3.7250mM NaAc pH3.7100mM sodium citrate pH3.7250mM sodium citrate pH3.7100mM sodium citrate pH3.75100mM sodium citrate pH3.751003.85100mM sodium citrate pH3.75100mM sodium citrate pH3.65	75.184.682.879.271.982.866.683.381.075.1	3.82218.78.8238.59.015.09.114.6	n/d34913318471023471576664n/d34902580
					141617	100mM sodium citrate pH3.75100mM sodium citrate pH3.75	44.747.150.7	18.415.89.4	378332172349
1819	100mM sodium citrate pH3.75100mM sodium citrate pH3.75	58.067.1	10.428.7	25502372

Operation #	Elution buffer	TACI-Fc production (%)	Aggregate (%)	HCPs(ppm)
					20	100mM sodium citrate pH3.75	65.6	17.5	2353
21222324	100mM sodium citrate pH3.75100mM sodium citrate pH3.75	75.657.151.958	19.420.718.411.5	1807246520301746
					25262728	100mM sodium citrate pH3.75100mM sodium citrate pH3.9100mM Na Ac pH4.1	41.839.431.028.3	22.96.08.825.0	3029242429363311
29303132	100mM sodium citrate pH3.9100mM NaAc pH4.1100mM sodium citrate pH3.75100mM NaAc pH4.2	46.442.857.538.1	9.113.426.510.1	n/dn/dn/dn/d
					33	100mM Na citrate pH3.9	43.3	8.3	2011
343536373839	100mM sodium citrate pH3.9100mM sodium citrate pH3.9	63.665.762.761.660.664.6	6.67.37.47.47.48.0	174916891609147916231497

Conclusion

TACI-Fc5 in the clarified pool was captured directly on a MabSelect Xtra column with a kinetic capacity of 15g total TACI-Fc5 protein per liter of packing resin at a flow rate of 350 cm/h. Optimizing elution conditions, especially pH, maximizes product recovery while significantly reducing coagulum levels. A0.1M sodium citrate buffer (pH3.9) was selected to reduce the level of aggregate in the clear collection from the initial level of about 25-40% to about 5-10% and no turbidity was observed. HCP levels were typically 1500-2000 ppm. HCP levels were determined by polyclonal antibody ELISA. A mixture of antibodies against host cell proteins in clarified and concentrated cell culture supernatants of non-transfected CHO cells was prepared.

Example 2: cation exchange chromatography

The eluate from the protein a capture step is dialyzed against an appropriate loading buffer and used as starting material for cation exchange chromatography.

This step was carried out using Fractogel EMD SO with a bed height of 10cm₃ ^-Column (Merck 1.16882.0010). Fractogel EMD SO with bed height of 15cm can also be used₃ ^-And (3) a column. The latter dynamic capacities and flow rates may need to be adapted, which is within the routine knowledge of the skilled person.

All operations were carried out at room temperature, the flow rate being kept constant at 150 cm/h. The 280nmUV signal was recorded for all time periods.

Washing step

The column was washed with at least 1BV of WRO (reverse osmosis water).

Cleaning of

The column was then cleaned in an upflow mode with at least 3BV of 0.5M NaOH +1.5M NaCl solution.

Leaching with water

The column was rinsed in a downflow mode with at least 4BV of WRO.

Balancing

The column was equilibrated with at least 4BV of 100mM sodium citrate solution (pH5.0) (or until the target conductivity of 12. + -.1 mS/cm and pH 5.0. + -. 0.1 was reached).

Loading

The column was loaded with a post-capture material at pH5.0(pH 5.0. + -. 0.1, conductivity 12. + -.1 mS/cm) and the capacity per ml of packed resin was not more than 50mg TACI-Fc as determined by SE-HPLC assay.

Washing step

The column was then washed with at least 5BV of 100mM sodium phosphate solution (pH 6.5).

Elution is carried out

The column was eluted under different conditions with different buffers as reported in tables II-IV below.

Regeneration and cleaning

The column was regenerated and cleaned in an upflow mode with 4BV of 0.5M NaOH +1.5M NaCl solution. The flow was then stopped for 30 minutes.

Leaching with water

The column was rinsed with at least 4BV of WRO.

Storage of

The column was stored in at least 3BV of 20% ethanol.

Results

TABLE II: effect of eluent pH and conductivity

HCP levels in loading fluid: 189ppm of

pH	Conductivity (mS/cm)	TACI-Fc recovery	HCPs(ppm)	HCP Clearance (x)
					6.57.38.07.37.37.37.37.36.3	15.022.515.022.533.022.522.512.022.5	25％100％95％100％98％96％97％54％83％	11850345613345537947	1.63.85.53.41.44.23.62.44.1

pH	Conductivity (mS/cm)	TACI-Fc recovery	HCPs(ppm)	HCP Clearance (x)
					8.08.26.57.37.3	30.022.530.022.522.5	96％97％91％93％95％	108461164840	1.84.21.63.94.8

Table III shows the recovery of TACI-Fc and the clearance of HCP at loading capacities of 10 and 32mg TACI-Fc per ml resin and elution with phosphate buffer of conductivity 12-33 mS/cm. Collecting protein peak components from the rising of the eluent UV, and collecting 10 +/-0.5 BV in total.

Table III:optimizing the effect of pH and conductivity of the eluent on loading capacity

HCP levels in loading fluid: 201 ppm.

Load capacity (mg/ml)	pH	Conductivity (mS/cm)	TACI-Fc recovery	HCPs(ppm)	HCP clearance (x)
						10	8.0	15.020.7	91％93％	6761	3.03.3
32	8.0	20.7	88％	54	3.7

Table IV shows the effect of washing steps with 50 or 100 or 150mM sodium phosphate solution (pH6.5) on TACI-Fc recovery and HCP clearance efficiency.

Table IV:effect of washing step conditions on column Performance

HCP levels in loading fluid: 190ppm, level of coagulum: 2.0 percent

The buffer used for the second wash contained 100mM sodium phosphate, pH6.5, conductivity 8.4 mS/cm.

FIG. 1 shows a silver stained non-reducing SDS-PAGE gel of samples obtained in experiments in which free Fc was eliminated using the three-step wash conditions shown in Table IV.

Figure 2 shows overlapping chromatograms of washing experimental steps with different concentrations of sodium phosphate solution.

The washing step was optimized at pH6.5 with increasing sodium phosphate concentration (from 50mM to 150 mM). As can be seen in FIG. 1, a 150mM concentration of wash buffer (Wash 3, lane 6) resulted in the loss of TACI-Fc. A50 mM concentration of wash buffer (Wash 1, lane 8) produced a pure TACI-Fc protein peak, however the eluate contained a trace amount of free Fc. The washing step with 100mM sodium phosphate solution (pH6.5) resulted in 98% recovery in the main peak of the eluate and only 2% loss in the wash (FIG. 2). HCP was 3.2-fold cleared. SDS-PAGE analysis of the wash and eluate fractions showed that with a buffer concentration of 100mM or more, the wash contained free Fc and some intact TACI-Fc protein (FIG. 1, lanes 4 and 6). Concentrations of 100mM or higher were necessary to completely remove free Fc from the eluate fractions (FIG. 1, lanes 5 and 6).

Conclusion

A cation exchange step was developed as a second purification step after the capture step. The captured eluate has a low pH (5.0) and low conductivity and can be applied directly to a cation exchange column. Selecting Fractogel EMD SO with loading capacity of 50mg/ml₃ ^-And (3) resin. The washing step uses 0.1M sodium phosphate solution (pH6.5) to effectively remove the biologically inactive degradation product free Fc. Optimization of elution conditions achieved optimal clearance of HCP and high recovery of TACI-Fc (179mM sodium phosphate solution, pH8.0, conductivity 20.7 mS/cm).

In addition, when the absorbance at 280nm started to increase, elution was performed with 10BV of 20mM sodium phosphate and 180mM NaCl solution (pH 8.0).

Example 3: anion exchange chromatography

The starting material used in this purification step was Fractogel SO₃ ^-(see example 2) the eluate from the cation exchange step is dialyzed or diluted with a suitable loading buffer.

This step of anion exchange chromatography was carried out on a SOURCE 30Q column (GE Healthcare 17-1275-01) with a bed height of 10 cm. A SOURCE 30Q chromatographic column with a column bed height of 15cm can also be used in this step. The latter dynamic capacity and flow rate may need to be adapted, as is conventional to those skilled in the art.

All manipulations were carried out at room temperature and a signal of 280nmUV was recorded. This step was carried out at a flow rate of 150 or 200 cm/h.

Leaching with water

The column was rinsed with at least 1BV of RO water at a flow rate of 150 cm/h.

Cleaning of

The column was then cleaned with at least 3BV of 0.5M NaOH +1.5M NaCl.

Washing step

The column is washed with at least 3BV, preferably 4-10BV, of 0.5M sodium phosphate solution (pH7.5) at a flow rate of 200 cm/h.

Balancing

The column is equilibrated with at least 5BV of 10, 15, 20, 25 or 30mM sodium phosphate solution (pH 7.5). Optionally pre-equilibrating the column with 3BV of 0.5M sodium phosphate (pH7.5),

Loading, washing and subsequent Collection of TACI-Fc protein in the effluent

The material obtained after cation exchange chromatography was diluted to a sodium phosphate concentration of 10-30mM (after pH7.5), applied to the column, less than 50mg of TACI-Fc capacity per ml of packed resin was measured by SE-HPLC, washed with 4. + -. 0.5BV of equilibration buffer, and the effluent was collected from the start of UV rise to the end of the washing step.

Regeneration/cleaning

The column was regenerated and cleaned with at least 3BV of 0.5M NaOH +1.5M NaCl solution at a flow rate of 150cm/h in countercurrent flow mode (until the UV signal returned to baseline). At the end of regeneration, the pump was stopped for 30 minutes.

Washing step

The column was washed with at least 3BV of RO water at a flow rate of 200 cm/h.

Storage of

The column was stored in 3BV 20% ethanol (v/v) at a flow rate of at least 150 cm/h.

Results

Table V below summarizes the results obtained with the purification process described above.

TABLE V: effect of sodium phosphate plus Carrier fluid concentration

pH of the charging liquid	Concentration of sodium phosphate in the Loading solution (mM)	Concentration of TACI-Fc in Carrier liquid (mg/L)	Load capacity (mg/ml)	TACI-Fc recovery	Agglomerates	HCP(ppm)
							7.57.57.57.57.5	3025201510	773639651437283	39394946n./d.	94％90％90％88％82％	10.4％6.9％5.6％3.4％2.8％	82.850.443.945.026.3

Conclusion

The anion exchange chromatography step on a Source 30Q column in flow-through mode was optimized to maximize HCP and condensate clearance. Dilution or dialysis of the eluate from the cation exchange chromatography loaded with 20mM sodium phosphate buffer (pH7.5) gave the best compromise between product recovery (90%), HCP clearance (decrease from about 2000ppm to 44ppm) and coagulum clearance (decrease from about 25% to 5.6%). The dynamic capacity of the TACI-Fc protein per ml of the packed resin was 50mg at a flow rate of 150-.

Example 4: chromatography on hydroxyapatite

The starting material used for this purification step was the flow-through of anion chromatography (see example 3).

A CHT type I ceramic hydroxyapatite 40 micron (particle) column (Biorad157-0040) with a bed height of 10cm was used.

All operations were carried out at room temperature, the flow rate being kept constant at 175 cm/h. The 280nmUV signal was recorded. All solutions were sterilized by filtration and were cleaned with sodium hydroxide solution before use. The column was stored in 0.5M NaOH solution when not in use.

Initial washing step (rinsing and Pre-equilibration)

The column was washed to reduce its pH with at least 1BV of 20mM sodium phosphate (pH7.5) buffer, followed by at least 3BV of 0.5M sodium phosphate pH7.5 buffer.

Balancing

The column was equilibrated with at least 5BV of 20mM sodium phosphate pH7.5 buffer (or until the goals of conductivity 3.0. + -. 0.3mS/cm and pH 7.5. + -. 0.1 were reached).

Loading

The effluent from the SOURCE 30Q column was taken and added with 0.5M calcium chloride stock solution to a final calcium chloride concentration of 0.1mM, and 85% orthophosphoric acid solution was added to adjust the pH to 7.0, and loaded on the column in such an amount that the TACI-Fc protein capacity per ml of the packed resin was 50mg NMT according to the SE-HPLC test. Alternatively, the SOURCE 30Q effluent, adjusted to pH7.0, was loaded onto a hydroxyapatite column without calcium chloride.

Washing step

With at least 4BV of 3, 4 or 5mM sodium phosphate, 10mM MES, 0.1mM CaCl₂The column was washed with pH6.5 buffer. In these steps, the same buffer without calcium chloride can also be used.

Elution is carried out

Starting from the UV increase with different BVs (see tables VI and VII), 5, 4, 3 or 2mM sodium phosphate (see Table VI), 10mM MES, 0.1mM CaCl₂And 0.6, 0.7, 0.8 or 0.9M KCl pH6.5 buffer (see Table VII). Elution can also be carried out using the same buffer without calcium chloride.

Leaching with water

The column was washed sequentially with the following buffers:

-at least 1BV of 20mM sodium phosphate ph7.5 buffer;

-at least 3BV of 0.5M sodium phosphate ph7.5 buffer; and

-at least 1BV of 20mM sodium phosphate ph7.5 buffer.

Preservation of

The column was kept in at least 3BV of 0.5M NaOH solution.

Results

Table VI shows the effect of sodium phosphate concentration (2-5mM) of the elution buffer on removal of aggregates and product recovery. The peak fractions were pooled and analyzed for TACI-Fc concentration and aggregate levels by SE-HPLC.

TABLE VI: effect of sodium phosphate concentration of elution buffer

Sodium phosphate concentration (mM) of buffer	BV of eluent	TACI-Fc yield	Agglomerates
				5	12131415161718	73％74％68％77％77％70％76％	0.49％0.52％0.65％0.67％0.70％0.73％0.85％

4	12131415161718	68％67％66％67％66％66％66％	0.34％0.29％0.36％0.39％0.38％0.32％0.40％
				3	12131415161718	70％76％73％71％69％69％70％	0.46％0.42％0.51％0.52％0.55％0.50％0.53％
2	12131415161718	65％66％66％68％66％71％65％	0.19％0.00％0.18％0.14％0.17％0.19％0.16％

Table VII shows the effect of KCl concentration of the elution buffer on aggregate clarity and product recovery. Two sodium phosphate concentrations were studied: 2mM and 3 mM. Pooled peak fractions were analyzed by SE-HPLC for TACI-Fc concentration and aggregate levels.

Table VII:effect of elution buffer Potassium chloride concentration

Phosphate concentration (mM)	KCl concentration (M)	BV of eluent	TACI-Fc yield	Agglomerates
					3	0.6	1011121314	102％109％106％105％103％	0.48％0.46％0.43％0.42％0.43％
3	0.7	1011121314	96％97％98％96％96％	0.42％0.40％0.41％0.40％0.43％
					3	0.8	1011121314	106％110％112％101％110％	0.58％0.55％0.57％0.59％0.57％
2	0.6	1011121314	71％79％80％80％81％	0.29％0.28％0.29％0.29％0.26％
					2	0.9	1011121314	64％72％73％70％66％	0.27％0.25％0.29％0.33％0.24％

Conclusion

Hydroxyapatite chromatography provides a reliable and effective method for reducing the level of TACI-Fc aggregates. The level of aggregates at the start of the anion exchange chromatography purified material (see example 3) was about 5-8%, and hydroxyapatite chromatography reduced these levels to below 0.8% with TACI-Fc recovery of 85-90%.

Total result

Our developed four-step purification method of TACI-Fc yielded highly pure TACI-Fc (fusion protein) with an overall reduction of the level of aggregates to below 1% (0.2-0.8%, 5 experiments). The HCP levels were reduced overall to about 5-10ppm and the free Fc levels were reduced overall to below 0.5% (0.2 and 0.1%, two experiments).

Reference to the literature

Akerstrom and Bjork, J Biol chem.1989Nov 25; 264(33): 19740-6.

Altschul S et al, J Mol Biol 215, 403-

Altschul S F et al, Nucleic Acids Res., 25: 389-3402, 1997

Armour KL et al, 1999 Recombinant human IgG molecules lacking Fc gamma receptor 1 binding and monocyte triggering activity (Recombinant human IgG molecules binding Fcgammateptor I binding and monocyte triggering activities) Eur J Immunol.29 (8): 2613-24

Black et al, US 2002/0115175

Bodmer et al, TIBS 27(1), 19-24

7.Boschetti，E.，Jungbauer，Sep.Sci.&Tech.2No.15，Acad.Press(2000)53

Boschetti et al, Genetic Engineering Vol.20, No.13, July, 2000

Bossen et al, JBC 281(2), 13964-

Bram and von Buow, U.S. Pat. No. 5,969,102(1999)

Bram et al, WO 98/39361

Carter PJ., 2006 design of a Potent antibody therapy (Power antibodies by design). Nature Reviews immunology. Advance, on-line (online publication)

Devereux J et al, Nucleic Acids Res, 12, 387-

Feng et al, Bioprocessing 2005, 1-7

15.Giovannini，Biotechnology and Bioengineering 73：522-529(2000)

Grantham et al, Science, Vol.185, pp.862-864(1974)

17 Gross et al, Nature 404(27), 995-999-

Gross et al, WO 00/40716

Engineered IgG antibodies with longer serum half-lives in primates (Engineered human IgG antibodies with a long range serum half-life in primates) J Biol chem.279 (8): 6213-6

Hymowitz et al, JBC 280(8), 7218-

Idusogene EE et al, 2000. C1q binding site map of an antibody chimeric to human IgG1 Fc (rituxan) (Mapping of the C1q binding site on rituxan, a polymeric antibody with a human IgG1 Fc.) J Immunol.164 (8): 4178-84

Idusogene EE et al, 2001. Engineered antibodies have increased complement recruitment activity (Engineered antibodies with increased activity to recovery complement.) jimmunol.166 (4): 2571-5

23.Ishihara，EP 1614693

Kabat, E.A., Wu, T.T., Perry, H.M., Gottesman, K.S. and Foeller, C. (1991), sequence of Proteins of immunological interest (Sequences of Proteins of immunological interest) 5 th edition, National Institutes of Health, Bethesda, MD

Kochanek et al, EP 1230354

Locksley et al, Cell 104, 487-501, 2001

27.Melchers，Ann.Rheum.Dis 2003，62，25-27.

Moore et al, Science 285: 260-3

29, Moreau et al, Blood 103(8), 3148-

Naismith and Sprang, TIBS 23, 74-79, 1998

Novak et al, Blood 103(2), 689-

Rixon et al, WO 02/094852

33.Pearson，Methods Enzymol.1990；183：63-98

Porth, J.Carlsson, I.Olsson and G.Belfrage, Nature (London)258, 598-

Porath and B.Olin, Biochemistry 22, 1621-

Puren et al, Proc Natl Acad Sci U S A.1999Mar 2; 96(5): 2256-61

37.Shepard，J.of Chromatography 891：93-98(2000)

38, Shields RL et al, 2001. high resolution mapping of the Fc γ RI, Fc γ II, Fc γ RIII and FcRn binding site of human IgG1 and design of the IgG1 variant with enhanced binding to Fc γ R (high resolution mapping of the binding site on human IgG1 for Fc gamma RI, Fcgamma RII, Fc gamma RIII, and FcRn and design of IgG1 variants with modified binding to the Fc γ R. J Biol chem.276 (9): 6591-604.

Smith et al, WO91/03553

Smith et al, WO94/06476

41.Stanker，J.Immunological Methods 76：157-169(1985)(10mM to30mM sodium phosphate elusion gradient)

Steurer W.1995 coating Ex vivo islet cell allografts with mouse CTLA4/Fc promotes graft tolerance (Ex vivo coating of islet cell allografts with murineC TLA4/Fc proteins graft tolerance) J Immunol.155 (3): 1165-74

43 Sun et al, WO 2005/044856

Takeda et al, EP 1561756

45.Tarditi，J.Chromatography 599：13-20(1992)

Engineered immunoglobulin Fc regions modulate antibody levels in vivo (Engineering the Fc region of immunoglobulin G to modulatein vivo) Nat biotechnol.23 (10): 1283-8

Vigers et al, Nature.1997Mar 13; 386(6621): 190-4

Vedantham et al, WO 03/59935

Vola et al, BioTechniques 14: 650-655(1993)

50 von Bulow and Bram, Science 228: 138(1997)

Xia et al, J.Exp.Med.2000, 137- & 143.

Sequence listing

<110> Ales trade, Inc

<120> method for purifying Fc-fusion protein

<130>1143WO/PCT

<150>EP06119610.1

<151>2006-08-28

<150>US60/842，682

<151>2006-09-06

<160>7

<170>PatentIn version 3.3

<210>1

<211>40

<212>PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/annotation ═ artificial sequence descriptions: consensus sequences in which particular amino acids at positions 3, 12, 14, 15, 18, 21, 25, 34 and 38 are separated by any amino acid

<220>

<221> variants

<222>(1)..(2)，(4)..(11)，13，(16)..(17)，

(19) .. (20), (22) (24), (26) (33), (35) (37) and (39) (40)

<223< Xaa can be any naturally occurring amino acid

<400>1

Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Xaa Leu Leu Xaa

1 5 10 15

Xaa Cys Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa

20 25 30

Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa

35 40

<210>2

<211>293

<212>PRT

<213> Intelligent people

<400>2

Met Ser Gly Leu Gly Arg Ser Arg Arg Gly Gly Arg Ser Arg Val Asp

1 5 10 15

Gln Glu Glu Arg Phe Pro Gln Gly Leu Trp Thr Gly Val Ala Met Arg

20 25 30

Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu Gly Thr Cys Met

35 40 45

Ser Cys Lys Thr Ile Cys Asn His GlnSer Gln Arg Thr Cys Ala Ala

50 55 60

Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp

65 70 75 80

His Leu Leu Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln His

85 90 95

Pro Lys Gln Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg Ser Pro Val

100 105 110

Asn Leu Pro Pro Glu Leu Arg Arg Gln Arg Ser Gly Glu Val Glu Asn

115 120 125

Asn Ser Asp Asn Ser Gly Arg Tyr Gln Gly Leu Glu His Arg Gly Ser

130 135 140

Glu Ala Ser Pro Ala Leu Pro Gly Leu Lys Leu Ser Ala Asp Gln Val

145 150 155 160

Ala Leu Val Tyr Ser Thr Leu Gly Leu Cys Leu Cys Ala Val Leu Cys

165 170 175

Cys Phe Leu Val Ala Val Ala Cys Phe Leu Lys Lys Arg Gly Asp Pro

180 185 190

Cys Ser Cys Gln Pro Arg Ser Arg Pro Arg Gln Ser Pro Ala Lys Ser

195 200 205

Ser Gln Asp His Ala Met Glu Ala Gly Ser Pro Val Ser Thr Ser Pro

210 215 220

Glu Pro Val Glu Thr Cys Ser Phe Cys Phe Pro Glu Cys Arg Ala Pro

225 230 235 240

Thr Gln Glu Ser Ala Val Thr Pro Gly Thr Pro Asp Pro Thr Cys Ala

245 250 255

Gly Arg Trp Gly Cys His Thr Arg Thr Thr Val Leu Gln Pro Cys Pro

260 265 270

His Ile Pro Asp Ser Gly Leu Gly Ile Val Cys Val Pro Ala Gln Glu

275 280 285

Gly Gly Pro Gly Ala

290

<210>3

<211>251

<212>PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Note-description of the artificial sequence: this is the heavy chain portion of the human immunoglobulin

<400>3

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

Val Leu Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro

20 25 30

Cys Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro

35 40 45

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

50 55 60

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

65 70 75 80

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

85 90 95

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

100 105 110

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

115 120 125

Asn Lys Ala Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys

130 135 140

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

145 150 155 160

Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

165 170 175

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

180 185 190

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

195 200 205

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

210 215 220

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

225 230 235 240

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

245 250

<210>4

<211>348

<212>PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Note-description of the artificial sequence: this is a fusion protein sequence containing a human TACI receptor portion and a human immunoglobulin heavy chain sequence

<400>4

Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly

1 5 10 15

Ala Val Phe Val Ser Leu Ser Gln Glu Ile His Ala Glu Leu Arg Arg

20 25 30

Phe Arg Arg Ala Met Arg Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro

35 40 45

Leu Leu Gly Thr Cys Met Ser Cys Lys Thr Ile Cys Asn His Gln Ser

50 55 60

Gln Arg Thr Cys Ala Ala Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu

65 70 75 80

Gln Gly Lys Phe Tyr Asp His Leu Leu Arg Asp Cys Ile Ser Cys Ala

85 90 95

Ser Ile Cys Gly Gln His Pro Lys Gln Cys Ala Tyr Phe Cys Glu Asn

100 105 110

Lys Leu Arg Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro

115 120 125

Pro Cys Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe

130 135 140

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

145 150 155 160

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

165 170 175

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

180 185 190

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

195 200 205

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

210 215 220

Ser Asn Lys Ala Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

225 230 235 240

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

245 250 255

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

260 265 270

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

275 280 285

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

290 295 300

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

305 310 315 320

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

325 330 335

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

340 345

<210>5

<211>879

<212>DNA

<213> Intelligent people

<400>5

atgagtggcc tgggccggag caggcgaggt ggccggagcc gtgtggacca ggaggagcgc 60

tttccacagg gcctgtggac gggggtggct atgagatcct gccccgaaga gcagtactgg 120

gatcctctgc tgggtacctg catgtcctgc aaaaccattt gcaaccatca gagccagcgc 180

acctgtgcag ccttctgcag gtcactcagc tgccgcaagg agcaaggcaa gttctatgac 240

catctcctga gggactgcat cagctgtgcc tccatctgtg gacagcaccc taagcaatgt 300

gcatacttct gtgagaacaa gctcaggagc ccagtgaacc ttccaccaga gctcaggaga 360

cagcggagtg gagaagttga aaacaattca gacaactcgg gaaggtacca aggattggag 420

cacagaggct cagaagcaag tccagctctc ccggggctga agctgagtgc agatcaggtg 480

gccctggtct acagcacgct ggggctctgc ctgtgtgccg tcctctgctg cttcctggtg 540

gcggtggcct gcttcctcaa gaagaggggg gatccctgct cctgccagcc ccgctcaagg 600

ccccgtcaaa gtccggccaa gtcttcccag gatcacgcga tggaagccgg cagccctgtg 660

agcacatccc ccgagccagt ggagacctgc agcttctgct tccctgagtg cagggcgccc 720

acgcaggaga gcgcagtcac gcctgggacc cccgacccca cttgtgctgg aaggtggggg 780

tgccacacca ggaccacagt cctgcagcct tgcccacaca tcccagacag tggccttggc 840

attgtgtgtg tgcctgccca ggaggggggc ccaggtgca 879

<210>6

<211>753

<212>DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Note-description of the artificial sequence: encoding the amino acid sequence of SEQ ID NO: 3 DNA of the protein

<400>6

atgaagcacc tgtggttctt cctcctgctg gtggcggctc ccagatgggt cctgtccgag 60

cccaaatctt cagacaaaac tcacacatgc ccaccgtgcc cagcacctga agccgagggg 120

gcaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 180

cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 240

tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 300

aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 360

aaggagtaca agtgcaaggt ctccaacaaa gccctcccat cctccatcga gaaaaccatc 420

tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 480

gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 540

atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 600

gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 660

tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 720

acgcagaaga gcctctccct gtctccgggt aaa 753

<210>7

<211>1044

<212>DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Note-description of the artificial sequence: encoding the amino acid sequence of SEQ ID NO: 4 in the presence of a DNA fragment encoding a protein represented by the formula

<400>7

atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggcgc cgtcttcgtt 60

tcgctcagcc aggaaatcca tgccgagttg agacgcttcc gtagagctat gagatcctgc 120

cccgaagagc agtactggga tcctctgctg ggtacctgca tgtcctgcaa aaccatttgc 180

aaccatcaga gccagcgcac ctgtgcagcc ttctgcaggt cactcagctg ccgcaaggag 240

caaggcaagt tctatgacca tctcctgagg gactgcatca gctgtgcctc catctgtgga 300

cagcacccta agcaatgtgc atacttctgt gagaacaagc tcaggagcga gcccaaatct 360

tcagacaaaa ctcacacatg cccaccgtgc ccagcacctg aagccgaggg ggcaccgtca 420

gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 480

acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 540

gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 600

taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 660

aagtgcaagg tctccaacaa agccctccca tcctccatcg agaaaaccat ctccaaagcc 720

aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc 780

aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 840

gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 900

tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag 960

gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 1020

agcctctccc tgtctccggg taaa 1044

Claims (28)

1.A method for purifying an Fc-fusion protein having an isoelectric point (pI) of 6.9 to 9.5, comprising the steps of:

a. subjecting the liquid containing the Fc-fusion protein to protein a or protein G affinity chromatography;

b. subjecting the eluate of step (a) to cation exchange chromatography;

c. subjecting the eluate of step (b) to anion exchange chromatography; and

d. and (c) carrying out hydroxyapatite chromatography on the effluent liquid of the step (c), and collecting the eluate to obtain the purified Fc-fusion protein.

2. The method of claim 1, wherein the elution of step (a) is performed at a pH in the range of 2.8-4.5.

3. The method of any preceding claim, wherein step (b) further comprises:

b1. washing the cation exchange resin with a buffer solution with pH6-7 and conductivity of 6-10 mS/cm; and

b2. eluting the column with buffer solution with pH7.3-8.2 and conductivity 15-22 mS/cm.

4. The method of any preceding claim, wherein the equilibration and loading of step (c) is performed with a buffer having a conductivity of 3-4.6mS/cm and a pH of less than one unit of the pI value of the Fc-fusion protein.

5. The process according to any of the preceding claims, wherein the elution of step (d) is carried out in the presence of sodium phosphate at a concentration of 3-10 mM.

6. The process according to any of the preceding claims, wherein the elution of step (d) is carried out in the presence of potassium chloride at a concentration of 0.4-1M.

7. The process according to any of the preceding claims, wherein the elution of step (d) is carried out at a pH of 6-7.

8. The method according to any of the preceding claims, wherein step (a) is performed on a resin containing cross-linked agarose modified with recombinant protein a or protein G.

9. The process of any preceding claim, wherein step (b) is carried out on a strong cation exchange resin.

10. The method of claim 9, wherein the resin comprises SO₃ ^-Group-modified crosslinked isobutyrate resins.

11. The process of any preceding claim, wherein step (c) is carried out on a strong anion exchange resin.

12. The method of claim 11, wherein the resin comprises N⁺(CH₃)₃A modified polystyrene/divinylbenzene resin.

13. The method according to any of the preceding claims, wherein step (d) is performed on a ceramic hydroxyapatite resin.

14. The method of claim 13, wherein the ceramic hydroxyapatite resin comprises particles having a particle size of 40 microns.

15. The method of any preceding claim, further comprising at least one ultrafiltration step.

16. The method of claim 15, wherein the ultrafiltration step is performed between steps (b) and (c) and/or after step (d).

17. The method of any preceding claim, further comprising formulating the Fc-fusion protein into a pharmaceutical composition.

18. The method of any preceding claim, wherein the Fc-fusion protein has an isoelectric point (pI) between 8-9.

19. The method of claim 18, wherein the Fc-fusion protein has an isoelectric point (pI) between 8.3-8.6.

20. The method of any preceding claim, wherein the Fc-fusion protein comprises a ligand binding portion of a member of the Tumor Necrosis Factor Receptor (TNFR) superfamily.

21. The method of claim 20, wherein the ligand binding moiety is selected from the group consisting of TNFR1, the extracellular domain of TNFR2, or a TNF binding fragment thereof.

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

23. The method of claim 22, wherein the Fc-fusion protein comprises a polypeptide selected from the group consisting of:

(a) the amino acid sequence of SEQ ID NO: 2 amino acids 34-66;

(b) the amino acid sequence of SEQ ID NO: 2 amino acids 71-104;

(c) the amino acid sequence of SEQ ID NO: 2 amino acids 34-104;

(d) the amino acid sequence of SEQ ID NO: 2 amino acids 30-110;

(e).SEQ ID NO：3：

(f).SEQ ID NO：4：

(g) a nucleic acid sequence capable of hybridizing to SEQ ID NO: 5 or 6 or 7;

(h) a mutein of any one of (c), (d), (e) or (f) having at least 80% or 85% or 90% or 95% sequence identity to a polypeptide of (c), (d), (e) or (f);

wherein the polypeptide binds to at least one of Blys or APRIL.

24. The method of any preceding claim, wherein the Fc-fusion protein comprises a heavy chain constant region of an immunoglobulin.

25. The method of claim 24, wherein the constant region is a constant region of a human immunoglobulin.

26. The method of claim 24 or 25, wherein the immunoglobulin is IgG₁。

27. The method of any one of claims 23-25, wherein the constant region comprises a hinge region, a CH2, and a CH3 region.

28. A purified Fc-fusion protein composition obtained by the method of any one of the preceding claims, wherein said Fc-fusion protein comprises a polypeptide selected from the group consisting of:

(a) the amino acid sequence of SEQ ID NO: 2 amino acids 34-66;

(b) the amino acid sequence of SEQ ID NO: 2 amino acids 71-104;

(c) the amino acid sequence of SEQ ID NO: 2 amino acids 34-104;

(d) the amino acid sequence of SEQ ID NO: 2 amino acids 30-110;

(e).SEQ ID NO：3：

(f).SEQ ID NO：4：

(g) a nucleic acid sequence capable of hybridizing to SEQ ID NO: 5 or 6 or 7;

(h) a mutein of any one of (c), (d), (e) or (f) having at least 80% or 85% or 90% or 95% sequence identity to a polypeptide of (c), (d), (e) or (f);

wherein the polypeptide binds to at least one of Blys or APRIL, and

wherein the composition contains less than 1% or less than 0.5% protein aggregates, and

wherein the composition contains less than 1% or less than 0.5% or less than 0.1% free Fc protein.