BRPI0716382B1 - method for reducing the content of free fc portions in a fluid comprising an fc-containing protein, and use of cation exchange chromatography - Google Patents

Abstract

A method for reducing the content of free fc portions in a fluid comprising a protein containing fc, and using cation exchange chromatography the present invention relates to a process for reducing the concentration of free fc portions in a fluid comprising a protein containing fc. fc comprising a cation exchange chromatography step.

Description

FIELD OF THE INVENTION

The present invention relates to the field of protein purification. More specifically, it refers to the purification of a protein containing Fc by means of cation exchange chromatography, in particular to reduce the amount of proteins without Fc portions in a protein preparation containing Fc.

BACKGROUND OF THE INVENTION

Proteins have become commercially important as drugs that are generally called biological. One of the biggest challenges is the development of processes that are cost-effective for protein purification on a commercial scale. Although many methods are available today for the production of proteins on a large scale, crude products, such as cell culture supernatants, contain not only the desired product, but also impurities, which are difficult to separate from the desired product. Although cell culture supernatants from cells expressing recombinant protein products may contain less impurities if cells are grown in serum-free medium, host cell proteins (HCPs) still remain, being eliminated during the purification process. In addition, health authorities request high standards of protein purity for human administration.

It is known that numerous chromatographic systems are widely used for protein purification.

Ion exchange chromatography systems are used to separate proteins mainly according to differences in charge.

Anionic exchangers can be classified as weak or strong. The charge group of a weak anion exchanger is a weak base, which becomes deprotonated and then loses its charge at a high pH. DEAE-sepharose is an example of a weak anion exchanger, where the amino group may be positively charged below pH ~ 9 and gradually loses

Petition 870180167443, of 12/26/2018, p. 12/21 your load at higher pH values. Diethylaminoethyl (DEAE) or diethyl- (2-hydroxy propyl) aminoethyl (QAE) has chloride as a counterion, for example. A strong anion exchanger, on the other hand, contains a strong base, which remains positively charged in all pH ranges normally used in ion exchange chromatography (pH 1 to 14). Q-sepharose (Q stands for quaternary ammonium) is an example of a strong anion exchanger.

Cationic exchangers can also be classified as weak or strong. A strong cation exchanger contains a strong acid (such as a sulfopropyl group) that remains charged from pH 1 to 14; while a weak cation exchanger contains a weak acid (such as a carboxymethyl group), which gradually loses its charge according to pH reductions below 4 or 5. Carboxymethyl (CM) and sulfopropyl (SP) have sodium as a counter -ion, for example.

A different chromatography resin is based on an insoluble hydroxylated calcium phosphate matrix called hydroxyapatite. Chromatography on hydroxyapatite is a protein purification method that uses an insoluble hydroxylated calcium phosphate (Ca ₅ (PO4) ₃ OH) 2, which forms the matrix and the ligand. Functional groups consist of pairs of positively charged calcium ions (C sites) and clusters of negatively charged phosphate groups (P sites). The interactions between hydroxyapatite and proteins are complex and multimodal. In an interaction method, positively charged amino groups on proteins associated with negatively charged P sites and protein carboxyl groups interact by complexing by coordinating C sites (Shepard et al., 2000).

Crystalline hydroxyapatite was the first type of hydroxyapatite used in chromatography. Chromatography in Hydroxyapatite Ceramics (CHA) is an additional development in chromatography in hydroxyapatite. Ceramic hydroxyapatite has high durability, good protein binding capacity, and can be used at higher flow rates and pressures than crystalline hydroxyapatite. (Vola et al., 1993).

Hydroxyapatite has been used in the chromatographic separation of proteins, nucleic acids, as well as antibodies. In hydro droxyapatite chromatography, the column is normally balanced, and the sample applied, at a low concentration of phosphate buffer and the adsorbed proteins are then eluted in a phosphate buffer concentration gradient (Giovannini et al., 2000).

In addition, an additional form of protein purification is based on the affinity of one protein of interest to another protein that is immobilized on a chromatography resin. Examples of such immobilized ligands are the proteins of the bacterial cell wall Protein A and Protein G, having specificity to the Fc portion of certain immunoglobulins. Although both Protein A and Protein G have a strong affinity for IgG antibodies, they have varying affinities to other immunoglobulin classes and isotypes as well.

Protein A is a 43,000 Dalton protein that is produced by Staphylcoccus aureus bacteria and contains four IgG Fc binding sites. Protein G is produced from the group Streptococci G and has two binding sites to the Fc region of IgG. Both proteins have been largely characterized by their affinity for various types of immunoglobulins. Another development is Protein A / G, a genetically engineered protein that combines the binding capabilities of Protein A and G. Protein L is an additional bacterial protein, which originates from Peptostreptococcus, binding to Immunoglobulins and fragments of them containing Ig light chains (Akerstrom and Bjork, 1989).

Protein A, Protein G, and Protein L affinity chromatographies are widely used for antibody isolation and purification.

Since the binding sites for Protein A and Protein G are present in the Fc region of an immunoglobulin, affinity chromatography with Protein A and Protein G (or Protein A / G), in addition, allows the purification of so-called fusion proteins Fc. Protein L binds to Ig light chains and can then be used for the purification of antibodies containing light chains.

Antibodies, or immunoglobulins (Igs) consist of light chains and heavy chains linked together by disulfide bonds. The first domain located at the terminal amino of each chain is variable in the amino acid sequence, providing the huge spectrum of antibody binding specificity. These domains are known as heavy variable (VP) and light variable (VL) regions. The other domains in each chain are relatively invariant in the amino acid sequence and are known as heavy constant (CP) and light constant (CL) regions.

The main classes of antibodies are IgA, IgD, IgE, IgG and IgM; and these classes can furthermore be divided into subclasses (isotypes). For example, the IgG class has four subclasses, that is, IgGi, lgG2, IgGa, and lgG ₄ .

The differences between the antibody classes are derived from differences in the heavy chain constant regions, containing between 1 and 4 constant domains (CH1 to CH4), depending on the immunoglobulin class. A region called a hinge is located between the CH1 and CH2 domains. The hinge region is especially sensitive to proteolytic divage; such proteolysis produces two or three fragments depending on the exact dividing site. The part of the heavy chain constant region that contains the CH2 and CH3 domains is also called the Fc part of the immunoglobulin. Antibodies are thus proteins containing Fc. Another type of proteins containing Fc are called Fc fusion proteins.

Several antibodies that are used as therapeutic proteins are known. Examples of recombinant antibodies on the market are, for example: Abciximab, Rituximab, Basiliximab, Daclizumab, Palivizumab, Infliximab, Trastuzumab, Alemtuzumab, Adalimumab, Cetuximab, Efalizumab, Ibritumomabe, Bevacizumabe or Omalizumabe or Omalizumabe.

Fc fusion proteins are chimeric proteins composed of the effector region of a protein, such as the Fab region of an antibody or the receptor binding region, fused to the Fc region of an immunoglobulin, which is often an immunoglobulin G (IgG). Fc fusion proteins are widely used as therapeutics, since they offer advantages conferred by the Fc region, such as:

- The possibility of purification using affinity chromatography with protein A or protein G with affinities that vary according to the IgG isotype. Human IgGi, IgG2 and IgG4 are strongly bound to Protein A and all human IgGs, including IgG ₃ , are strongly bound to Protein G;

- An increased half-life in the circulatory system, since the Fc region binds to the circulating FcRn receptor that protects from lysosomal degradation;

- Depending on the medical use of the Fc fusion protein, effector functions Fc may be desirable. Such effector functions include antibody-dependent cell cytotoxicity (ADCC) through interactions with Fc receptors (FcyRs) and complement-dependent cytotoxicity (CDC) by binding to complement component 1q (C1q). IgG isoforms exert different levels of effector functions. Human IgGi and IgG ₃ have strong ADCC and CDC effects, while human IgG ₂ has weak ADCC and CDC effects. Human IgG ₄ demonstrates weak ADCC effects and no CDC.

The serum half-life and effector functions can be modulated by engineering in the Fc region to increase or decrease its binding to FcRn, FcyRs and C1q respectively, depending on the therapeutic use for the Fc fusion protein.

In ADCC, the Fc region of an antibody binds to Fc receptors (FcyRs) on the surface of immune effector cells, such as natural killers and macrophages, leading to phagocytosis or lysis of the target cells.

In CDC, antibodies kill target cells by activating the complement cascade on the cell surface. IgG isoforms exert different levels of effector functions that increase in the order of lgG ₄ <lgG2 <lgG-i <lgG ₃ . Human IgGi shows high ADCC and CDC, and is best suited for therapeutic use against pathogens and cancer cells.

Under certain circumstances, for example, when depletion of the target cell is undesirable, abolition or decreased effector functions may be necessary. On the contrary, in the case of antibodies intended for cancer use, the increase in effector functions can improve its therapeutic activity (Carter et al., 2006).

The modification of effector functions can be carried out by engineering in the Fc region to improve or reduce the binding of FcyRs or complement factors.

IgG binding to activation (FcyRI, FcyRlla, FcyRllla and FcyRlllb) and inhibitory (FcyRllb) or to the first complement component (C1q) depends on residues located in the hinge region and in the CH2 domain. Two regions of the CH2 domain are critical for the binding of FcyRs and complement C1 q, and have unique sequences in lgG ₂ and lgG ₄ . For example, substitution of IgG2 residues at positions 233 to 236 in human IgGi greatly reduces ADCC and CDC (Armor et al., 1999 and Shields et al., 2001).

Numerous mutations have been produced in the Ig2 CH2 domain and their effect on ADCC and CDC has been tested in vitro (Shields et al., 2001, Idusogie et al., 2001 and 2000, Steurer et al., 1995). In particular, a mutation in alanine at E333 for an increase in ADCC and CDC has been reported (Idusogie et al., 2001 and 2000).

Increasing the serum half-life of a therapeutic antibody is another way to improve its effectiveness, allowing for higher circulating levels, less frequent administration and reduced doses. This can be achieved by increasing the connection of the Fc region to the neonatal FcR (FcRn). FcRn, which is expressed on the surface of endothelial cells, binds IgG in a pH-dependent manner and protects them from degradation. Several mutations located at the interface between the CH2 and CH3 domains have been shown to increase IgGi half-life (Hinton et al., 2004 and Vaccaro et al., 2005).

Table 1 summarizes some known mutations in the IgG Fc region (extracted from the Invitrogen website).

applications Vaccination; therapeutic use Vaccination; therapeutic use Therapeutic use without cell depletion Therapeutic use with cell depletion Vaccination; therapeutic use Potential Benefits Improved target location; increased effectiveness; reduced dose or frequency of administration Improved target location; increased effectiveness; reduced dose or frequency of administration Reduced adverse events Increased effectiveness Reduced adverse events properties Increased plasma half-life Increased plasma half-life Reduced ADCC and CDC ADCC and CDC increased of Reduced CDC Mutations T250Q / M428L M252Y / S254T / T256E + H433K / N434F E233P / L234V / L235A / AG236 + A327G / A330S / P331S E333A K322A IgG isotype human lgG1 human lgG1 human lgG1 human lgG1 human lgG2 Projected FC hlgG1e1 CXI φ vO _O) -C hlgG1e3 hlgG1e4 T— Φ Csl O σ>

A class of Fc fusion proteins that has therapeutic utility, the Fc regions have been fused to extracellular domains of certain receptors forming part of the tumor necrosis factor receptor (TNF-R) superfamily (Locksley et al., 2001, Bodmer et al., 2002, Bossen et al., 2006). A characteristic of the members of the TNFR family is the presence of pseudo-repetitions rich in cysteine in the extracellular domain, as described, for example, by Naismith and Sprang, 1998.

Two TNF receptors, p55 (TNFR1) and TNFR p75 (TNFR2) are examples of such members of the TNFR superfamily. Etanercept is an Fc fusion protein containing the soluble part of TNFR p75 (for example, WO91 / 03553, WO 94/06476). Under the brand name Enbrel®, it is sold 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 is a fusion protein that contains the extracellular components of the human p55 TNF receptor and the Fc portion of human IgG, and is intended for the potential treatment of severe sepsis and multiple sclerosis.

0X40 is also a member of the TNFR superfamily. OX40-lgG1 and OX40-hlG4mut fusion proteins have been prepared for the treatment of inflammatory and autoimmune diseases, such as Crohn's disease.

A BAFF-R Fc fusion protein, also called BR3, called BR3-Fc, is a soluble attraction receptor for a series of BAFF inhibitors (TNF family B cell activation factor), is being developed for the potential treatment of autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE).

BCMA is an additional receptor belonging to the TNFR superfamily. A BCMA-lg fusion protein has been described for inhibiting autoimmune disease (Melchers, 2006).

Another receptor of the TNF-R superfamily is TACI, the transmembrane activator and interacting CAML (von Bulow and Bram, 1997; US 5,969,102, Gross et al., 2000), which has an extracellular domain containing two cysteine-rich pseudo-repetitions. TACI binds to two members of the tumor necrosis factor (TNF) ligand family. The first linker is called BLyS, BAFF, neutrocin-α, TALL-1, zTNF4, or THANK (Moore et al., 1999). The other linker was designated as APRIL, TNRF-1 or ZTNF2 death linker (Hahne et al., J. Exp. Med. 188: 1185 (1998).

Fusion proteins containing soluble forms of the TACI receptor fused to an IgG Fc region are also known and have been 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 for the treatment of multiple sclerosis (MS). In addition, TACI-Fc is being developed in multiple myeloma (MM) (Novak et al., 2004; Moreau et al., 2004) and non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL) and Waldenstrom's macroglobulemia ( WM).

Considering the therapeutic utility of the Fc-containing protein, in particular antibodies and Fc fusion proteins, there is a need for significant amounts of the highly purified protein, which is suitable for human administration.

SUMMARY OF THE INVENTION

One of the problems that can be encountered during the production of the Fc-containing protein is the presence of free Fc moieties, that is, fragments of polypeptides derived from the Fc-containing protein, which do not contain a substantial moiety derived from a variable region of the antibody or another specific protein or domain normally present in the Fc fusion protein.

The present invention addresses this problem. It is based on the development of a fluid purification process, composition or preparation of a protein containing Fc, by which the amount of free Fc portions that can be present as an impurity can be reduced.

Accordingly, the invention relates to a method for reducing the concentration of free Fc portions in a fluid comprising a protein containing Fc, the method comprising subjecting said fluid to cation exchange chromatography.

In a second aspect, the invention relates to the use of cation exchange chromatography to reduce free Fc in a protein preparation containing Fc.

In a third aspect, the invention relates to a purified protein containing Fc, comprising less than 5% or less than 2% or less than 1% or less than 0.5% or less than 0.2% or less than 0 , 1% of free Fc portions.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 - shows a non-reduced silver-colored SDS-PAGE of different fractions resulting from the cation exchange chromatography described in Example 2. Column 1: molecular weight markers, Column 2: purified TACI-Fc, column 3: load, Column 4: wash 2, Column 5: elute 2, Column 6: wash 3, Column 7: elute 3, Column 8: wash 1, Column 9: elute 1, Column 10: purified free Fc;

Figure 2 - shows the chromatographic profile of the cation exchange chromatography described in Example 2.

BRIEF DESCRIPTION OF THE SEQUENCE LIST

SEQ ID NO: 1 is a cysteine fingerprint sequence (cysteine-rich pseudo-repetition) common to members of the TNFR superfamily;

SEQ ID NO: 2 is the entire human TACI receptor sequence (for example, described in WO 98/39361);

SEQ ID NO: 3 is an example of a human Fc sequence of the invention (for example, described in WO 02/094852);

SEQ ID NO: 4 is a preferred Fc fusion protein of the invention, comprising sequences derived from the extracellular portion of TACI and an Fc portion of human IgG1 (for example, described in WO 02/094852);

SEQ ID NO: 5 is a polynucleotide encoding a polypeptide of SEQ ID NO: 2;

SEQ ID NO: 6 is a polynucleotide encoding a polypeptide of SEQ ID NO: 3;

SEQ ID NO: 7 is a polynucleotide encoding a polypeptide of SEQ ID NO: 4.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the finding that cation exchange chromatography can reduce the amount or size of free Fc portions that may be present in a fluid or protein composition containing Fc.

The invention, therefore, relates to a method for reducing the concentration of free Fc portions in a fluid comprising an Fc-containing protein, the method comprising subjecting said fluid to cation exchange chromatography.

The fluid comprising the Fc-containing protein can be any composition or preparation, such as, for example, a body fluid derived from a human or animal, or a fluid derived from a cell culture, such as, for example, a culture supernatant cell phone. It can also be a fluid derived from another purification step, such as, for example, the eluate or continuous flow - through a capture step or any other suitable purification step, such as those explained in more detail below.

The term Fc-containing protein, as used in this application, refers to any protein that has at least one immunoglobulin constant domain selected from the CH1 domain, hinge, CH2, CH3, CH4, or any combination thereof, and preferably a domain of hinge, CH2 and CH3. The immunoglobulin constant domain can be derived from any of the IgG, IgA, IgE, IgM, or combination or isotypes thereof. Preferably, it is IgG, such as, for example, IgG-ι, IgG2, IgGa or IgG4. More preferably, it is IgG-i.

An Fc-containing protein according to the present invention can thus be, for example, an antibody or a fusion protein

Fc, or variants thereof, such as fragments, muteins or functional derivatives of antibodies or Fc fusion proteins.

The Fc-containing protein of the invention can be a monomer, dimer or multimer. The Fc-containing protein can also be a pseudodimer (sometimes called a monomer), containing a dimeric Fc portion (for example, a dimer of two CH2CH3 hinge constructs with disulfide bridge), of which only one is fused to a portion additional, such as an immunoglobulin variable domain, a coordinating compound and optionally inhibiting a receptor fragment, or any other protein. An example of such a pseudodimer is an Fc fusion protein that has interferon-β fused to one of the two IgG-CH2-CH3 hinge constructs such as, for example, that described in WO 2005/001025.

The Fc-containing protein can also be a heterodimer, containing two different non-immunoglobulin moieties or immunoglobulin variable domains, or a homodimer, containing two copies of a single non-immunoglobulin moiety or immunoglobulin variable domain.

Preferably, the Fc-containing protein is a dimer. It is also preferred that the Fc-containing protein of the invention is a homodimer.

According to the present invention, the Fc portion of the Fc-containing protein can also be modified in order to modulate effector functions.

For example, the following Fc mutations, according to EU index positions (Kabat et al., 1991), can be introduced if the Fc portion is derived from IgG- ,:

T250Q / M428L

M252Y / S254T / T256E + H433K / N434F

E233P / L234V / L235A / AG236 + A327G / A330S / P331S E333A; K322A.

Additional Fc mutations can be, for example, substitutions at selected EU index positions of 330, 331, 234, or 235, or combinations thereof. An amino acid substitution at EU index 297 located in the CH2 domain can also be introduced into the Fc portion in the context of the present invention, eliminating a potential N-linked carbohydrate binding site. In addition, the cysteine residue at position EU index 220 can also be replaced with a serine residue, eliminating the cysteine residue that normally forms disulfide bonds with the immunoglobulin light chain constant region.

According to the present invention, it is preferred that the Fc portion comprises or consists of SEQ ID NO: 3 or is encoded by a polynucleotide comprising or consisting of SEQ ID NO: 6.

In a preferred embodiment, the Fc-containing protein comprising an immunoglobulin variable region, for example, one or more heavy chain variable domains and / or one or more light chain variable domains. Preferably, the antibody contains one or two heavy chain variable domains. More preferably, the antibody additionally contains one or two constant and / or variable light chain domains.

It is preferred that the Fc-containing protein is an antibody.

The term antibody refers to an immunoglobulin or fragment thereof, and includes any polypeptide comprising an antigen binding site. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, polyspecific, non-specific, humanized, human, chimeric, single-chain, synthetic, recombinant, hybrid, mutated, grafted or created in vitro antibodies. The antibody can be selected from any of the known antibody isotypes, for example, IgA, IgG, IgD, IgE, IgM. The antibody can be a monomer, dimer, or multimer, such as a trimer or pentamer.

Examples of antibodies that can be purified in accordance with the present invention are Abciximab, Rituximab, Basiliximab, Daclizumab, Palivizumab, Infliximab, Trastuzumab, Alemtuzumab, Adalimumab, Cetuximab, Efalizumab, Ibritumomabe, Bevacizumabe or Omalizumab. Additional examples of antibodies that can be subjected to cation exchange chromatography in accordance with the present invention are antibodies directed against:

CD2, CD3, CD4, CD8, CD11a, CD11b, CD14, CD18, CD20, CD22, CD23, CD25, CD33, CD40, CD44, CD52, CD80, CD86, CD147, CD164, IL-2 receptor, IL-receptor 4, IL-6 receptor, IL12 receptor, IL-18 receptor subunits (IL-18R-alpha, IL-18R-beta), TACI, BCMA, BAFF-R, EGF receptor, VEGF receptor, integrin α4β7, VLA4 integrin, B2 integrins, TRAIL 1, 2, 3, and 4 receptors, RANK, RANK ligand, epithelial cell adhesion molecule (EpCAM), intercellular adhesion molecule 3 (ICAM-3), CTLA4, receptor Fc-gamma-l, II or III, HLA-DR 10 beta, HLA-DR antigen or L-selectin.

Antibodies directed against TNF, Blys, or interferon-γ are additional examples of therapeutically interesting antibodies.

Fc fusion proteins are also Fc-containing proteins that are preferably subjected to the method of the invention.

The term Fc fusion protein, as used in this application, is intended to encompass proteins, in particular therapeutic proteins, comprising a portion derived from immunoglobulin, which will be referred to in this application as an Fc portion, and a portion derived from a second non-protein immunoglobulin that will be called in this application a therapeutic portion, regardless of whether or not it is intended for the treatment of disease.

Therapeutic Fc fusion proteins, that is, Fc fusion proteins intended for the treatment or prevention of disease in an animal or preferably for human treatment or administration, are especially suitable for purification according to the invention.

Any Fc fusion protein can be purified according to the present invention, such as, for example, an interferon-β containing fusion protein. Preferably, the method of the invention is for purification of an Fc fusion protein comprising a ligand-binding fragment, such as all or part of an extracellular domain, of a member of the tumor necrosis factor receptor (TNFR) superfamily.

The therapeutic portion of an Fc fusion protein can, for example, be or be derived from EPO, TPO, Growth Hormone, Interferon-alpha, Interferon-beta, Interferon-gamma, PDGF-beta, VEGF, IL-1alpha, IL1beta, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12, IL-18, IL-18 binding protein, TGF-beta, TNF-alpha or TNF-beta.

The therapeutic portion of an Fc fusion protein can also be derived from a receptor, for example, a transmembrane receptor, preferably being or being derived from the extracellular domain of a receptor, and in particular, a ligand-binding fragment of the part or extracellular domain of a given receptor. Examples of therapeutically interesting receptors are CD2, CD3, CD4, CD8, CD11a, CD11b, CD14, CD18, CD20, CD22, CD23, CD25, CD33, CD40, CD44, CD52, CD80, CD86, CD147, CD164, IL-receptor 2, IL-4 receptor, IL-6 receptor, IL-12 receptor, IL-18 receptor subunits (IL-18R-alpha, IL-18R-beta), EGF receptor, VEGF receptor, integrin alpha 4 10 beta 7, integrin VLA4, integrins B2, TRAIL 1, 2, 3, and 4 receptors, RANK, RANK ligand, epithelial cell adhesion molecule (EpCAM), intercellular adhesion molecule 3 (ICAM-3) , CTLA4 (which is a cytotoxic T-cell-associated antigen), Fc-gamma-1, II or III receptor, HLA-DR 10 beta, HLA-DR antigen, L-selectin.

It is highly preferred that the therapeutic portion is derived from a receptor belonging to the TNFR superfamily. The therapeutic portion can, for example, be or be derived from the extracellular domain of TNFR1 (p55), TNFR2 (p75), 0X40, Osteoprotegerin, CD27, CD30, CD40, RANK, DR3, Fas ligand, TRAIL-R1, TRAIL- R2, TRAIL-R3, TAIL-R4, NGFR, AlTR, BAFFR, BCMA, TACL

According to the present invention, the therapeutic portion derived from a member of the TNFR superfamily preferably comprises or consists of all or part of the extracellular domain of the TNFR member, and more preferably comprises a ligand-binding fragment of such a TNFR member.

The following table 5 lists members of the TNFR superfamily from which a therapeutic moiety according to the present invention can be derived, and their respective ligands. A ligand-binding fragment of a member of the TNFR family can be easily determined by the person skilled in the art, for example, in a simple in vitro assay measuring the link between the protein fragment of a given receptor and the respective ligand. Such an assay can be, for example, a simple RIA or ELISA sandwich in vitro assay in which one of the proteins, for example, the receptor fragment, is immobilized in a carrier (for example, an ELISA plate) and is incubated, after the appropriate blocking of the protein binding sites in the carrier, with the second protein, for example, the ligand. After incubation, binding to the ligand is detected, for example, by radiolabeling the ligand and determining the bound radioactivity, after appropriate washing, in a scintillation counter. Binding to the linker can also be determined with a labeled antibody, or a primary antibody specific for the linker and a labeled secondary antibody, directed against the constant part of the primary antibody. The binding to the ligand can thus be easily determined, depending on the marker used, for example, in a color reaction.

Preferably, the method of the present invention is for purification of an Fc fusion protein comprising a therapeutic portion derived from a member of the TNFR superfamily selected from those listed in Table 5.

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

Member of the TNFR superfamily Binder NGFR NGF AND GIVE EDA-A1 XEDAR EDA-A2 CD40 CD40L

Member of the TNFR superfamily Binder Fas FasL 0x40 OX40L AITR AITRL GITR GITRL CD30 CD30L CD40 CD40L HveA LIGHT, LT-alpha 4-1 BB 4-1BBL TNFR2 TNF-alpha, LT-alpha, LT-alpha-beta LT-betaR LIGHT, LT-alpha, LT-alpha-beta DR3 TL1A CD27 CD27L TNFR1 TNF-alpha, LT-alpha, LT-alpha-beta LTBR LT-beta RANK RANKL TACI BlyS, APRIL BCMA BlyS, APRIL BAFF-R BAFF (= BlyS) TRAILR1 TRAIL

Member of the TNFR superfamily Binder 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 portion selected from an extracellular domain of TNFR1, TNFR2, or a TNF-binding fragment thereof.

In an additional preferred embodiment, the fusion protein

Fc comprises a therapeutic portion selected from an extracellular domain of BAFF-R, BCMA, or TACI, or a fragment thereof, binding to at least Blys or APRIL.

An assay to test the ability to bind to Blys or APRIL is described, for example, in Hymowitz et al., 2006.

Still in an additional preferred embodiment, the therapeutic portion of an Fc fusion protein comprises the cysteine-rich pseudo-repetition of SEQ ID NO: 1.

If it is more preferable, the therapeutic portion should be derived from TACI. TACI is preferably human TACI. SEQ ID NO: 2, which cor15 responds to the entire amino acid sequence of the TACI receptor (also entry 014836 in SwissProt). More preferably, the therapeutic portion comprises a soluble portion of TACI, preferably derived from the extracellular domain of TACI. Preferably, the TACI-derived therapeutic moiety comprises at least 33 to 67 amino acids of SEQ ID NO: 2 and / or 70 to 104 amino acids of SEQ ID NO: 2. In a preferred embodiment, the TACI extracellular domain included in the therapeutic moiety, according to the invention, comprises or consists of 1 to 166 amino acids of SEQ ID NO: 2 or 30 to 166 amino acids of SEQ ID NO: 2, or 30 to 119 amino acids of SEQ ID NO: 2, or 30 to 110 amino acids of SEQ ID NO: 2. All those therapeutic portions are preferred for the preparation of the Fc fusion protein to be purified using the method of the invention and are combined with the Fc portions described in detail above, and in particular with an Fc portion comprising or consisting of SEQ ID NO: 3. A highly preferred Fc fusion protein to be purified according to the present invention which comprises or consists of SEQ ID NO: 4 or is encoded by the polynucleotide of SEQ ID NO: 7.

Therefore, it is highly preferred that the Fc fusion protein comprises a polypeptide selected from

The. 34 to 66 amino acids of SEQ ID NO: 2;

B. 71 to 104 amino acids of SEQ ID NO: 2;

ç. 34 to 104 amino acids of SEQ ID NO: 2;

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

and. SEQ ID NO: 3;

f. SEQ ID NO: 4;

g. a polypeptide encoded by a polynucleotide hybridizing to the complement of SEQ ID NO: 5 or 6 or 7 under highly stringent conditions; and

H. a mutein of either (c), (d), (e), or (f) having at least 80% or 85% or 90% or 95% sequence identity to the polypeptide (c), (d), ( e) or (f);

wherein the polypeptide binds to at least Blys or APRIL.

According to the present invention, the Fc-containing protein is subjected to cation exchange chromatography in order to reduce, decrease, or eliminate free Fc portions, preferably at least 50, 40, 30, 20 or 10% of the total protein concentration , or more preferably less than 10%.

The term free Fc portions, free Fc portion, or simply free Fc, as used in this application, is intended to encompass any part of the Fc-containing protein to be purified according to the present invention, which is derived from the constant domain or domains immunoglobulin without understanding additional complete domains. Thus, if the Fc-containing protein comprises variable domains of immunoglobulin, free Fc does not contain significant portions of the variable domains. If the Fc-containing protein is an Fc fusion protein, free Fc does not contain significant portions of the therapeutic portion of the Fc fusion protein. Free Fc may contain, for example, dimers of the IgG hinge, CH2 and CH3 domains that are not linked or coupled to significant portions of a therapeutic portion or immunoglobulin variable domains, such as, for example, the Fc portion that is generated by papain divage. A significant portion may be, for example, not less than 80, 85, 90, 95, 98 or 99% of the entire variable domain or therapeutic portion present in the Fc-containing protein.

Monomers derived from the Fc portion may also be contained in the free Fc fraction. It is understood that free Fc may also contain a number of amino acid residues from the therapeutic portion or from the variable domains of Ig, such as, for example, one for fifty or one for twenty, or one for ten, or one for five amino acids, or one or two simple amino acids, belonging to the therapeutic portion or variable domain, still fused to the Fc portion.

Cation exchange chromatography can be performed on any suitable cation exchange resin, such as, for example, weak or strong cation exchangers as explained above in the Background to the Invention.

Preferably, cation exchange chromatography is performed on a strong cation exchange resin. More preferably, the cation exchange material comprises a cross-linked methacrylate modified with SO ₃ 'groups. A commercially available column under the name Fractogel EMD SO3 · (from Merck) is an example of a cation-exchange resin that is especially suitable in the context of the present method.

Preferably, the fluid or composition comprising the Fc-containing protein is loaded into a cation-exchange resin at a pH of at least one unit below the isoelectric point (p1) of said Fc fusion protein.

In a preferred embodiment, the cation exchange resin is washed with a buffer that has a conductivity of 6 to 10 mS / cm and a pH of 5.5 to 7.5.

The buffer can have, for example, a conductivity of 6,1,6,2,

6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1.9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, or 9.9 mS / cm.

More preferably, the conductivity ranges from 7.6 to 9.2, i.e., 8.4 + 0.8 mS / cm. The washing step is preferably carried out at a pH ranging from 5.5 to 7.5, preferably from 6.0 to 7.0.

The pH of the buffer can be, for example, 5.5, 5.6, 5.7, 5.8,

5.9, 6.0, 6.1.6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7, 1.7.2, 7.3, 7.4, or 7.5.

In an additional preferred embodiment, the washing step is carried out in a buffer comprising 60 to 140 sodium phosphate, preferably 70 to 130, more preferably 75 to 125 mM. The buffer may comprise sodium phosphate, for example, at 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135 or 140 mM.

In an additional preferred embodiment, the cation exchange column is eluted in a pH range of 7.0 to 8.5, preferably 7.25 or 7.3 or 7.35 or 7.4 or 7.45 or 7, 5 or 7.55 or 7.6 or 7.65 or 7.7 or 7.75 or 7.8 or 7.85 or 7.9 or 7.95 or 8.0 or 8.05 or 8.1 or 8.15 or 8.2 or 8.25 or 8.3 or 8.35 or 8.4 or 8.45 or 8.5.

Elution can preferably be carried out in a conductivity range of 15 to 22 mS / cm. For example, conductivity can be selected from 15, 16, 17, 18, 19, 20, 21 or 22 mS / cm.

A preferred elution buffer is a phosphate buffer.

According to the present invention, cation exchange chromatography can preferably be used for elimination or reduction of free Fc in the range of 5 to 15 times. Thus, a reduction in the concentration of free Fc in the Fc-containing protein comprising fluid, preparation or composition to less than 20% or less than 15% or less than 10% or less than 5% or less than 2% or less than 1% or less than 0.8% or less than 0.5% or less than 0.3% or less than 0.2% or less than 0.1% of the total protein concentration, can be achieved.

In a preferred embodiment, cation exchange chromatography can be used in a purification method that has one or more additional steps, preferably selected from affinity chromatography, anion exchange chromatography and hydroxyapatite chromatography.

In a highly preferred embodiment, the method of the invention is used as a second step in an Fc-containing protein purification scheme, comprising the following steps:

The. Submitting a fluid comprising said protein containing Fc to affinity chromatography with Protein A or Protein G or Protein L;

B. Submission of step eluent (a) to cation exchange chromatography;

ç. Submission of step eluent (b) to anion exchange chromatography;

d. Submission of the continuous flow from step (c) to hydroxyapatite chromatography and collection of the eluate to obtain the protein containing purified Fc.

According to the present invention, a fluid comprising an Fc-containing protein is first subjected to Protein A or Protein G or Protein L or Protein A / G affinity chromatography. The fluid may preferably be cell culture material, for example, solubilized cells, more preferably the cell culture supernatant. The term cell culture supernatant, as used in this application, refers to a medium in which cells are cultured and in which proteins are secreted as long as they contain appropriate cellular signals, called signal peptides. It is preferred that cells expressing the Fc-containing protein are cultured under serum-free culture conditions. Thus, preferably, the cell culture supernatant is devoid of components derived from animal serum. Most preferably, the cell culture medium is a chemically defined medium.

Protein A, G, A / G or L used for affinity chromatography can be, for example, recombinant. It can also be modified to improve its properties (such as, for example, in the resin called MabSelect SuRe, commercially available from GE Healthcare). In a preferred embodiment, step (a) is carried out on a resin comprising cross-linked agarose modified with recombinant Protein A. A commercially available column under the name Mabselect Xtra (from GE Healthcare) is an example of an affinity resin that is especially suitable for step (a) of the present method.

Protein A or G or L affinity chromatography is preferably used as a capture step, and thus serves to purify the Fc-containing protein, in particular, to eliminate host cell proteins and protein aggregates containing Fc, and for the concentration of the protein preparation containing Fc.

The term aggregates, as used in this application, is intended to refer to protein aggregates, and encompasses multimers (such as dimers, tetramers or higher-order aggregates) of the Fc-containing protein to be purified and may result in, for example, high molecular weight aggregates.

Affinity chromatography has the additional advantage of reducing aggregate levels 2 to 4 times.

When using Protein A or G or A / G or L affinity chromatography, the host cell's protein levels can be reduced from 100 to 300 times.

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

4.5 to 2.8.

In an additional preferred embodiment, the elution in step (a) is carried out in a buffer selected from sodium citrate or sodium acetate. Suitable buffer concentrations are, for example, selected from 50 mM or 100 mM or 150 mM or 200 mM or 250 mM.

In accordance with the present invention, the chromatography eluate with Protein A or Protein G or Protein A / G or Protein L is subjected to cation exchange chromatography, as explained in detail above.

Preferably, the elution from the cation exchange chromatography, that is, from step (b), is diluted or dialyzed in an appropriate loading buffer before loading it into the anion exchange column. The anion exchange column is also preferably balanced with the loading buffer.

A preferred pH of the loading buffer is one unit below the pH. Suitable pH values range from 6.0 to 8.5, preferably from 7.0 to 8.0, for example, 7.0, 7.05, 7.1, 7.15, 7.2, 7, 25, 7.3, 7.35,

7.4, 7.45, 7.5, 7.55, 7.6, 7.65, 7.7, 7.75, 7.8, 7.85, 7.9, 7.95 or 8, 0. A preferred conductivity of the loading buffer is in the range of 3.0 to 4.6 mS / cm.

An appropriate balance / loading buffer can be, for example, sodium phosphate in a concentration range of 5 to 35, preferably 20 to 30 mM. The concentration of the buffer can be, for example, 10, 15, 20, 25, 30 mM. Within the scope of the present invention, a continuous flow (called a rupture curve) of anion exchange chromatography, comprising the protein containing Fc of interest, is collected.

Step (c) of the method of the invention, furthermore, reduces aggregates 3 to 5 times and host cell proteins 30 to 70 times.

According to the present invention, the continuous flow of the anion exchange chromatography of step (c) is then used for further purification by hydroxyapatite chromatography. Any hydroxyapatite resin can be used to carry out step (d) of the method according to the invention. In a preferred embodiment, step (d) is carried out on a ceramic hydroxyapatite resin, such as a type I or type II hydroxyapatite resin. The hydroxyapatite resin can have particles of any size, such as 20, 40 or 80 pm. In a highly preferred embodiment, the ceramic hydroxyapatite resin comprises particles that are 40 µm in size. A hydroxyapatite resin that is especially suitable for step (d) of the present method is a commercially available column under the name CHT Hydroxyapatite Ceramic Type I, 40 pm.

In a preferred embodiment, the continuous flow of step (c) is directly loaded onto the hydroxyapatite resin, that is, without prior dilution or dialysis, in an appropriate loading buffer. The loading is preferably carried out at a pH of 6.5 to 7.5, such as 6.6, 6.7, 6.8,

6.9, 7.1, 7.2, 7.3, or 7.4, and preferably 7.0.

In an additional preferred embodiment, the elution in step (d) is carried out in the presence of sodium phosphate ranging from 2 to 10 mM, preferably in the 2.75 to 5.25 mM ranges, such as, for example, in 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5.

In yet another preferred modality, the elution in step (d) is carried out in a pH range of 6.0 to 7.0, for example, in 6.1, 6.2, 6.3,

6.4, 6.5, 6.6, 6.7, 6.8, 6.9.

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

According to the present invention, the hydroxyapatite chromatography elute is collected, containing the protein preparation containing finally purified Fc.

Suitable matrix materials, that is, carrier materials for the chromatographic resins used in steps (a) to (c), which can be used in connection with the present invention can be, for example, agarose (sepharose, superose), dextran ( sefadex), polypropylene, cellulose methacrylate, polystyrene / divinyl benzene, or similar. The resin materials can be present in different lattice forms, depending on the specific use.

The volume of the resin, the length and diameter of the column to be used, as well as the dynamic capacity and flow speed depend on several parameters, such as, volume of fluid to be treated, concentration of protein in the fluid to be subjected to the process of the invention, etc. The determination of these parameters at each stage is well known within the average skills of the person skilled in the art.

In a preferred embodiment of the present purification process, one or more ultrafiltration steps are performed. Ultrafiltration is useful for removing small organic molecules and salts in the eluate resulting from the previous chromatrographic steps, for balancing the Fc-containing protein by volume of buffer volume, or for concentrating the Fc-containing protein to the desired concentration. Such ultrafiltration can be, for example, performed on ultrafiltration membranes, with pores of sizes that allow the removal of components that have molecular weights less than 5, 10, 15, 20, 25, 30 or more kDa.

Preferably, ultrafiltration is carried out between steps (b) and (c), and / or after step (d). More preferably, two ultrafiltration steps are performed, one between steps (b) and (c) and one after step (d).

If the purified protein according to the process of the invention is intended for administration to humans, it is advantageous to include one or more virus removal steps in the process. Preferably, a filtering step for virus removal is performed after step (d). More preferably, the virus removal filtration step is a nanofiltration step where the filter has a nominal pore size of 20nm. The method of the present invention, and in particular steps (a), (c), (d) in combination with nanofiltration effectively eliminates the viral load at a combined LRV (log reduction value) of up to approximately 15 to 25.

In order to facilitate storage or transport, for example, the material can be frozen and thawed before and / or after any purification step of the invention.

In accordance with the present invention, the recombinant Fc-containing protein can be produced in eukaryotic expression systems, such as yeast, insects, or mammalian cells, resulting in glycosylated Fc-containing proteins.

According to the present invention, the most preferred is to express the Fc-containing protein in mammalian cells, such as animal cell lines, or in human cell lines. Chinese hamster ovary (CHO) cells or the murine myeloma cell line NSO are examples of cell lines that are especially suitable for the expression of the Fc-containing protein to be purified. The Fc-containing protein can also preferably be produced in human cell lines, such as, for example, the human fibrosarcoma cell line HT1080, the human retinoblastoma cell line PERC6, or the human kidney embryonic cell line 293, or a cell line of permanent amniocyte as described, for example, in EP 1 230 354.

If the Fc-containing protein to be purified is expressed by mammalian cells that secrete it, the starting material of the purification process of the invention is the cell culture supernatant, also called harvest or crude harvest. If cells are grown in a medium containing animal serum, the cell culture supernatant also contains serum proteins as impurities.

Preferably, cells expressing and secreting the Fc-containing protein are cultured under serum-free conditions. The Fc-containing protein can also be produced in a chemically defined medium. In that case, the starting material of the purification process of the invention is the cell culture supernatant without serum which contains mainly host cell proteins as impurities. If growth factors are added to the cell culture medium, such as insulin, for example, this protein will also be eliminated during the purification process.

In order to create soluble protein containing secreted Fc, which is released into the cell culture supernatant, the natural signal peptide of the therapeutic portion of the Fc-containing protein is used, or preferably a heterologous signal peptide, that is, a signal peptide derived of another secreted protein that is efficient in the particular expression system used, such as, for example, the bovine or human Growth Hormone signal peptide, or immunoglobulin signal peptide.

As mentioned above, a preferred Fc-containing protein to be purified, according to the present invention, is a fusion protein that has a therapeutic portion derived from human TACI (SEQ ID NO: 2), and in particular a fragment derived from its extracellular domain (1 to 165 amino acids of SEQ ID NO: 2). A preferred fragment comprises 30 to 110 amino acids of SEQ ID NO: 2. Thereafter, the therapeutic moieties derived from the TACI extracellular domain will be called soluble TACI or sTACI. A preferred Fc moiety comprises SEQ ID NO: 3, resulting in an Fc fusion protein according to SEQ ID NO: 4, subsequently called TACI-Fc. The term TACI-Fc, as used in this application, also encompasses TACI-Fc muteins.

The term muteins, as used in this application, refers to analogs of sTACI or TACI-Fc, in which one or more amino acid residues of sTACI or TACI-Fc are replaced by different amino acid residues, or are deleted, or one or more amino acid residues are added to the original sTACI or TACI-Fc sequence without significantly modifying the activity of the resulting products when compared to the original sTACI or TACI-Fc. These muteins are prepared using known syntheses and / or by site-directed mutagenesis techniques, or any other known technique suitable for this purpose.

Muteins in accordance with the present invention include proteins encoded by a nucleic acid, such as DNA or RNA, which hybridize to the complement of a DNA or RNA, which encodes a sTACI or TACIFc according to any of SEQ ID NO : 2 or 4 under stringent conditions. An example of a DNA sequence encoding a TACIFcéaSEQIDNO: 7.

The term stringent conditions refers to the subsequent hybridization and washing conditions, to which those skilled in the conventional art conventionally refer to as stringent. See Ausubel et al., Current Protocols in Molecular Biology, supra, Interscience, N.Y., §§6.3 and 6.4 (1987, 1992). Without restriction, examples of stringent conditions include washing conditions 12 to 20 ° C below the calculated Tm for the hybrid under study in, for example, SSC 2x and 0.5% SDS for 5 minutes, SSC 2x and SDS 0.1 % for 15 minutes; 0.1 x SSC and 0.5% SDS at 37 ° C for 30 to 60 minutes and then a 0.1 x SSC and 0.5% SDS at 68 ° C for 30 to 60 minutes. Those of ordinary skill understand that stringent conditions also depend on the size of DNA sequences, oligonucleotide probes (such as 10 to 40 bases) or mixed oligonucleotide probes. If mixed probes are used, it is preferable to use tetramethylammonium chloride (TMAC) instead of SSC. See Ausubel, supra.

In another embodiment, such muteins have at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity or homology to these.

Identity reflects a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by comparing the sequences. In general, identity refers to an exact nucleotide to nucleotide or amino acid to amino acid match of the two polynucleotides or two polypeptide sequences, respectively, over the length of the sequences being compared.

For strings where there is no exact match, an identity percentage can be determined. In general, the two sequences to be compared are aligned to provide a maximum correlation between the sequences. This may include inserting gaps in one or both sequences to improve the degree of alignment. A percentage of identity can be determined over the entire length of each of the strings being compared (called global alignment), which is particularly suitable for sequences of the same or very similar length, or over shorter defined lengths, (called local alignment) , which is best suited for strings of uneven length.

Methods for comparing the identity and homology of two or more sequences are well known in the art. Thus, for example, programs available in the Wisconsin Sequence Analysis Package, version 9.1 (Devereux J et al., 1984), for example, the BESTFIT and GAP programs, can be used to determine the percentage of identity between two polynucleotides and the percentage of identity and the percentage of homology between two polypeptide sequences. BESTFIT uses the local homology algorithm of Smith and Waterman (1981) and finds the best region of unique similarity between two sequences. Other programs for determining similarity and / or identity between sequences are also known in the art, for example, the BLAST family of programs (Altschul SF et al, 1990, Altschul SF et al, 1997, accessible through the NCBI home page at www.ncbi.nlm.nih.gov) and FASTA (Pearson WR, 1990).

Such muteins preferably have an amino acid sequence suitably duplicated from that of sTACI or TACI-Fc, as well as have ligand-binding activity substantially similar to a protein of SEQ ID NO: 2 or 4. For example, TACI activity is its ability to link to Blys or APRIL (Hymowitz et al., 2006). While mutein has substantial APRIL or Blys binding activity, it can be considered to have activity substantially similar to TACI. In this way, it can be easily determined by the person skilled in the art whether any mutein supplied has substantially the same activity as a protein of SEQ ID NO: 2 or 4 through usual experimentation.

Preferred modifications of muteins in accordance with the present invention are what are known as conservative substitutions. Conservative amino acid substitutions of sTACI or TACI-Fc may include synonymous amino acids within a group that has suitably similar physico-chemical properties so that substitution between members of the group preserves the biological function of the molecule (Grantham, 1974). It is clear that insertions and deletions of amino acids can also be made in the sequences defined above without changing their function, in particular if the insertions or deletions involve only a few amino acids, for example, below thirty, below twenty, or preferably below ten , and do not remove or displace amino acids that are critical for functional conformation, for example, cysteine residues. Proteins and muteins produced by such deletions and / or insertions are within the scope of the present invention.

Preferably, the conservative amino acid groups are those defined in Table 2. More preferably, the synonymous amino acid groups are those defined in Table 3; and most preferably the synonymous amino acid groups are those defined in Table 4.

TABLE 2

Preferred Groups of Amino Acids Synonyms

Amino Acid Synonym Group To be Ser, Thr, Gly, Asn Arg Read Arg, Gin, Lys, Glu, His He, Phe, Tyr, Met, Vai, Leu Pro Gly, Ala, Thr, Pro Thr Pro, Ser, Ala, Gly, His, Gin, Thr Allah Gly, Thr, Pro, Wing Go Met, Tyr, Phe, He, Leu, Vai Gly lie Wing, Thr, Pro, Ser, Gly Met, Tyr, Phe, Vai, Leu, He Phe Trp, Met, Tyr, He, Vai, Leu, Phe Tyr Trp, Met, Phe, He, Vai, Leu, Tyr

TABLE 2 - Continuation Preferred Groups of Amino Acids Synonyms Amino Acid Synonym Group Cys Ser, Thr, Cys His Glu, Lys, Gin, Thr, Arg, His Gin Glu, Lys, Asn, His, Thr, Arg, Gin Asn Gin, Asp, Ser, Asn Lys Glu, Gin, His, Arg, Lys Asp Glu, Asn, Asp Glu Asp, Lys, Asn, Gin, His, Arg, Glu Met Phe, He, Vai, Leu, Met Trp Trp TABLE 3 Most Preferred Groups of Amino Acids Synonyms Amino Acid Synonym Group To be To be Arg His, Lys, Arg Read Leu, He, Phe, Met Pro Wing, Pro Thr Thr Allah Pro, Wing Go Go, Met, He Gly Gly lie He, Met, Phe, Go, Read Phe Met, Tyr, He, Leu, Phe Tyr Phe, Tyr Cys Cys, Ser His His, Gin, Arg Gin Glu, Gin, His Asn Asp, Asn Lys Lys, Arg Asp Asp, Asn

TABLE 3 - Continuation

Most Preferred Groups of Amino Acids Synonyms Amino Acid Group Synonym

Glu Glu, Gin

Met Met, Phe, He, Go, Read

Trp Trp

TABLE 4

Most Preferred Groups Yet Amino Acids Synonyms

Amino Acid Synonym Group To be To be Arg Read Arg Leu, lie, Met Pro Pro Thr Thr Allah Allah Go Go Gly lie Gly He, Met, Leu Phe Phe Tyr Cys His Tyr Cys, Ser His Gin Gin Asn Asn Lys Asp Glu Lys Asp Glu Met Met, He, Leu Trp Met

A functional derivative can be prepared from a purified Fc fusion protein according to the present invention. Functional derivatives, as used in this application, include protein derivatives containing Fc to be purified, according to the present invention, which can be prepared from the functional groups that occur as side chains in the residues or N- or C-terminal groups , by means known in the art, and are included in the invention while remaining pharmaceutically acceptable, that is, they do not destroy the activity of the protein which is substantially similar to the activity of the unmodified Fc-containing protein, as defined above, and do not impart toxic properties to compositions that contain them.

The functional derivatives of an Fc-containing protein can, for example, be conjugated to polymers to improve protein properties, such as stability, half-life, bioavailability, human body tolerance, or immunogenicity. To achieve this goal, the Fc-containing protein can be linked, for example, to polyethylene glycol (PEG). Pegylation can be carried out by known methods, described in WO 92/13095, for example.

Functional derivatives may also include, for example, aliphatic esters of the carboxyl groups, amides of the carboxyl groups by reaction with ammonia or with primary or secondary amines, N-acyl derivatives of amino groups free from the amino acid residues formed with acyl moieties (for example , alkanoyl or carbocyclic aromayl groups) or O-acyl derivatives of hydroxyl-free groups (for example, those seryl or threonyl residues) formed with acyl moieties.

In a third aspect, the invention relates to a protein purified by the purification process according to the invention. Subsequently, such a protein is also called a protein containing purified Fc.

Such a purified Fc-containing protein is preferably the highly purified Fc-containing protein. Highly purified Fc fusion protein is determined, for example, by the presence of a single band on an unreduced silver-colored SDS-PAGE gel after loading the protein in the amount of 2 mcg per column. The purified Fc fusion protein can also be defined as a single HPLC elution peak.

The Fc-containing protein preparation obtained from the purification process of the invention can contain less than 20% impurities, preferably less than 10%, 5%, 3%, 2% or 1% impurities, or can be purified to homogeneity , that is, be free of any detectable protein contaminants as determined, for example, by silver-stained SDSPAGE or HPLC, as explained above.

The purified Fc-containing protein can be intended for therapeutic use, in particular for administration to human patients. If the purified Fc-containing protein is administered to patients, it is preferably administered systemically, and preferably subcutaneously or intramuscularly, or topically, that is, locally. Rectal or intrathecal administration may also be appropriate, depending on the specific medical use of the purified Fc-containing protein.

For this purpose, in a preferred embodiment of the present invention, the purified Fc-containing protein can be formulated in a pharmaceutical composition, that is, together with a pharmaceutically acceptable carrier, excipients or the like.

The definition of pharmaceutically acceptable is intended to encompass any vehicle, which 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 (s) can be formulated in a unit dosage form for vehicle injection, such as saline, glucose solution, albumin serum and Ringer's solution.

The active ingredients of the pharmaceutical composition, according to the invention, can be administered to an individual in several ways. Routes of administration include intradermal, transdermal (e.g., in slow-release formulations), intramuscular, intraperitoneal, intravenous, subcutaneous, oral, intracranial, epidural, topical, rectal and intranasal routes. Any other therapeutically effective route of administration can be used, for example, absorption by epithelial or endothelial tissues or by gene therapy in which a DNA molecule encoding the active agent is administered to the patient (for example, via a vector), which induces the expression and secretion of the active agent in vivo. In addition, the protein (s) according to the invention can be administered in conjunction with other components of biologically active agents, such as pharmaceutically acceptable surfactants, excipients, carriers, diluents and vehicles.

For parenteral administration (for example, intravenous, subcutaneous, intramuscular), the active protein (s) can be formulated as a solution, suspension, emulsion or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle (for example , water, saline, glucose solution) and additives that maintain isotonicity (eg mannitol) or chemical stability (eg preservatives and buffers). The formulation is sterilized by commonly used techniques.

The therapeutically effective amounts of the active protein (s) will be a function of many variables, including the type of protein containing Fc, the affinity of the protein containing Fc to its ligand, the route of administration, the clinical condition of the patient.

A therapeutically effective amount is such that when administered, the Fc-containing protein results in the inhibition of its ligand from the therapeutic portion of the Fc fusion protein, as explained above and referring in particular to Table 5 above.

The dosage administered, such as single or multiple doses, to an individual will be modified depending on a variety of factors, including pharmacokinetic properties of the Fc fusion protein, route of administration, patient conditions and characteristics (gender, age, body weight, health, size), extension of symptoms, simultaneous treatments, frequency of treatment and the desired effect. Adjustment and manipulation of established dosage ranges are within the competence of those skilled in the art, as well as in vitro and in vivo methods for determining the inhibition of the natural ligand of the therapeutic portion in an individual.

The purified Fc-containing protein can be used in an amount of 0.001 to 100 mg / kg or 0.01 to 10 mg / kg of body weight, or

0.1 to 5 mg / kg body weight or 1 to 3 mg / kg body weight or 2 mg / kg body weight.

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

Daily doses are usually given in divided doses or in the form of an effective prolonged release to obtain the desired results. Auxiliary or subsequent administrations can be performed at a dosage that is the same, lesser or greater than the initial or previous dose administered to the individual. An auxiliary or subsequent administration can be made during or before the onset of the disease.

The present invention, moreover, relates to the use of cation exchange chromatography to reduce the concentration of free Fc portions in a composition comprising a protein containing Fc.

In a preferred embodiment, the concentration of free Fc is reduced to less than 20% or less than 15% or less than 10% or less than 5% or less than 2% or less than 1% or less than 0.8% or less than 0.5% or less than 0.2% or less than 0.1% of the total protein concentration of said composition.

Having now fully described this invention, it will be appreciated by those skilled in the art that it can be performed within a wide range of parameters, concentrations and equivalent conditions without departing from the spirit and scope of the invention and without undue experimentation.

While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses or adaptations of the following invention, in general, principles of the invention and even such departures from the present description as it comes within the known or usual practice within the technique to which the invention belongs and how it can be applied to the essential features presented above as follows in the scope of the attached claims.

All references cited in this application, including newspaper articles or summaries, published or unpublished foreign or United States patent applications, foreign or published patents in the United States or any other references, are fully incorporated by reference in this application, including all data , tables, numbers and text presented in the cited references. In addition, the complete contents of the references cited within the references cited in this application are also fully incorporated by reference.

Reference to known method steps, conventional method steps, known methods or conventional methods is by no means an admission that any aspect, description or embodiment of the present invention is described, taught or suggested in the related art.

The foregoing description of specific modalities will fully describe the general nature of the invention, which others can, by applying knowledge within the skill of the art (including the contents of the references cited in this application), promptly modify and / or adapt such specific modalities to various applications, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning of a variety of described modal equivalents, based on the teaching and guidance presented in this application. It should be understood that the phraseology or terminology in this application is for the purpose of description and not limitation, such that the terminology or phraseology of this report should be interpreted by the person skilled in the art in light of the teachings and guidance presented in this application, in combination with the knowledge of a person skilled in the art.

EXAMPLES:

PURIFICATION OF RECOMBINANT HUMAN TACI-Fc, FROM

CHO CELL SUPERVISOR WITHOUT SERUM

Glossary used in all Examples

BV: Bed volume CHO: Chinese Hamster Ovary DSP: Downstream process EDTA: Diamino Tetracetic Ethylene Acid ELISA: Enzyme-linked immunosorbent assay HAC: Hydroxyapatite chromatography HCP: Host Cell Protein HPLC: High Performance Liquid Chromatography id: internal diameter K: potassium kD: kilo Dalton MONTH: 2-Morpholinoethanesulfonic acid At: sodium NaAc: Sodium acetate n / a: not determined PA-SE-HPLC: High Performance Liquid Chromatography by Size Exclusion in Protein A ppm: parts per million RO: Reverse osmosis RT: Room temperature SDS-PAGE: Polyacrylamide gel electrophoresis with sodium dodecyl sulfate SE-HPLC: High Performance Liquid Chromatography by Size Exclusion T ° C: Temperature TMAC: Tetramethylammonium chloride UV: Ultraviolet WFI: Injection Water

WRO Reverse Water Osmosis

Example 1: Capture Step: Protein A affinity purification

The starting material was purified from the harvest of a CHO cell clone grown under serum-free conditions expressing TACI-Fc and stored frozen until use.

The capture step in a MabSelect Xtra ™ column (GE Healthcare 17-5269-03) was performed according to the following protocol, in a column that has a bed height of 17 cm. All operations were performed at room temperature, except the loading solution, which was maintained at a temperature below 15 ° C. The UV signal was recorded at 280 nm.

Sanitization

The column was sanitized with at least 3BV of 0.1 M acetic acid + 20% ethanol in reverse flow at 250 cm / h. The flow was interrupted for 1 hour.

Wash step

The column was washed with at least 2BV of RO water in reverse flow at 250cm / h.

Balance

The column was equilibrated with at least 5BV of sodium phosphate at 25 mM + NaCI at 150 mM pH 7.0 (until the conductivity and pH parameters are within the specified range: pH 7.0 ± 0.1, conductivity 18 ± 2 mS / cm) in downward flow at 450 cm / h.

Loading

The column was loaded with the purified crop maintained at a temperature below 15 ° C for a capacity of up to 15 mg of total TACI-Fc per ml of packaged resin as determined by the Biacore assay at a flow rate of 350 cm / h.

Wash step

Wash the column with at least 2BV of the balance buffer at 350 cm / h and then with at least 4BV of the balance buffer (until the UV signal returns to the baseline) at 450 cm / h.

Elution

The material was eluted with different elution buffers as shown in Table I at a flow rate of 350 cm / h. The eluted fraction was collected from the beginning of the increase in the UV signal at 6.0 ± 0.5BV of elution. The eluate was incubated for 1 hour at room temperature at a pH below 4.1 (adjusted by adding the citric acid solution, if necessary) and then the pH was adjusted to 5.0 ± 0.1 by adding the solution of citric acid. 32% NaOH.

Regeneration

The column was regenerated with at least 3BV of NaOH at 50 mM + NaCI at 1M in reverse flow at 450cm / h, the flow stopped for 15 minutes, then resumed the flow at 450cm / h at least 3BV (until the UV signal returns to the baseline).

From that stage, the column was operated in reverse flow mode.

Wash step

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

Sanitization

The column was sanitized with at least 3BV of the sanitizing buffer at 250 cm / h, the flow stopped and the column incubated for 60 minutes. Final washing steps

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

Finally, the column was stored after rinsing with at least 3BV of 20% ethanol at 250cm / h.

Results

Table I: Results using different elution buffers

Race number Elution Buffer Yield of TACI-Fc (%) Aggregates (%) HCPs (ppm) 1 50mM sodium acetate pH3.7 47.7 30.3 5558 2 sodium acetate at 100mM pH3.8 55.7 25.2 n / a 3 200mM sodium acetate pH3.8 58.0 28.2 n / a 4 sodium acetate at 100mM pH3.7 68 30.0 n / a 5 0.2M sodium acetate + 150mM sodium chloride pH4 75.1 3.8 n / a 6 sodium acetate at 100mM pH3.7 84.6 22 3491 7 250mM sodium acetate pH3.7 82.8 18.7 3318 8 sodium citrate at 100mM pH3.7 79.2 8.8 4710 9 sodium citrate at 250mM pH3.7 71.9 23 2347 10 sodium citrate at 100mM pH 3.75 82.8 8.5 1576 11 sodium citrate at 100mM pH 3.75 66.6 9.0 664 12 sodium acetate at 100mM pH3,85 83.3 15.0 n / a 13 sodium citrate at 100mM pH 3.75 81.0 9.1 3490 14 sodium citrate at 100mM pH3,65 75.1 14.6 2580 14 sodium citrate at 100mM pH 3.75 44.7 18.4 3783 16 sodium citrate at 100mM pH 3.75 47.1 15.8 3217 17 sodium citrate at 100mM pH 3.75 50.7 9.4 2349 18 sodium citrate at 100mM pH 3.75 58.0 10.4 2550 19 sodium citrate at 100mM pH 3.75 67.1 28.7 2372 20 sodium citrate at 100mM pH 3.75 65.6 17.5 2353 21 sodium citrate at 100mM pH 3.75 75.6 19.4 1807 22 sodium citrate at 100mM pH 3.75 57.1 20.7 2465 23 sodium citrate at 100mM pH 3.75 51.9 18.4 2030 24 sodium citrate at 100mM pH 3.75 58 11.5 1746 25 sodium citrate at 100mM pH 3.75 41.8 22.9 3029 26 sodium citrate at 100mM pH3.9 39.4 6.0 2424 27 sodium citrate at 100mM pH3.9 31.0 8.8 2936 28 sodium acetate at 100mM pH4.1 28.3 25.0 3311

Race number Elution Buffer Yield of TACI-Fc (%) Aggregates (%) HCPs (PPm) 29 sodium citrate at 100mM pH3.9 46.4 9.1 n / a 30 sodium acetate at 100mM pH4.1 42.8 13.4 n / a 31 sodium citrate at 100mM pH 3.75 57.5 26.5 n / a 32 sodium acetate at 100mM pH4.2 38.1 10.1 n / a 33 sodium citrate at 100mM pH3.9 43.3 8.3 2011 34 sodium citrate at 100mM pH3.9 63.6 6.6 1749 35 sodium citrate at 100mM pH3.9 65.7 7.3 1689 36 sodium citrate at 100mM pH3.9 62.7 7.4 1609 37 sodium citrate at 100mM pH3.9 61.6 7.4 1479 38 sodium citrate at 100mM pH3.9 60.6 7.4 1623 39 sodium citrate at 100mM pH3.9 64.6 8.0 1497

Conclusions

Purified TACI-Fc5 at harvest was captured directly on a MabSelect Xtra column at a total dynamic capacity of 15g of TACl-Fc5 per L of the packaged resin at a flow rate of 350 cm / h. Elution conditions, especially pH, have been optimized to maximize product recovery by providing a significant reduction in aggregate levels. A 0.1 M sodium citrate elution buffer pH 3.9 was selected providing aggregate levels of approximately 5 to 10% starting from approximately 25 to 40% purified at harvest and with no turbidity observed. HCP levels were typically at 1500 to 2000ppm. HCP levels were measured by ELISA using polyclonal antibodies. The antibody mixture was generated against host cell proteins derived from the purified supernatant and concentrated from cell cultures of untransfected CHO cells.

Example 2: Cation exchange chromatography

The elution from the protein A capture step, dialyzed in the appropriate loading buffer, was used as a starting material for cation exchange chromatography.

A Fractogel EMD SO ₃ 'column (Merck 1.16882.0010) having a bed height of 10 cm was used in this step. A Fractogel column

SO3 · with a bed height of 15 cm can also be used. In the latter case, dynamic capacity and flow velocity may need adaptation, which is within the routine knowledge of the person skilled in the art.

All operations were performed at room temperature and the flow rate was kept constant at 150 cm / h. The UV signal at 280 nm was recorded at all times.

Wash step

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

Sanitization

Then, the column was sanitized with at least 3BV of 0.5M NaOH + 1.5M NaCI in downflow mode.

Rinse

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

Balance

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

Loading

The column was loaded with the post-capture material at pH 5.0 (pH at 5.0 ± 0.1, conductivity at 12 ± 1 mS / cm) and at a capacity of no more than 50 mg of TACI- Fc, per ml of the packaged resin as determined by the SE-HPLC assay.

Wash step

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

Elution

The column was eluted with different buffers and under different conditions as shown in tables II to IV below.

Regeneration and sanitation

The column was regenerated and sanitized with 4BV 0.5M NaOH + 1.5M NaCI in the upflow mode. Then, the flow was stopped for 30 minutes.

Rinse

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

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

Table II: Effect of pH and conductivity on elution

HCP levels at load: 189ppm

pH Conductivity (mS / cm) TACI-Fc recovery HCPs (ppm) HCP debugging (x) 6.5 15.0 25% 118 1.6 7.3 22.5 100% 50 3.8 8.0 15.0 95% 34 5.5 7.3 22.5 100% 56 3.4 7.3 33.0 98% 133 1.4 7.3 22.5 96% 45 4.2 7.3 22.5 97% 53 3.6 7.3 12.0 54% 79 2.4 6.3 22.5 83% 47 4.1 8.0 30.0 96% 108 1.8 8.2 22.5 97% 46 4.2 6.5 30.0 91% 116 1.6 7.3 22.5 93% 48 3.9 7.3 22.5 95% 40 4.8

Table III shows the recovery of TACI-Fc and the clearance of

HCP when loaded to a capacity of 10 and 32 mg of TACI-Fc per ml of resin and elution in a phosphate buffer at a conductivity between 15 12 to 33 mS / cm. Peak collection was done at the beginning of the UV increase by + 0.5 BV.

Table III: Effect of pH and conductivity on optimum elution when loaded to capacity

HCP levels at load: 201 ppm

Loading capacity (mg / ml) PH Conductivity (mS / cm) TACI-Fc recovery HCPs (ppm) HCP debugging (x) 10 8.0 15.0 20.7 91% 93% 67 61 3.0 3.3 32 8.0 20.7 88% 54 3.7

Table IV shows the effect of a washing step with sodium phosphate at 50, 100 or 150 mM pH 6.5 on TACI-Fc recovery and HCP clearance.

Table IV: Effect of washing stage conditions on column performance

HCP levels in the load: 190ppm and aggregate levels: 2.0%

Concentration of sodium phosphate in the wash (mM) Yield of TACI-Fc in washing Yield of TACI-Fc in the eluate Aggregates in the eluate HCPs in the eluted (PPm) wash 1 50 0.7% 99% 2.8% 62 wash 2 100 2.1% 98% 2.9% 59 wash 3 150 9.1% 90% 2.7% 49

The buffer used in wash 2, containing 100 mM sodium phosphate pH 6.5, had a conductivity of 8.4 mS / cm.

Figure 1 shows an unreduced silver-colored SDS-PAGE gel from samples derived from experiments using the three wash step conditions shown in Table IV in free Fc clearance.

Figure 2 shows overlapping chromatograms of experiments from the washing step with sodium phosphate in different concentrations.

The washing step was optimized to pH 6.5 with increasing concentrations of sodium phosphate a (50 to 150 mM). As can be seen in Figure 1, a concentration of the 150 mM wash buffer (wash 3, column 6) resulted in losses of TACI-Fc. A concentration of the 50 mM wash buffer (wash 1, column 8) resulted in a peak of pure TACI-Fc, however, the eluate contained traces of free Fc. A washing step with sodium phosphate at 100 mM pH 6.5, resulted in 98% recovery at the main elution peak and losses of only 2% on washing (Figure 2). HCP clearance was 3.2 times. SDS-PAGE analysis of the wash and eluted fractions shows that the wash step contained free Fc with some intact TACIFc at 100 mM buffer concentration or above (Figure 1, columns 4 and 6). A concentration of 100 mM or more is required to completely remove free Fc from the eluted fraction (Figure 1, columns 5 and 7). Conclusions

A cation exchange step was developed as a second purification step, after the capture step. The capture was eluted at low pH (5.0) and low conductivity and could be directly loaded in a cationic exchanger. A Fractogel EMD SO ₃ 'resin was selected with a loading capacity of 50 mg / ml. The free Fc non-bioactive degradation product could be efficiently removed in a washing step with 0.1 M sodium phosphate pH 6.5. The elution conditions were optimized for better HCP clearance and high TACI-Fc recovery (179 mM sodium phosphate pH 8.0, 20.7 mS / cm conductivity).

On the other hand, elution can be performed on 10 BV of 20 mM sodium phosphate and 180 mM NaCI pH8.0 from the beginning of the increase in absorbance at 280 nm.

Example 3: Anion exchange chromatography

The starting material used for this purification step was eluted from the cation exchange step in Fractogel SOa '(see Example 2), dialysed or diluted in the appropriate loading buffer.

This anion exchange chromatography step was performed on a SOURCE 30Q column (GE Healthcare 17-1275-01) with a bed height of 10 cm. A SOURCE 30Q column with a bed height of 15 cm can also be used in this step. In the latter case, dynamic capacity and flow velocity may need adaptation, which is within the routine knowledge of the person skilled in the art.

All operations were performed at room temperature and the UV signal was recorded at 280 nm. The steps were performed at a flow speed of 150 or 200 cm / h.

Rinse

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

Sanitization

Then, the column was sanitized with at least 3BV of 0.5M NaOH + 1.5M NaCla.

Wash step

The column was washed with at least 3BV, preferably 4 to 10BV of 0.5M sodium phosphate pH 7.5 at a flow rate of 200cm / h. Balance

The column was equilibrated with at least 5 BV of sodium phosphate at 10, 15, 20, 25, or 30 mM pH 7.5. Optionally, the column can be pre-equilibrated with 3 BV of 0.5 M sodium phosphate pH 7.5.

Concomitant loading, washing and collection of TACI-Fc in continuous flow

The column was loaded with diluted post-cation exchange material to obtain a phosphate concentration of 10 to 30 mM, pH 7.5, in a capacity no more than 50 mg of TACI-Fc per ml of the packaged resin as determined by the SE-HPLC test, collecting the continuous flow from the beginning of the UV increase until the end of the washing step, which is performed in 4 ± 0.5 BV of the equilibration buffer.

Regeneration / Sanitization

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

Wash step

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

Storage

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

Results

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

Table V: Effect of phosphate concentration on loading

Loading pH Phosphate concentration in loading (mM) Concentration of TACI-Fc in loading (mg / L) Loading capacity (mg / ml) TACI-Fc recovery Aggregates HCPs (PPm) 7.5 30 773 39 94% 10.4% 82.8 7.5 25 639 39 90% 6.9% 50.4 7.5 20 651 49 90% 5.6% 43.9 7.5 15 437 46 88% 3.4% 45.0 7.5 10 283 n./d. 82% 2.8% 26.3

Conclusions

The anion exchange step in a Source 30Q column in continuous flow mode has been optimized to maximize the clearance of HCPs and aggregates. The elution of the diluted or diafiltered cation exchange loading in 20 mM sodium phosphate buffer at pH 7.5 provided the best fit between product recovery (90%) and HCPs clearance (from approximately 2000ppm to 44ppm) and aggregates ( approximately 25% to 5.6%). The dynamic capacity of 50 mg of TACI-Fc per ml of packaged resin at a flow rate of 150 to 200 cm / h was used.

Example 4: Chromatography on hydroxyapatite

The starting material used for this purification step was the continuous flow of anion exchange chromatography (see Example 3).

A 40 pm column of CHT Type I Ceramic Hydroxyapatite (Biorad 157-0040) with a bed height of 10 cm was used.

All operations were performed at room temperature. The flow rate was kept constant at 175 cm / h and the UV signal was recorded at 280 nm. All solutions were filtered under sterile conditions and the equipment sanitized with sodium hydroxide before use. The column was stored in 0.5M NaOH solution when not used.

Initial washing steps (Rinse and pre-balance)

The column was washed with at least 1BV of 20 mM sodium phosphate buffer pH 7.5, and then with at least 3BV of 0.5M sodium phosphate buffer pH 7.5 to reduce the pH.

balance

The column was equilibrated with at least 5BV of sodium phosphate at 20 mM pH 7.5 (or until the target conductivity of 3.0 ± 0.3 mS / cm and pH 7.5 ± 0.1 was reached).

Loading

The column was loaded with SOURCE 30Q continuous flow with calcium chloride added to the final concentration of 0.1 mM of a 0.5M stock solution and pH adjusted to 7.0 by the addition of 85% orthophosphoric acid, in an NMT capacity of 50 mg of TACI-Fc per ml of the packaged resin as determined by the SE-HPLC assay. It is also possible to load the SOURCE 30Q continuous flow without calcium chloride, adjusted to pH 7.0, in the hydroxyapatite column.

Washing steps

The column was washed with at least 4BV of 3, 4 or 5 mM sodium phosphate, 10 mM MES, 0.1 mM CaCb pH 6.5. It is also possible to use the same buffer without calcium chloride.

Elution

The column was eluted with sodium phosphate at 5, 4, 3 or 2 mM (see Table VI), 10 mM MES, 0.1 mM CaCl, KCI buffer and 0.6, 0.7, 0.8 or 0.9 M pH 6.5 (see Table VII) of the beginning of the UV increase for different BV (see Tables VI and VII). It is also possible to use the same buffer without calcium chloride for elution.

Rinse

The column was rinsed with:

- at least 1BV of sodium phosphate buffer at 20 mM pH 7.5;

- at least 3BV of 0.5M sodium phosphate buffer pH 7.5; and

- with at least 1BV of 20 mM sodium phosphate buffer pH 7.5.

Storage

The column was stored in at least 3BV of 0.5M NaOH. Results

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

Phosphate concentration (mM) Elution BV Yield of TACI-Fc Aggregates 12 73% 0.49% 13 74% 0.52% 14 68% 0.65% 5 15 77% 0.67% 16 77% 0.70% 17 70% 0.73% 18 76% 0.85% 12 68% 0.34% 13 67% 0.29% 14 66% 0.36% 4 15 67% 0.39% 16 66% 0.38% 17 66% 0.32% 18 66% 0.40% 12 70% 0.46% 13 76% 0.42% 14 73% 0.51% 3 15 71% 0.52% 16 69% 0.55% 17 69% 0.50% 18 70% 0.53%

Phosphate concentration (mM) Elution BV Yield of TACI-Fc Aggregates 12 65% 0.19% 13 66% 0.00% 14 66% 0.18% 2 15 68% 0.14% 16 66% 0.17% 17 71% 0.19% 18 65% 0.16%

Table VII shows the effect of KCI concentration in the elution buffer on aggregate clearance and product recovery. Two concentrations of sodium phosphate were investigated: at 2 and 3 mM. The peak elution fractions were pooled and analyzed by SE-HPLC for 5 TACI-Fc concentration and aggregate levels.

Table VII: Effect of potassium chloride concentration in the Elution buffer

Phosphate concentration (mM) KCI concentration (M) BVda elution Yield of TACI-Fc Aggregates 10 102% 0.48% 11 109% 0.46% 3 0.6 12 106% 0.43% 13 105% 0.42% 14 103% 0.43% 10 96% 0.42% 11 97% 0.40% 3 0.7 12 98% 0.41% 13 96% 0.40% 14 96% 0.43% 10 106% 0.58% 11 110% 0.55% 3 0.8 12 112% 0.57% 13 101% 0.59% 14 110% 0.57%

Phosphate concentration (mM) KCI concentration (M) BVda elution Yield of TACI-Fc Aggregates 10 71% 0.29% 11 79% 0.28% 2 0.6 12 80% 0.29% 13 80% 0.29% 14 81% 0.26% 10 64% 0.27% 11 72% 0.25% 2 0.9 12 73% 0.29% 13 70% 0.33% 14 66% 0.24%

Conclusions:

Hydroxyapatite chromatography provides a reliable, efficient way to reduce aggregate levels in TACI-Fc. Starting with anion exchange chromatography, the material was purified (see Example 3) with aggregate levels of approximately 5 to 8%, hydroxyapatite chromatography can reduce these levels to less than 0.8% with a TACI-Fc recovery of 85 to 90%.

Reference Listing

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

2. Altschul S F et al, J Mol Biol, 215, 403-410, 1990.

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

4. Armor KL. et al., 1999. Recombinant human IgG molecules lacking Fcgamma receptor I binding and monocyte triggering activities. Eur J Immunol. 29 (8): 2613-24.

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

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

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

8. Bossen et al., JBC 281 (2), 13964-13971.

9. Bram and von Bulow, US 5,969,102 (1999).

10. Bram et al., WO 98/39361.

11. Carter PJ., 2006. Potent antibody therapeutics by design. Nature Reviews Immunology. Advance online publication.

12. Devereux J et al, Nucleic Acids Res, 12, 387-395, 1984.

13. Feng et al., Bioprocessing 2005, 1-7.

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

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

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

17. Gross et al., WO 00/40716.

18. Hinton PR. et al., 2004. Engineered human IgG antibodies with longer 10 serum half-lives in primates. J Biol Chem. 279 (8): 6213-6.

19. Hymowitz et al., JBC 280 (8), 7218-7227.

20. Idusogie EE. et al., 2000. Mapping of the C1q binding site on rituxan, a chimeric antibody with a human lgG1 Fc. J Immunol. 164 (8): 4178-84.

21. Idusogie EE. et al., 2001. Engineered antibodies with increased activity 15 to recruit complement. J Immunol. 166 (4): 2571-5.

22. Kabat, E. A., Wu, T. T., Perry, Η. M., Gottesman, K. S., and Foeller, C. (1991), Sequences of Proteins of Immunological Interest, 5th Ed., National Institutes of Health, Bethesda, MD.

23. Kochanek et al., EP 1 230 354.

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

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

26. Moore et al., Science 285: 260-3.

27. Moreau et al., Blood 103 (8), 3148-3157.

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

29. Novak et al., Blood 103 (2), 689-694.

30. Parker et al., WO 02/094852.

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

32. Porath, J. Carlsson, I. Olsson, and G. Belfrage, Nature (London) 258, 598-599 (1975).

33. Porath and B. Olin, Biochemistry 22, 1621-1630 (1983).

34. Puren et al., Proc Natl Acad Sei USA. 1999 Mar 2; 96 (5): 2256-61

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

36. Shields RL. et al., 2001. High resolution mapping of the binding site on human lgG1 for Fc gamma RI, Fc gamma RH, Fc gamma Rlll, and FcRn and design of lgG1 variants with improved binding to the Fc gamma R. J Biol Chem. 276 (9): 6591-604.

37. Smith et al., WO 91/03553.

38. Smith et al., WO 94/06476.

39. Stanker, J. Immunological Methods 76: 157-169 (1985) (10 mM to 30 mM sodium phosphate elusion gradient).

40. Steurer W. et al., 1995. Ex vivo coating of islet cell allografts with muri10 ne CTLA4 / Fc promotes graft tolerance. J Immunol. 155 (3): 1165-74.

41. Sun et al., WO 2005/044856.

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

43. Vaccaro C. et al., 2005. Engineering the Fc region of immunoglobulin G to modulate in vivo light antibodies. Nat Biotechnol. 23 (10): 1283-8.

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

45. Vedantham et al., WO 03/59935.

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

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

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

Claims (19)

1. Method to reduce the content of free Fc portions in a fluid comprising a protein containing Fc, characterized by the fact that it comprises subjecting that fluid to cation exchange chromatography on a cation exchange resin at a pH of one unit or more below the isoelectric point (pI) of the protein containing Fc, and wash the cation exchange resin with a buffer that has a conductivity of 8.2 to 8.6 mS / cm and a pH of 5.5 to 7.5. 2. Method according to claim 1, characterized by the fact that the washing step is conducted at a pH of 6.0 to 7.0. Method according to claim 1 or 2, characterized in that the washing step is carried out in a buffer comprising 75 to 125 mM sodium phosphate. Method according to any one of claims 1 to 3, characterized by the fact that it comprises the stage of: elute the protein containing Fc at a pH ranging from 7.0 to 8.5, where the elution step is conducted at a conductivity of 15 to 22 mS / cm. Method according to any one of claims 1 to 4, characterized by the fact that cation exchange chromatography is conducted on a cation exchange resin that comprises SO3 ^- groups. 6. Method according to claim 5, characterized by the fact that the resin comprises a cross-linked methacrylate matrix. Method according to any one of claims 1 to 6, characterized by the fact that it also comprises a purification step selected from affinity chromatography, anion exchange chromatography and hydroxyapatite chromatography. 8. Method according to any one of claims 1 to 7, characterized by the fact that the Fc-containing protein comprises an immunoglobulin (Ig) constant region. 9. Method according to claim 8, characterized by the fact that the constant region is a human constant region. 10. Method according to claim 8 or 9, characterized Petition 870190062858, of 07/05/2019, p. 13/24 by the fact that immunoglobulin is an IgGi. A method according to any one of claims 8 to 10, characterized by the fact that the constant region comprises a CH2 and a CH3 domain. 12. Method according to any one of claims 1 to 11, characterized by the fact that the Fc-containing protein comprises an immunoglobulin variable region. 13. Method according to claim 12, characterized by the fact that the Fc-containing protein is an antibody. 14. Method according to any one of claims 1 to 11, characterized in that the protein containing Fc is an Fc fusion protein. 15. Method according to claim 14, characterized in that the Fc fusion protein comprises a ligand binding portion of a member of the tumor necrosis factor receptor (TNFR) superfamily. 16. Method according to claim 15, characterized in that the ligand-binding portion is selected from an extracellular domain of TNFR1 or TNFR2. 17. Method according to claim 16, characterized in that the ligand-binding portion is selected from an extracellular domain of BAFF-R, BCMA or TACI. 18. Method according to claim 17, characterized by the fact that the Fc fusion protein comprises a polypeptide selected from The. amino acids 34 to 66 of SEQ ID NO: 2; B. amino acids 71 to 104 of SEQ ID NO: 2; ç. amino acids 34 to 104 of SEQ ID NO: 2; d. amino acids 30 to 110 of SEQ ID NO: 2; and. SEQ ID NO: 3; and f. SEQ ID NO: 4; where the polypeptide binds to at least one of Blys or Petition 870190062858, of 07/05/2019, p. 14/24 APRIL. 19. Use of cation exchange chromatography as defined in any one of claims 1 to 18, characterized in that it is for reducing the concentration of free Fc portions in a composition 5 comprising a protein containing Fc, in which the concentration of Free Fc is reduced to less than 5% of the total protein concentration of said composition.