WO2016031932A1 - METHOD FOR PRODUCING PROTEIN HAVING Fc REGION BY ALKALI WASHING - Google Patents

Abstract

　The purpose of the present invention is to provide a method for purifying a protein having an Fc region having reduced problems with properties when purifying a protein having an Fc region having problems with properties such as free thiol content, self-association, and cohesiveness. The free thiol content is decreased and self-association and cohesiveness are also decreased by conducting a step for washing by an alkali wash solution in a state in which a protein having an Fc region is bonded on a carrier for affinity chromatography and refining the protein having an Fc region.

Description

Method for producing protein having Fc region by alkaline washing

The present invention relates to a method for purifying a protein having an Fc region, and a method for producing a protein having an Fc region including the purification method. The present invention also relates to a method for reducing the free thiol content, self-association and aggregation properties of a protein having an Fc region, and a method for enhancing storage stability. Furthermore, the present invention relates to a method for producing a human anti-IL-33 monoclonal antibody.

Proteins having an Fc region such as antibodies are widely used in general, such as therapeutic drugs, diagnostic drugs, and research reagents. These proteins are produced by culturing genetically modified cells into which a gene of a protein having an Fc region has been introduced, hybridomas that produce antibodies, or the like. The protein having the Fc region is mainly purified from the culture supernatant obtained by culturing these cells.

There is affinity chromatography as a method for purifying a protein having an Fc region. In particular, a carrier for affinity chromatography on which Protein A, Protein G, or a modified form thereof is immobilized is widely used because it binds to the Fc region.

When a protein having an Fc region such as an antibody is purified by affinity chromatography using Protein A etc., problems such as aggregation and precipitation occur frequently (Non-patent Document 1: Shukla et al., J. Chromatography, 2007, Vol. 848, p28). Aggregation and precipitation reduce the purification yield of proteins with Fc regions and increase the contamination of impurities. Antibody aggregates can cause inflammation when administered in vivo as a pharmaceutical composition and can enhance the antigenicity of the antibody itself. In addition, antibody aggregates can give erroneous results due to non-specific binding that does not depend on specific binding to an antigen when used as a diagnostic agent or a research reagent. As one of the causes of antibody aggregation and precipitation, it is considered that cysteine residues in the antibody molecule involve free thiol residues that are generated due to the inability to properly form disulfide bonds.

In order to remove high molecular weight impurities (HMW) that are aggregates of antibodies, there is a method of washing with a washing solution containing arginine in the washing step in affinity chromatography. However, HMW is still mixed and removal of HMW is sufficient. No (Patent Document 1). In addition, a washing solution containing a divalent cation salt is used in the washing step in affinity chromatography in order to wash away and remove the underdisulfide bond species (UDB) in which the formation of the disulfide bond of the protein having the Fc region is insufficient. The removal of UDB is not sufficient (Patent Document 2). UDB has free thiol residues and can therefore cause aggregation. Therefore, problems such as aggregation when purifying a protein having an Fc region remain extremely inconvenient.

International Publication No.2012 / 164046 International Publication No. 2006/138553

Proteins having an Fc region have problems with physical properties such as free thiol content, self-association and aggregation. Such physical property problems affect the storage stability of a protein having an Fc region. In addition, problems with physical properties such as aggregation affect the yield and purity in the purification process due to loss due to precipitation of proteins with Fc regions, and contamination by impurities bound to protein aggregates with Fc regions. To do. In recent years, high-concentration subcutaneous injections are becoming the mainstream of antibody drugs that have been developed in recent years, and physical properties such as self-association and aggregation are regarded as important. Therefore, there is a need for a method for purifying a protein having an Fc region with reduced physical property problems.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have obtained a method for purifying a protein having an Fc region, comprising a step of washing the protein having an Fc region with an alkaline washing solution in a state of being bound on an affinity chromatography carrier. As a result, the present invention has been completed. The present invention thus relates to the following inventions:

[1] A method for purifying a protein having an Fc region,
(a) contacting a solution containing a protein having an Fc region with an affinity chromatography support, and binding the protein having an Fc region to the affinity chromatography support;
(b) washing with an alkaline washing liquid having a pH value in the range of 10.1 to 12, and
(c) eluting a protein having an Fc region,
A method for purifying a protein having an Fc region, comprising:
[2] The method according to item 1, wherein the alkaline cleaning liquid has a pH value of 10.5 to 11.5.
[3] The method according to item 1 or item 2, wherein the alkaline cleaning solution does not contain 0.2 M or more sodium chloride.
[4] The method according to any one of items 1 to 3, wherein the alkaline cleaning solution does not contain arginine.
[5] The method according to any one of items 1 to 4, wherein the carrier for affinity chromatography is a carrier having Protein A, Protein G, or a modified form thereof.
[6] The method according to any one of items 1 to 5, wherein the protein having an Fc region is an antibody.
[7] The method according to any one of items 1 to 6, wherein the protein having an Fc region is a human monoclonal antibody, a humanized monoclonal antibody, or a chimeric monoclonal antibody.
[8] The method according to any one of items 1 to 7, wherein the protein having an Fc region is an antibody having a λ chain.
[9] A method for producing a protein having an Fc region, comprising the method according to any one of items 1 to 8.
[10] A protein having an Fc region produced by the method according to item 9.
[11] A method for producing a human monoclonal antibody,
(I) contacting a solution containing a human monoclonal antibody with an affinity chromatography carrier and binding the human monoclonal antibody to the affinity chromatography carrier;
(ii) washing with an alkaline wash having a pH value in the range of 9 to 12, and
(iii) a step of eluting the human monoclonal antibody;
Wherein the light chain complementarity determining region 1 (LCDR1), the light chain complementarity determining region 2 (LCDR2), the light chain complementarity determining region 3 (LCDR3), and the heavy chain complementarity of a human monoclonal antibody A combination of amino acid sequences of the determination region 1 (HCDR1), the heavy chain complementarity determination region 2 (HCDR2), and the heavy chain complementarity determination region 3 (HCDR3),
(a) LCDR1: TGSSSNIGAVYDVH (SEQ ID NO: 1 in the sequence listing), LCDR2: RNNQRPS (SEQ ID NO: 2 in the sequence listing), LCDR3: QTYDSSRWV (SEQ ID NO: 3 in the sequence listing), HCDR1: DYYMN (SEQ ID NO: 4 in the sequence listing) , HCDR2: SISRYSSYIYYADSVKG (SEQ ID NO: 5 in the sequence listing), and HCDR3: DIGGMDV (SEQ ID NO: 6 in the sequence listing)
(b) LCDR1: SGSSSNIGNNAVS (SEQ ID NO: 7 in the sequence listing), LCDR2: ASNMRVI (SEQ ID NO: 8 in the sequence listing), LCDR3: GAWDDSQKALV (SEQ ID NO: 9 in the sequence listing), HCDR1: NYYMH (SEQ ID NO: 10 in the sequence listing) , HCDR2: SISARSRYHYYADSVKG (SEQ ID NO: 11 in the sequence listing), and HCDR3: LATRHNAFDI (SEQ ID NO: 12 in the sequence listing)
(c) LCDR1: SGSSSNIGRNAVN (SEQ ID NO: 13 in the sequence listing), LCDR2: ASNMRVS (SEQ ID NO: 14 in the sequence listing), LCDR3: WAWDDSQKVGV (SEQ ID NO: 15 in the sequence listing), HCDR1: NYYMH (SEQ ID NO: 10 in the sequence listing) , HCDR2: SISARSSYIYYADSVKG (SEQ ID NO: 16 in the sequence listing), and HCDR3: LATRNNAFDI (SEQ ID NO: 17 in the sequence listing)
(d) LCDR1: SGSSSNIGRNAVN (SEQ ID NO: 13 in the sequence listing), LCDR2: ASNMRRS (SEQ ID NO: 18 in the sequence listing), LCDR3: SAWDDSQKVVV (SEQ ID NO: 19 in the sequence listing), HCDR1: RYYMH (SEQ ID NO: 20 in the sequence listing) HCDR2: SISAQSSHIYYADSVEG (SEQ ID NO: 21 of the sequence listing) and HCDR3: LATRQNAFDI (SEQ ID NO: 22 of the sequence listing), or
(e) LCDR1: SGSSSNIGNNAVN (SEQ ID NO: 23 in the sequence listing), LCDR2: ASNMRRP (SEQ ID NO: 24 in the sequence listing), LCDR3: EAWDDSQKAVV (SEQ ID NO: 25 in the sequence listing), HCDR1: NYYMH (SEQ ID NO: 10 in the sequence listing) , HCDR2: SISARSSYLYYADSVKG (SEQ ID NO: 26 in the sequence listing), and HCDR3: LATRHVAFDI (SEQ ID NO: 27 in the sequence listing)
A method for producing a human monoclonal antibody, which is a human monoclonal antibody.
[12] A combination of amino acid sequences of the light chain variable region (VL) and the heavy chain variable region (VH) of a human monoclonal antibody,
(A) VL: QSVLTQPPSASGTPGQRVTISCTGSSSNIGAVYDVHWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCQTYDSSRWVFGGGTKLTVL and VH (SEQ ID NO: 28 of Sequence Listing): EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMNWVRQAPGKGLEWVSSISRYSSYIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDIGGMDVWGQGTLVTVSS (SEQ ID NO: 29 of the Sequence Listing)
(B) VL: QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNAVSWYQQLPGTAPKLLIYASNMRVIGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGAWDDSQKALVFGGGTKLTVL (SEQ ID NO: of the Sequence Listing 30) and VH: EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYYMHWVRQAPGKGLEWVSSISARSRYHYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLATRHNAFDIWGQGTLVTVSS (SEQ ID NO: 31 of the Sequence Listing)
(C) VL: QSVLTQPPSASGTPGQRVTISCSGSSSNIGRNAVNWYQQLPGTAPKLLIYASNMRVSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCWAWDDSQKVGVFGGGTKLTVL (SEQ ID NO: 32 of the Sequence Listing) and VH: (SEQ ID NO: 33 of the Sequence Listing) IbuikyuerueruiesujijijierubuikyuPijijiesueruarueruesushieieiesujiefutiefuesuenuwaiwaiemueichidaburyubuiarukyueiPijikeijieruidaburyubuiesuesuaiesueiaruesuesuwaiaiwaiwaieidiesubuikeijiaruefutiaiesuarudienuesukeienutieruwaierukyuemuenuesueruarueiiditieibuiwaiYCARLATRNNAFDIWGQGTLVTVSS
(D) VL: (SEQ ID NO: 34 of Sequence Listing) KyuesubuierutikyuPiPiesueiesujitiPijikyuarubuitiaiesushiesujiesuesuesuenuaijiaruenueibuienudaburyuwaikyukyueruPijitieiPikeierueruaiwaieiesuenuemuaruaruesujibuiPidiaruefuesujiesukeiesujitiesueiesuerueiaiesujieruaruesuidiieiDYYCSAWDDSQKVVVFGGGTKLTVL and VH: (SEQ ID NO: 35 of Sequence Listing) EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYYMHWVRQAPGKGLEWVSSISAQSSHIYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLATRQNAFDIWGQGTLVTVSS, or (e) VL: QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNAVNWYQQLPGTAPKLLIYASNMRRPGVPDRFSGSKSGTSASLAISGLRSEDEADYYCEAWDDSQKAVVFGGGTKLTVL and VH (SEQ ID NO: 36 of Sequence Listing): EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYYMHWVRQAPGKGLEWVSSISARSSYLYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLATRHVAFDIWGQGTLVTVSS (the sequence listing SEQ ID NO: 37),
The manufacturing method of item 11 which is.
[13] The production method according to item 11 or item 12, wherein the alkaline cleaning liquid has a pH value of 10.5 to 11.5.
[14] The production method according to any one of items 11 to 13, wherein the alkaline cleaning solution does not contain 0.2 M or more sodium chloride.
[15] The production method according to any one of items 11 to 14, wherein the alkaline cleaning liquid does not contain arginine.
[16] The production method according to any of items 11 to 15, wherein the carrier for affinity chromatography is a carrier having Protein A, Protein G, or a modified form thereof.
[17] A human monoclonal antibody produced by the method according to any one of items 11 to 16.
[18] A method for improving the physical properties of a protein having an Fc region contained in a solution,
(a) contacting a solution containing a protein having an Fc region with an affinity chromatography support, and binding the protein having an Fc region to the affinity chromatography support;
(b) contacting an alkaline solution having a pH value in the range of 9.1 to 12, and (c) eluting a protein having an Fc region,
Including a method.
[19] The method according to item 18, wherein the method for improving the physical properties of a protein having an Fc region is a method for reducing the free thiol content of a protein having an Fc region.
[20] The method according to item 18, wherein the method for improving the physical properties of a protein having an Fc region is a method for reducing the self-association property of a protein having an Fc region.
[21] The method according to item 18, wherein the method for improving the physical properties of a protein having an Fc region is a method for reducing the aggregation property of a protein having an Fc region.

[22] The method according to item 18, wherein the method for improving the physical properties of a protein having an Fc region is a method for further enhancing the storage stability of a protein having an Fc region.
[23] The method according to any of items 18 to 22, wherein the alkaline solution has a pH value of 10.6 to 11.5.

[24] The method according to any one of items 18 to 23, wherein the alkaline solution does not contain 0.2 M or more sodium chloride.
[25] The method according to any one of items 18 to 24, wherein the alkaline solution does not contain arginine.

[26] The method according to any of items 18 to 25, wherein the carrier for affinity chromatography is a carrier having Protein A, Protein G, or a modified form thereof.
[27] The method according to any of items 18 to 26, wherein the protein having an Fc region is an antibody.
[28] The method according to any of items 18 to 27, wherein the protein having an Fc region is a human monoclonal antibody, a humanized monoclonal antibody, or a chimeric monoclonal antibody.

[29] The method according to any of items 18 to 28, wherein the protein having an Fc region is an antibody having a λ chain.

The method for purifying a protein having an Fc region of the present invention can reduce at least one of the free thiol content, self-association property, and aggregation property of the protein having an Fc region. In addition, a protein having an Fc region purified by the present invention is excellent in storage stability. Furthermore, the purification method of the present invention can increase the purity and yield of a protein having an Fc region. Therefore, according to the present invention, a protein having an Fc region having excellent physical properties can be produced with high purity at low cost.

In addition, the human anti-IL-33 monoclonal antibody produced in the present invention is safe due to reduced free thiol content, self-association and aggregation, and further reduced contamination of impurities. Is excellent. Therefore, the human anti-IL-33 monoclonal antibody produced by the present invention can be used as a new diagnostic, preventive, therapeutic or alleviating agent for diseases related to IL-33.

It is the graph which evaluated the reduction | decrease of the aggregation property by the alkali treatment of the protein which has Fc area | region. It is the graph which evaluated the reduction of the self-association property by the alkali treatment of the protein which has Fc area | region. It is the graph which evaluated the improvement of the storage stability by the alkali treatment of the protein which has Fc area | region. It is the graph which showed the antibody yield when A10-1C04 antibody was refine | purified using the washing | cleaning liquid of various pH from a culture supernatant. It is the photograph which showed the result of having analyzed the washing | cleaning fraction obtained by wash | cleaning Protein A column which captured A23-1A05 antibody using the washing | cleaning liquid of various pH by SDS-PAGE. It is the graph which showed the time-dependent change of the light absorbency of the washing | cleaning liquid which passed through the column obtained by wash | cleaning Protein A column which captured A23-1A05 antibody using the washing | cleaning liquid of various pH. It is the graph which showed the free thiol content when A23-1A05 antibody was refine | purified using the washing | cleaning liquid of various pH from a culture supernatant. When the protein ST2-Fc having the Fc region is purified by adding an alkaline washing step on a Protein A column, and when the protein is purified without an alkaline washing step, the aggregation property of ST2-Fc after purification is shown by dynamic light. It is the graph measured by the scattering method.

In order to facilitate understanding of the present invention, terms used in the present invention will be described below. The terminology used herein is not intended to be limiting in particular.

[Protein with Fc region]
In the present invention, the protein having an Fc region refers to a protein having an Fc region of an immunoglobulin, a variant thereof, or a fragment thereof. The Fc region of an immunoglobulin refers to a region of a fragment on the constant site side of the heavy chain when the immunoglobulin is decomposed with papain, and is usually a region composed of C _H 2 and C _H 3 regions of the heavy chain. The modified Fc region may include those in which a part of the Fc region is substituted, deleted, and / or added. Proteins with Fc region include not only immunoglobulins such as IgG, IgM, IgD, IgE and IgA, multispecific antibodies such as bispecific antibodies, but also immunoglobulin Fc regions (including variants and fragments thereof). Fusion proteins (Fc fusion proteins) bound to other proteins and peptides. In addition, the term also includes an immunoglobulin, a multispecific antibody, or an Fc fusion protein in which a small molecule drug, an imaging compound such as a radioactive substance or a fluorescent dye, or a polymer such as PEG is bound.

[Purification]
In the present invention, purification refers to various mixed solutions containing a protein having an Fc region, which is the target protein (for example, animal cell-derived culture supernatant, microorganism-derived culture supernatant, microbial disruption product, cell-free expression system solution, gene set This refers to a technique for separating a target protein and other impurities by subjecting a sample derived from a modified animal / plant to a separation step. Impurities include medium-derived components (for example, proteins such as saccharides, lipids, growth factors and peptides thereof), host cell-derived components (for example, host cell-derived proteins, peptides, nucleic acids, lipids and saccharides), and target-derived impurities. (Cleaved body, chemically modified body and aggregate).

[Affinity chromatography]
It refers to chromatography using a substance having affinity for the target protein, for example, a carrier having a ligand. By performing affinity chromatography using a carrier having affinity for the Fc region, a protein having the Fc region can be purified and / or produced. A carrier commonly used for antibody purification can be used as a carrier for affinity chromatography having binding properties to a protein having an Fc region. As a carrier used for antibody purification, a carrier to which a ligand that binds to the Fc region is bound can be used. In the present invention, for example, Protein A, Protein G, or a variant thereof can be used as a ligand that binds to the Fc region.

[Alkaline cleaning solution]
In the present invention, the alkaline washing solution refers to a basic solution used for separating impurities from a target protein bound to an affinity chromatography carrier. The alkaline washing solution not only removes impurities, but also has an effect of reducing the free thiol content, self-association property, and / or aggregation property of the target protein. Furthermore, the recovery rate of the protein having the Fc region is improved by performing a washing step with an alkaline washing solution. The reason for this is not clear, but free thiol groups contained in proteins with Fc regions form appropriate intramolecular disulfide bonds when exposed to alkaline washings on the column, resulting in aggregation or precipitation of proteins with Fc regions. It is thought that there is no loss due to However, it is not intended to be limited to that theory.

[Protein A]
Protein A exists in the cell wall of S. aureus and has a high affinity for the Fc region of immunoglobulin. Protein A variant is Protein A in which a part of the amino acid sequence of Protein A is modified to modify the binding specificity and stability with the Fc region. ProteinA variants are commercially available, and for example, MabSelect SuRe (GE Healthcare), TOYOPEARL AF-rProtein A HC-650F (Tosoh Corp.), KANEKA KanCap (Kaneka Corp.), etc. can also be used. Protein A and a variant thereof used in the present invention may be a natural protein derived from Staphylococcus aureus, or may be produced by a gene recombination technique. From the viewpoint of carrying out the alkali washing step, it is preferable to use an alkali-durable variant.

[Protein G]
Protein G is a protein that exists in the cell wall of group G streptococci and has high affinity for the Fc region of immunoglobulin. Protein G variant is Protein G in which a part of the amino acid sequence of Protein G is modified to modify the binding specificity and stability with the Fc region. ProteinG and its variants used in the present invention may be natural proteins derived from group G streptococci, or may be produced by gene recombination techniques. From the viewpoint of carrying out the alkali washing step, it is preferable to use an alkali-durable variant.

[antibody]
In the present invention, an antibody refers to an immunoglobulin that is a glycoprotein capable of binding to a specific antigen, or a fragment thereof having an Fc region. The term also includes monoclonal antibodies, polyclonal antibodies, and multispecific antibodies.

[Human monoclonal antibody]
A human monoclonal antibody refers to a monoclonal antibody having a variable region and a constant region derived from a human germline immunoglobulin sequence, and refers to a human-derived antibody for both light and heavy chains. Depending on the constant region of the heavy chain, IgG having IgG heavy chain (including IgG1, IgG2, IgG3 and IgG4), IgM having μ heavy chain, IgA having α heavy chain (IgA1, IgA2) ), IgD having a heavy chain of δ chain, or IgE having a heavy chain of ε chain. In principle, the light chain includes either a kappa chain or a lambda chain.

[Humanized monoclonal antibody]
The humanized antibody refers to a monoclonal antibody consisting of a variable region composed of a complementarity determining region of a non-human mammal-derived antibody and a framework region derived from a human antibody, and a constant region derived from a human antibody.

[Chimeric monoclonal antibody]
A chimeric antibody refers to a monoclonal antibody in which the light chain, the heavy chain, or both are composed of a variable region derived from non-human and a constant region derived from human.

[Λ chain]
Lambda chain refers to one type of immunoglobulin light chain. The amino acid sequence of the variable region of the λ chain is diverse, and immunoglobulins containing the light chain of the λ chain can bind to various antigens. Another type of immunoglobulin light chain is the kappa chain.

[Complementarity determination region]
The complementarity determining region refers to a region that forms an antigen-binding site in the variable region of an immunoglobulin molecule, and is also referred to as a hypervariable region, and refers to a portion that has a particularly large change in amino acid sequence for each immunoglobulin molecule. In the complementarity determining region, there are three complementarity determining regions (complementarity determining region 1, complementarity determining region 2 and complementarity determining region 3) in each of the light chain and the heavy chain. In this application, the complementarity-determining regions of immunoglobulin molecules are determined according to the Kabat numbering system (Kabat et al., 1987, Sequences of Immunological Interstitut, US Department of Health and Human Services, NIH, US).

[Variable area]
The variable region of an antibody refers to a portion that is located on the N-terminal side of the light and heavy chains and determines the antigen-binding property of the antibody. The variable region is composed of three complementarity determining regions and four framework regions surrounding it.

[Self-association]
In the present invention, self-association means a temporary and reversible interaction between the same kind of protein molecules and a temporary and reversible multimer formation. Such multimer formation easily returns to the monomer by an operation such as dilution. Self-association refers to the ease of self-association. Although a small amount of impurities may affect the self-association of any protein, the association of any protein in the presence of such impurities is also included in the term self-association. Examples of the parameter indicating self-association include a second virial coefficient and an interaction parameter. The interaction parameter is an index of self-association calculated using the concentration dependence of the diffusion coefficient obtained by an experimental method. If the value is −12.4 g / mL or more, repulsive force between molecules If it is less than that, it is reported to be an attractive (self-associative) interaction (Saito et al., Pharm. Res., 2013. Vol. 30 p1263). The diffusion coefficient is an index of the ease of diffusion of molecules in a solution and can be measured by a dynamic light scattering method or the like.

[Free thiol]
In the present invention, free thiol refers to a free thiol group contained in a cysteine residue in a protein having an Fc region. In a protein having an Fc region, a disulfide bond that is supposed to be formed by cross-linking two cysteine residues between the light chain and heavy chain of an antibody or within the heavy chain may not be formed, and exists as a free thiol. The free thiol content refers to the number of moles (molar ratio) of free thiol groups per mole of protein having an Fc region.

[Cohesiveness]
In the present invention, aggregation refers to an irreversible interaction that occurs between a plurality of protein molecules, and indicates multimer formation that does not dissociate even by manipulation such as dilution. Aggregates are formed by physicochemical interactions or chemical reactions, but are not limited by the formation route. Aggregates can be confirmed by a dynamic light scattering method as a peak having a particle size larger than that of monomers (nonaggregates) that do not dissociate by dilution.

[Storage stability]
Storage stability refers to stability over a storage period under specific storage conditions. The storage stability can be evaluated by an arbitrary index related to the change occurring within the storage period. In the present invention, any index for evaluating the storage stability of a protein having an Fc region includes various factors such as appearance, antigen binding property, degradability, self-association property, free thiol content, aggregation property, and biological activity. However, it is not limited by these.

[yield]
In the present invention, the yield indicates the ratio of the amount of the target protein obtained after the purification step to the amount of the target protein contained in the mixed solution before the purification step.

[IL-33]
IL-33 is a cytokine belonging to the IL-1 family, and has a chromatin-binding domain on the N-terminal side and an IL-1-like cytokine domain with a molecular weight of 18 kDa having 12 β-strands on the C-terminal side. . IL-33 is cleaved by enzymes such as esterase, cathepsin G, or proteinase 3 in the process of necrosis in cells, and becomes mature IL-33 consisting only of the C-terminal fragment, and is thought to function as a cytokine. ing. When IL-33 is released extracellularly as a cytokine, it binds to IL-33 receptors (ST2 and IL-1RAcP) and initiates intracellular signal transduction in cells that express the IL-33 receptor. It has the function. Signal transduction induced by IL-33 includes, but is not limited to, the NF-κB pathway and the MAPKKs pathway, and ultimately induces production of various cytokines, chemokines, and inflammatory mediators. Examples of cytokines induced by IL-33 include TNF-α, IL-1β, IL-3, IL-4, IL-5, IL-6, IL-13, and particularly IL-5, IL -6 and IL-13 are induced. Examples of chemokines induced by IL-33 include CXCL2, CCL2, CCL3, CCL6, CCL17, and CCL24. Examples of inflammatory mediators induced by IL-33 include PGD2 and LTB4. IL-33-induced cytokines, chemokines, and inflammatory mediators are involved in immune system cell migration, cytokine production, and degranulation to cause inflammation. In the present invention, IL-33 may refer to either full-length IL-33 or mature IL-33, as long as it acts by binding to the IL-33 receptor, or a derivative or mutation thereof. It may be a body. Moreover, human IL-33 or IL-33 derived from other organisms may be used.

Hereinafter, embodiments of the present invention will be described. In addition, the following embodiment is an illustration for demonstrating this invention, and this invention is not limited only to the embodiment.

In one embodiment of the present invention, a method for purifying a protein having an Fc region is provided. This method is characterized in that a protein having an Fc region is bound to an affinity chromatography support and then washed with an alkaline washing solution. Impurities are removed with an alkaline washing solution, and the free thiol content, self-association and aggregation properties of the protein containing the Fc region bound to the affinity chromatography support are reduced. Next, the protein having the Fc region is purified by eluting the protein having the Fc region using the eluate. The cleaning with the cleaning liquid may be performed once, or may be performed a plurality of times using the same or different cleaning liquid.

In the present invention, the solution containing a protein having an Fc region includes, but is not limited to, a body fluid containing a protein having an Fc region such as blood, ascites, and milk, and a protein encoding the protein so as to produce a protein having an Fc region. B cells that have been immortalized by cell disruption solutions such as transgenic E. coli, genetically modified yeast, and genetically modified plants transformed with the gene to be transformed, and hybridomas that produce proteins having the Fc region, Epstein-Barrvirus (EBV), etc. Examples include culture supernatants obtained by culturing cells and transgenic animal cells, and culture supernatants containing proteins having an Fc region are preferred. In the present invention, as a solution containing a protein having an Fc region, a solution obtained by re-dissolving the solution after further precipitation, a concentrated solution, a diluted solution, a dialyzed solution, and a solution subjected to a rough purification process by chromatography or the like Etc.

In the present invention, the solution containing the protein having the Fc region may contain impurities other than the protein having the target Fc region. Impurities include components derived from body fluids and host cells (proteins, nucleic acids, lipids, sugar chains, etc.), contaminated viruses, bacteria, fungi and components constituting them, components derived from culture media, and degradation products of proteins having an Fc region , Aggregates, and sub-visible particles.

In various embodiments, the present invention includes the steps of contacting a protein containing an Fc region with an affinity chromatography support and binding the protein having an Fc region to the affinity chromatography support. As the carrier for affinity chromatography, Protein A, Protein G, Protein A / G, Protein L, anti-immunoglobulin antibody, Fc receptor extracellular domain and fragments thereof, and carriers on which these variants are immobilized are used. As mentioned above, it is possible to bind a protein having a desired Fc region, to retain a protein having an Fc region even in a washing step with an alkaline washing solution, and to elute a protein having an Fc region in an elution solution. There is no particular limitation as long as it is possible. A preferred embodiment of the carrier for affinity chromatography of the present invention is a carrier having Protein A, Protein G, or a modified form thereof.

The purification method in the present invention can be performed using a column packed with a carrier for affinity chromatography. Moreover, it can also carry out by a batch process, without using a column. A preferred embodiment of the present invention is a purification method using a column. When a column is used, a contact step, a washing step, and a solution containing a protein having an Fc region are passed through a column packed with an affinity chromatography carrier, then an alkaline washing solution, and finally an elution solution. An elution step is performed. Each process may be performed continuously or at intervals. Further, a cleaning step with a cleaning solution other than the alkaline cleaning solution, a column equilibration step, a regeneration step, and the like may be further added.

In order to separate the protein containing the Fc region bound to the carrier for affinity chromatography from impurities, the column packed with the carrier can be washed with one or a plurality of washing solutions. Examples of general cleaning solutions include physiological saline and Phosphate buffered saline (PBS), and a surfactant may be added to these cleaning solutions. Examples of the surfactant include polysorbate (Tween 20, Tween 40, Tween 60, Tween 80 (above trade name), etc.) and Triton X-100 (trade name). The amount of the surfactant added may be not less than the critical micelle concentration, and is preferably in the range of 0.01% (V / V) to 1% (V / V).

In various embodiments, the present invention includes a step of washing an affinity chromatography support to which a protein having an Fc region is bound with an alkaline solution. The pH of the alkaline cleaning solution is in the range of 9 to 12, preferably 9.5 to 12, more preferably 10 to 12, even more preferably 10.1 to 12, 10.5 to 12, 10.6 to 12, 10.5 to 11.5, Most preferably from 10.6 to 11.5. The lower limit of the pH of the alkaline cleaning solution is, for example, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10, 7, 10.8, 10.9 and 11 are mentioned, and the upper limit of the pH of the alkaline cleaning liquid is 11, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12. Buffering agents can be added to maintain the pH of the wash solution. The buffer may be appropriately selected from those commonly used by those skilled in the art depending on the pH to be used. Phosphate, citrate, glycine, trishydroxyaminomethane (Tris), borate, carbonate , Bicarbonate, and good buffer buffers such as CHES, CAPS, Bicine, and TAPS. A preferred embodiment of the alkaline washing solution of the present invention is an alkaline washing solution containing sodium carbonate or sodium phosphate, and the concentration of these buffering agents is preferably 10 mM to 1000 mM, more preferably 50 mM to 500 mM, most preferably 100 mM. 250 mM.

In the alkaline cleaning liquid of the present invention, amino acids (for example, arginine, histidine, leucine, isoleucine, valine), divalent cations (for example, divalent cations of manganese, nickel, copper, magnesium, calcium, barium), surfactants (for example, , Polysorbate, Triton X-100, etc.), protein denaturing agents (eg, urea, guanidine hydrochloride), redox agents (eg, dithiothreitol, 2-mercaptoethanol, TCEP, copper sulfate, cystamine, cystine, glutathione) Can be added. A preferred alkaline cleaning liquid in the present invention is an alkaline cleaning liquid to which these are not added.

A salt that is not a buffer (for example, sodium chloride or potassium chloride) may be added to the alkaline washing solution in affinity chromatography. Salts that are not buffering agents are effective in washing impurities bound to the affinity chromatography support by ionic bonds, and are less than 0.05M, less than 0.1M, less than 0.15M, less than 0.2M, less than 0.5M, less than 1M, 2M It can be added to the alkaline cleaning liquid of the present invention at a concentration of less than. A preferred alkaline cleaning solution in the present invention is an alkaline cleaning solution that does not contain a salt that is not a buffer, such as sodium chloride.

In various embodiments, the present invention includes a step of eluting a protein having an Fc region from an affinity chromatography support. The eluting step is performed using an appropriate eluent. Any eluate may be used as long as the protein having the Fc region can be eluted from the bound affinity chromatography support, for example, a low pH, preferably 2 to 6.5, more preferably 2.5 to 4.0. Can be used. The preferred eluent of the present invention is 100 mM acetate buffer or glycine hydrochloride solution.

The protein having the Fc region recovered from the eluate after the alkali washing step has a reduced free thiol content, self-association property, and aggregation property, and is excellent in storage stability. In addition, additional purification steps may be performed to remove contaminating impurities. Typical purification steps include ion exchange chromatography, hydrophobic chromatography, hydroxyapatite chromatography, affinity chromatography, size exclusion chromatography, ammonium sulfate precipitation, ethanol precipitation, reverse phase HPLC, chromatofocusing, mixed mode chromatography, Depth filters, ultrafiltration and diafiltration.

In a preferred embodiment of the purification method of the present invention, the protein having an Fc region is an antibody. Particularly preferred embodiments are human monoclonal antibodies, humanized monoclonal antibodies, and chimeric monoclonal antibodies. These antibodies are useful as therapeutic drugs because they have low antigenicity to humans and can be administered to humans. The most preferred embodiment is a human antibody.

In another preferred embodiment of the purification method of the present invention, the protein having an Fc region is an antibody having a λ chain. An antibody having a λ chain is generally more aggregated and difficult to handle than an antibody having a κ chain. Therefore, the purification method of the present invention including an alkali washing step capable of reducing the aggregation property is extremely useful for purifying an antibody having a λ chain.

In another aspect, the present invention relates to a method for producing a protein having an Fc region. By washing the protein having an Fc region on an affinity chromatography support using an alkaline washing solution, the protein having an Fc region with reduced aggregation, self-association and free thiol content and excellent storage stability is obtained. It can be manufactured inexpensively, easily and in high yield.

In another aspect, the present invention relates to a protein having an Fc region produced by the production method of the present invention. The protein having the Fc region produced by the production method of the present invention is characterized in that it has a predetermined self-association property, free thiol content, and aggregation property. Such proteins have excellent non-aggregation properties characterized by having an interaction parameter of -12.4 mL / g or higher, a free thiol content of 0.1 mol or less per mol of protein, and a particle size of greater than 50 nm. Characterized by one or more, preferably 2, and more preferably 3 properties.

A preferred embodiment of the present invention is a method for producing a human monoclonal antibody capable of binding to IL-33. Human anti-IL-33 monoclonal antibody is washed on an affinity chromatography carrier with an alkaline washing solution to reduce aggregation, self-association, and free thiol content, and human anti-IL-33 has excellent storage stability Monoclonal antibodies can be produced inexpensively, easily and in high yield.

In another preferred embodiment, the present invention relates to a human anti-IL-33 monoclonal antibody produced by the production method of the present invention. The human anti-IL-33 monoclonal antibody produced by the production method of the present invention is characterized in that it has a predetermined self-association property, free thiol content, and aggregation property. Such proteins have excellent non-aggregation characteristics characterized by having an interaction parameter of -12.4 mL / g or more, a free thiol content of 0.1 mol or less per mol of antibody, and a particle size of more than 50 nm. Characterized by one or more, preferably 2, and more preferably 3 properties. A human anti-IL-33 monoclonal antibody having such properties has excellent storage stability and is useful as a pharmaceutical product.

In another aspect, the present invention relates to a method for reducing the self-association property of a protein having an Fc region contained in a solution. Self-association can be evaluated by analyzing dynamic light scattering and calculating interaction parameters, as described in the Examples of the present application. If the interaction parameter is high, the self-association property is low, and the interaction parameter is preferably −12.4 mL / g or more. As a preferred embodiment of the present invention, the interaction parameter in the solution of the protein having the Fc region subjected to the alkaline solution treatment is increased by 1 mL / g or more as compared with the protein having the Fc region not subjected to the alkaline solution treatment. An embodiment is an increase of 5 mL / g or more, and a still more preferable embodiment is an increase of 10 mL / g or more, thereby reducing self-association.

In yet another aspect, the present invention relates to a method for reducing the free thiol content of a protein having an Fc region contained in a solution. Free thiol has high reactivity, and a protein having an Fc region aggregates irreversibly by forming a disulfide bond or a thioether bond between molecules of the protein having the Fc region. The free thiol content can be measured by a chemical method as described in the Examples of the present application. As a preferred embodiment of the present invention, the free thiol content in the solution of the protein having the Fc region subjected to the alkaline solution treatment is reduced by 0.01 or more compared to the protein having the Fc region not subjected to the alkaline solution treatment. Is reduced by 0.05 or more, and more preferably, it is reduced by 0.1 or more.

In still another aspect, the present invention relates to a method for reducing the aggregation property of a protein having an Fc region contained in a solution. As described in Examples of the present application, by measuring dynamic light scattering or the like, it is possible to confirm a peak of an aggregate having a particle size larger than that of a protein monomer having an Fc region. As a preferred embodiment of the present invention, when a solution having a constant concentration (for example, 1 mg / mL, 10 mg / ml, 50 mg / ml) is measured, an alkaline solution is compared with a protein having an Fc region that is not treated with an alkaline solution. The peak area (dynamic light scattering, light intensity distribution) of the protein aggregate having the Fc region to be treated is reduced by 65% or more, more preferably 80% or more, and even more preferably 90% or more, most preferably A preferred embodiment is a reduction of 95% or more.

In still another aspect, the present invention relates to a method for enhancing the storage stability of a protein having an Fc region contained in a solution. As described in Examples of the present application, evaluation can be performed by storing a protein having an Fc region at a constant temperature for a certain period. For example, proteins with Fc regions can be stored at 4 ° C for 1 month, 2 months, 6 months, 1 year, 2 years, 3 years, or 40 ° C for 3 days, 4 weeks, 8 weeks, The storage stability of a protein having a region can be evaluated by measuring the aforementioned self-association property, free thiol content, and aggregation property. In addition, the presence or absence of a degradation product of a protein having an Fc region can also be examined by size exclusion chromatography, SDS polyacrylamide electrophoresis or mass spectrum analysis generally performed by those skilled in the art. As a preferred embodiment of the present invention, as compared with a protein having an Fc region that is not treated with an alkaline solution, a peak of an aggregate when a protein having an Fc region in the case of alkaline solution treatment is stored at a constant temperature for a certain period of time. The area (dynamic light scattering, light intensity distribution) is reduced by 80% or more, more preferably 90% or more, and still more preferably 95% or more.

In another aspect, the present invention relates to a purification method capable of simultaneously inactivating a virus contained in a solution containing a protein having Fc. Viruses such as retroviruses are known to be rapidly inactivated under alkaline conditions (Biotechnol Prog. 2003. １９ 19: 538-43.). Usually, virus inactivation is carried out as a separate step after purification with an affinity column. However, in the present invention, it is possible to reduce the process time and cost by carrying out simultaneously with the purification. Viruses such as retroviruses can be inactivated by using an alkaline washing solution during affinity column purification.

In another aspect, the present invention relates to a reduction in the cleaning-in-place (CIP) of the chromatography carrier. In the production of biopharmaceuticals, various impurities are adsorbed on the chromatographic carrier during the purification process using chromatography, and carry over to the next purification cycle. In order to maintain the quality of the target protein obtained by purification, it is necessary to wash the chromatographic support at fixed intervals. In-situ cleaning is often performed under harsh conditions such as sodium hydroxide solution or a combination of sodium hydroxide and a high concentration salt (Journal of Chromatography A, 1308 (2013) 86- 95). In the present invention, the impurities adsorbed on the chromatography carrier can be removed by washing under mild alkaline conditions for each cycle. Therefore, by using the present invention, it is possible to eliminate the stationary cleaning under severe conditions or reduce the frequency of stationary cleaning.

The pH of the alkaline solution in the present invention is in the range of 9 to 12, preferably 9.5 to 12, more preferably 10 to 12, even more preferably 10.1 to 12, 10.5 to 12, 10.6 to 12, 10.5 to 11.5. And most preferably from 10.6 to 11.5. The lower limit of the pH of the alkaline solution is, for example, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10, 7, 10.8, Examples of the upper limit of the pH of the alkaline solution include 11, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12. Buffering agents can be added to maintain the pH of the alkaline solution. The buffer may be appropriately selected from those commonly used by those skilled in the art depending on the pH to be used. Phosphate, citrate, glycine, trishydroxyaminomethane (Tris), borate, carbonate , Bicarbonate, and good buffer buffers such as CHES, CAPS, Bicine, and TAPS. The buffer used in the alkaline solution of the present invention is preferably a non-amino acid buffer. A preferred embodiment of the alkaline solution of the present invention is an alkaline solution containing sodium carbonate or sodium phosphate, and the concentration of these buffering agents is preferably 10 mM to 1000 mM, more preferably 50 mM to 500 mM, most preferably 100 mM. 250 mM.

In the alkaline solution of the present invention, amino acids (for example, arginine, histidine, leucine, isoleucine, valine), divalent cations (for example, manganese, nickel, copper, magnesium, calcium, barium), surfactants (for example, polysorbate, Triton) X-100 etc.), protein denaturing agents (eg urea, guanidine hydrochloride), redox agents (eg dithiothreitol, 2-mercaptoethanol, TCEP, copper sulfate, cystamine, cystine, glutathione) may be added. it can. A preferred alkaline solution in the present invention is an alkaline solution to which these are not added. In particular, the alkaline solution in the present invention preferably does not contain arginine and its derivatives.

A salt that is not a buffer (eg sodium chloride, potassium chloride) is added to the alkaline solution of the present invention at a concentration of less than 0.05M, less than 0.1M, less than 0.15M, less than 0.2M, less than 0.5M, less than 1M, less than 2M. can do. A preferred alkaline solution in the present invention is an alkaline solution that does not contain a salt that is not a buffer, such as sodium chloride.

In one embodiment of the present invention, the protein having an Fc region is an antibody. The antibody in the present invention may be a polyclonal antibody or a monoclonal antibody, and may be an antibody derived from any animal such as a mouse antibody, a human antibody, a rat antibody, a rabbit antibody, a goat antibody, or a camel antibody. Furthermore, the antibody in the present invention may be a chimeric antibody or a humanized antibody in which these animal-derived antibodies are combined.

Another embodiment of the protein having the Fc region of the present invention includes an Fc fusion protein. Proteins that fuse with the Fc region may be, but are not limited to, extracellular domains of membrane-bound proteins such as TNF receptors and CTLA-4, including cytokines, chemokines, enzymes, growth factors, coagulation factors, hormones and these It may be an agonist or an antagonist whose function is altered. It may also be an artificial antigen-binding protein using the extracellular domain of human fibronectin or the Kunitz domain of a serine protease inhibitor (Clifford Mintz et.al BioProcess International, 2013, Vol.11 (2) , Pp40-48).

The Fc region of the protein having the Fc region of the present invention is a C-terminal region of an IgG antibody, preferably a human-derived IgG1, IgG2, IgG3, IgG4 Fc region, more preferably a human IgG1 Fc region. is there. The sugar chain bound to the Fc region may be modified, and in particular, by adjusting the fucose content, the antibody-dependent cytotoxic activity of the antibody can be regulated (WO 2005/035586, International Publication No. 2002/31140, International Publication No. 00/61739). Further, the amino acid sequence of the Fc region may be modified as long as it can bind to the carrier for affinity chromatography. For example, human IgG1 and human IgG3 chimeric Fc region, Fc with enhanced binding to Fc receptor (FcRn) The area can be raised (Shouhei Hashiguchi, Biochemistry, 2010, Vol.82 (8), p710).

In one embodiment of the present invention, the protein having an Fc region is an antibody having a λ chain. The light chain of an antibody is composed of a λ chain or a κ chain, but an antibody having a λ chain is useful because it has diversity as well as an antibody having a κ chain and can bind to various antigens.

In a preferred embodiment, the protein having the Fc region to be purified according to the present invention is a human anti-IL-33 monoclonal antibody. The human anti-IL-33 monoclonal antibody is described in Japanese Patent Application No. 2014-078223 filed on April 4, 2014.

Hereinafter, the method for producing a protein having an Fc region of the present invention will be described. Using a genetic engineering technique, DNA containing a DNA sequence encoding a protein having a desired Fc region is incorporated into an expression vector, transformed into a host cell, and the host cell is cultured. A protein having an Fc region can be produced (for example, Borrebaeck C. A. K. and Larrick J. W. THERAPEUTIC MONOCLONAL ANTIBODIES, Publicized in the United KingdomMIBLISSL When the protein having the Fc region is an Fc fusion protein, the entire length of the protein having the Fc region is encoded by linking the DNA encoding the Fc region downstream of the DNA encoding the protein to be fused with the Fc region. A DNA sequence can be generated.

A chimeric antibody, which is a preferred embodiment of the present invention, is produced by ligating a DNA encoding a non-human-derived antibody variable region with a DNA encoding a human antibody constant region, incorporating this into an expression vector, and transforming it into a host cell. (See European Publication No. 125023, International Publication No. 92/197559).

A humanized antibody which is a preferred embodiment of the present invention is a ligation of a complementarity determining region of an antibody derived from a non-human mammal and a DNA encoding the other part of the human antibody region, which is incorporated into an expression vector and introduced into a host. It is obtained by making it produce.

A human antibody which is a preferred embodiment of the present invention can be obtained, for example, using DNA encoding the light chain and heavy chain variable regions of a human antibody obtained by screening a human antibody phage library. Human antibodies include trioma technology, human B-cell hybridoma technology (Kozbor et al., 1983 Immunol Today 4: p72), and EBV hybridoma technology for generating human monoclonal antibodies (Cole et al., 1985, MONOCLONAL ANTIBODIES ANDANDANCE CANCER R. Liss, Inc., p. 77) etc. can also be used. Furthermore, a human antibody can also be produced by immunizing a transgenic mouse introduced with a human antibody gene with an antigen protein to produce a hybridoma. Examples of the transgenic mice include HuMab (registered trademark) mice (Medarex, Princeton NJ), KMTM mice (Kirin Pharma Company, Japan), KM (FCγRIIb-KO) mice and the like.

A DNA sequence encoding the full length of the heavy chain and the full length of the light chain of the human anti-IL-33 monoclonal antibody preferable in the present invention is, for example, IgG1 having a λ chain as the light chain, and is represented in Table 1 below:

An in vitro production system can be used as a production system for producing a protein having an Fc region. Examples of in vitro production systems include production systems using eukaryotic cells such as animal cells, plant cells or fungal cells, and production systems using prokaryotic cells such as bacterial cells such as Escherichia coli and Bacillus subtilis. As animal cells used, mammalian cells such as CHO, COS, myeloma, BHK, HeLa, Vero, 293, NS0, Namalwa, YB2 / 0, commonly used cells, insect cells, etc. may be used. However, 293 cells and CHO cells are preferred.

As described above, when a protein having an Fc region is produced in an in vitro production system, DNA encoding the protein having the Fc region is incorporated into an expression vector to transform the host. Preferred examples of vectors that can be used in animal cells include, but are not limited to, pConPlus, pcDM8, pcDNA I / Amp, pcDNA3.1, and pREP4.

A hybridoma can be used as an in vitro production system for producing a protein having an Fc region. A hybridoma can be obtained by fusing lymphocytes and a myeloma cell line according to a general method (for example, Kohler et al. Nature 256, 495 (1975), Gelfre et al. Nature 266, 55052 (1977). Examples of strains include P3-NS1 / 1-Ag4-1, P3-x63-Ag8.653 and Sp2 / O-Ag14.

Another aspect of the present invention resides in a pharmaceutical composition comprising a human anti-IL-33 monoclonal antibody produced by the production method of the present invention. This pharmaceutical composition can be used for diagnosis, treatment, prevention or alleviation of IL-33 related diseases. Further, a method for diagnosis, treatment, prevention or alleviation of an IL-33 related disease comprising administering the human anti-IL-33 monoclonal antibody of the present invention, and a medicament for diagnosis, treatment, prevention or alleviation of an IL-33 related disease To the use of the monoclonal antibodies of the invention for the production of

Examples of IL-33 related diseases include, but are not limited to, asthma, atopic dermatitis, urticaria, hay fever, anaphylactic shock, eosinophilic sinusitis, hypereosinophilic syndrome, Churg-Strauss syndrome , Allergic encephalomyelitis, polymyalgia rheumatica, rheumatic heart disease, multiple sclerosis, arthritis (eg, rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, osteoarthritis, Reiter syndrome), systemic Erythromatodes (including discoid lupus), psoriasis, ankylosing spondylitis, hepatitis (eg, autoimmune hepatitis, chronic active hepatitis, etc.), inflammatory bowel disease (eg, ulcerative colitis, Crohn's disease, gluten) Susceptible bowel disease, etc.), systemic lupus erythematosus, Sjogren's syndrome, Behcet's disease, pemphigus, pemphigoid, autoimmune hemolytic anemia, autoimmune inflammatory eye disease, autoimmune neonatal blood Platelet reduction, autoimmune neutropenia, autoimmune ovitis and testicularitis, autoimmune thrombocytopenia, autoimmune thyroiditis, polymyositis, dermatomyositis, myasthenia gravis, adrenergic agonist Tolerance, alopecia greata, antiphospholipid syndrome, autoimmune diseases of the adrenal glands (eg, autoimmune Addison disease), celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), cold agglutinin Disease, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis (eg, IgA nephropathy), Graves' disease, hyperthyroidism (ie, Hashimoto's thyroiditis), idiopathic platelets Reduced purpura (ITP), mixed connective tissue disease, type 1 or immune-mediated diabetes, pernicious anemia, polychrondritis itis), multi-gland syndrome, stiff man syndrome, vitiligo, sarcoidosis, multi-endocrine disorders, other endocrine insufficiency, arteriosclerosis, liver fibrosis (eg primary biliary cirrhosis, etc.), pulmonary fibrosis (eg , Idiopathic pulmonary fibrosis, etc.), chronic obstructive pulmonary disease, scleroderma (including CREST syndrome, Raynaud phenomenon, etc.), tubulointerstitial nephritis, dense deposit disease, acute kidney injury, myocarditis, cardiomyopathy, Neuritis (eg, Guillain-Barre syndrome), nodular polyarteritis, cardiotomy syndrome, chronic inflammatory demyelinating polyneuropathy, IgA neuropathy, lichen planus, Meniere's disease, post-myocardial Infarction (post-MI), uveitis, uveitis ophthalmitis (Uveitis Optalmia), vasculitis, primary agammaglobulinemia, cancer ( For example, brain tumor, laryngeal cancer, oral cavity cancer, hypopharyngeal cancer, thyroid cancer, esophageal cancer, breast cancer, lung cancer, stomach cancer, adrenocortical cancer, bile duct cancer, gallbladder cancer, liver cancer, pancreatic cancer, bladder cancer, colon cancer, uterus Cancer, ovarian cancer, prostate cancer, testicular cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, Ewing tumor, Hodgkin disease, non-Hodgkin lymphoma, melanoma, mesothelioma, multiple myeloma, etc.), immune system exclusion Infection (eg, severe acute respiratory syndrome (SARS)), lethal cytokine storm associated with highly toxic influenza infection, and sepsis, preferably asthma, atopic dermatitis, hay fever, Anaphylactic shock, scleroderma, Crohn's disease, ulcerative colitis, arthritis, systemic lupus erythematosus, ankylosing spondylitis, liver fibrosis, pulmonary fibrosis, acute kidney injury, vasculitis and cancer It is.

Other preferred proteins having an Fc region include proteins having an Fc region that is recognized for therapeutic use in humans. Examples of such proteins having an Fc region include antibodies that bind to tumor cell antigens, cytokines, cytokine receptors or adhesion proteins, and Fc fusion proteins. Thus, in various embodiments, a protein having an Fc region may be an antibody or Fc fusion protein that binds to an antigen selected from the group consisting of: CD3 (eg, OKT3), CD52 (eg, alemtuzumab) Campath®), VEGF (eg, bevacizumab; Avastin®, EGFR (eg, cetuximab; Erbitux®), CD33 (eg, gemtuzumab; Mylotarg®), CD20 (eg, rituximab); Rituxan®, tositumomab; Bexxar®, ibritumomab; Zevalin®), HER-2 (eg, trastuzumab; Herceptin®), TNFα (eg, infliximab; Remicade®) Adalimumab: Humira®, etanercept; Embrel®, Golimumab; Simponi®), CD25 (Eg, daclizumab; Zenapax®, basiliximab; Simulect®), RSV (eg, palivizumab; Synagis®), IgE (eg, omalizumab; Xolair®), gpIIb / IIIa ( For example, abciximab; Reopro®), CD11a (eg, efalizumab; Raptiva®), α4 integrin (eg, natalizumab; Tysabri®), IL-6 receptor (eg, tocilizumab; Actemra) Registered trademark)), CD80 / 86 (eg, abatacept; Orencia®), CTLA-4 (eg, ipilimumab; YERVOY®), BLys (eg, belimumab; Benlysta®), IL- 17A (eg, secukinumab; Cosentyx®), PD-1 (eg, nivolumab; Opdivo®, pembrolizumab; KEY TRUDA (registered trademark)).

Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to the following unless otherwise specified.

The examples use seven different human anti-IL-33 monoclonal antibodies (A10-1C04, A23-1A05, A25-2C02, A25-3H04, A26-1F02, A00-0070, A00-0036). An expression vector for mammalian cells expressing IgG was constructed by inserting DNA encoding the light chain and heavy chain amino acid sequences of each antibody downstream of the CMV promoter. The DNA sequence of the light chain of the human anti-IL-33 monoclonal antibody is SEQ ID NO: 38, 40, 42, 44, 46, 48 and 50, respectively, and the heavy chain DNA sequence is respectively SEQ ID NO: 39, 41, 43, 45, 47, 49 and 51 were used. The expression vector was introduced into FreeStyle 293-F cells (Life Technologies) using the gene introduction reagent NeoFection 293-1 (Aspec). The culture supernatant was obtained after culturing for 5 days after gene introduction. A stable expression strain by CHO cells was established by a GS system (Lonza) using pConPlus vector and CHO K1SV cells. The CHO cell stable expression strain was cultured from 0.3 × 10 ⁶ cells / mL using WAVE Bioreactor SYSTEM 20/50 EHT (GE Healthcare), and the culture supernatant containing secreted IgG was collected. The following examples were carried out using these culture supernatants.

Example 1: Antibody purification by Protein A chromatography Purification 1 (without alkaline washing step):
A Protein A column (HiTrap MabSelect SuRe, GE Healthcare) was connected to AKTA explorer 100 (GE Healthcare). PBS (pH 7.2) was sent to HiTrap MabSelect SuRe to equilibrate the column, and each culture supernatant was added. PBS (pH7.2) was fed to the column in 6 Column Volume (CV) (6 min) and the column was washed. Then, 100 mM glycine hydrochloride buffer (pH3.2) was sent in 10 CV (10 min), The bound antibody was eluted and the desired fraction was recovered from the chromatogram. Note that 1.0 M Tris-HCl buffer (pH 8.8) was previously added to the collection tube so that the pH was around neutral.

Purification 2 (with alkali cleaning process):
A Protein A column (HiTrap MabSelect SuRe) was connected to AKTA explorer 100. PBS (pH 7.2) was sent to HiTrap MabSelect SuRe to equilibrate the column, and each culture supernatant was added. PBS (pH 7.2) was fed to the column at 6 CV (6 min), the column was washed, and then 100 mM sodium carbonate buffer (pH 11.0) was fed at 6 CV (6 min). PBS (pH 7.2) was sent to the column at 8 CV (8 min) and the column was washed, then 100 mM glycine hydrochloride buffer (pH 3.2) was sent at 10 CV (10 min) to elute the antibody bound in the column. The desired fraction was recovered from the chromatogram. Note that 1.0 M Tris-HCl buffer (pH 8.8) was previously added to the collection tube so that the pH was around neutral.

Dialysis:
The antibody solution obtained by the purification 1 or the purification 2 was centrifuged and the supernatant was collected, followed by dialysis with Slide-A-Lyzer G2 10,000 MWCO (Thermo Scientific). PBS (pH 7.2) was used as the dialysis external solution, and the dialysis external solution was exchanged halfway. After collecting the dialyzed sample, it was filtered with 0.45 μm PES filter GD / X (GE Healthcare) to prepare a purified antibody.

Concentration measurement:
The human antibody concentration contained in the culture supernatant was measured by BLItz (Bio-Layer Interferometry) method using BLItz (Pall forte BIO).
The concentration of the purified antibody was measured with NanoDrop ND-1000 (Thermo Scientific). The concentration value was calculated by using E ^1% ₂₈₀ = 13.7 as the extinction coefficient at that time.

From the result of measuring the concentration of the purified antibody and the result of measuring the antibody concentration in the culture supernatant, the recovery rate under the purification conditions for the presence or absence of the alkaline washing step for each of the five types of antibodies was calculated. In addition, the recovery rate ratio (recovery rate with an alkali cleaning step / recovery rate without an alkali cleaning step) with or without an alkali cleaning step was also calculated using this recovery rate. As a result, 4 of the 5 antibodies showed an effect of improving the recovery rate by the alkali washing step in the Protein A purification step. In particular, in A25-2C02, the recovery rate was improved about 20 times. Of 20 antibodies (22 purified batches), 17 antibodies (19 purified batches) showed a yield improvement effect, and the recovery rate was also improved in trastuzumab having κ chains as light chains. The average recovery ratio of 20 antibodies (22 purified batches) was 2.7 times.

Example 2: Measurement of free thiol content Reduced glutathione was sequentially diluted with 0.1 M sodium phosphate buffer (pH 6.0) containing 1 M EDTA to prepare a standard solution. After adding 50 μL of 10 mM 4,4 ′ Dithiodipyridine (4DTP) per 950 μL of the standard solution and allowing to stand at room temperature for 10 minutes, the absorbance at 324 nm was measured with a spectrophotometer to prepare a calibration curve.
The purified antibody was diluted 10-fold with 0.1 M sodium phosphate buffer (pH 6.0) containing 1 mM EDTA to obtain a measurement sample. The color was developed with 4DTP as in the standard solution, and the amount of free thiol was calculated from the calibration curve. The free thiol content (molar ratio) per mole of antibody was calculated from the amount of free thiol obtained and the antibody concentration calculated by separately measuring the absorbance at 280 nm.

As a result, the free thiol content of the antibody obtained by the purification method according to the purification 1 without the alkali washing step is higher than the free thiol content of the antibody obtained by the purification method according to the purification 2 after the alkaline washing step. (Table 3). Since the variable region of the antibody subjected to the test does not contain a cysteine residue, the detected free thiol is considered to be a thiol group of a cysteine residue present in the constant region. Since such a free thiol group is highly reactive and may cause aggregation by forming a disulfide bond between molecules, it is possible to obtain an antibody with low risk such as aggregation by adding an alkali washing step. did it. The reason why the free thiol content is reduced by performing the alkali washing step is that the free thiol group in the cysteine residue that could not form an appropriate disulfide bond in the antibody molecule is converted into an appropriate intramolecular disulfide under alkaline conditions. It is thought to form a bond, but is not intended to be bound by this theory.

Example 3: Measurement of aggregation property The purified antibody solution dissolved in PBS (pH 7.2) was concentrated to several tens mg / mL using an ultrafiltration membrane (Vivaspin turbo 50000 MWCO 15 mL, Sartorius). . The concentration of the antibody was 45 mg / mL in A00-0070 by Purification 2 with an alkali washing step, 21 mg / mL in A00-0070 by Purification 1 without an alkali washing treatment, and the purification with an alkali washing step. In A00-0036 according to No. 2, the concentration was 47 mg / mL, and in A00-0036 according to Purification 1 without the alkali washing step, it was 44 mg / mL. The concentrated solution was subjected to particle size measurement using a dynamic light scattering measurement device (Nanotrac UPA UT151 Nikkiso). In addition, in order to confirm whether the aggregate formation was reversible, the particle size of a sample diluted two-fold sequentially with PBS (pH 7.2) was measured. As shown in FIG. 1, it was found that A00-0070 and A00-0036 that did not undergo the alkali washing step formed aggregates and formed irreversible aggregates that did not return even after dilution. On the other hand, it was shown that A00-0070 and A00-0036 that had undergone the alkali washing step hardly formed aggregates. From this, it was possible to obtain an antibody that does not aggregate by using an alkaline washing step even for an antibody that forms an aggregate.

Example 4: Measurement of self-association The interaction parameter representing the concentration dependence of the diffusion coefficient (inversely proportional to the particle diameter) is an important index that is also used in the formulation design of high-concentration protein preparations such as antibodies. If the value of the interaction parameter is higher than −12.4 mL / g, it is reported that it is a repulsive interaction and excellent in colloidal stability and low in self-association (Saito et al., Pharm. Res., 2013. Vol.30 p1263).

The purified antibody solution dissolved in PBS (pH 7.2) is concentrated to a few tens mg / mL with an ultrafiltration membrane, and the particle size measurement of a sample diluted twice with the same solvent is performed. (Nanotrac UPA UT151 Nikkiso). The diffusion coefficient was calculated from the obtained particle diameter by the following Stokes-Einstein equation.

{Where D is the diffusion coefficient (cm ² / sec), K _B is the Boltzmann constant, T is the thermodynamic temperature (° C.), π is the circumference, η is the dilution viscosity P (poise), d is the particle size ( cm). }
The concentration dependence of the diffusion coefficient was plotted, and the interaction parameter was obtained by fitting with the following calculation formula.

From this equation, the interaction parameter k _D which is the slope of the fitting line was obtained.

As shown in FIG. 2, the interaction parameters of A23-1A05, A26-1F02, and A25-3H04 by the purification 1 without passing through the alkali washing step are low, and they have an attractive (self-associative) interaction. On the other hand, the interaction parameters of A23-1A05, A26-1F02, and A25-3H04 by the purification 2 after the alkali washing step were high, and the self-association property was reduced. By using an alkali washing treatment step, it was possible to obtain an antibody with reduced self-association properties.

Example 5: Improving storage stability About 10 mg / mL A10-1C04 antibody dissolved in pH 6.3 citrate buffer (50 mM citrate, 150 mM NaCl) was stored at 4 ° C or 40 ° C for 3 days . As shown in FIG. 3, when the particle size was measured by a dynamic light scattering method (Zetasizer μV, Malvern), when the A10-1C04 antibody purified by the above-mentioned purification 1 without being subjected to an alkali washing step was stored at 40 ° C. for 3 days. In contrast, aggregates were observed, whereas no aggregate formation was observed with the A10-1C04 antibody obtained by the purification 2 after the alkali washing step. The storage stability could be improved by the alkali washing step.

Example 6: pH Dependence of Yield In order to confirm the pH dependency of the yield improvement effect by the alkali washing step, purification was carried out using seven kinds of washing solutions having different pHs. A Protein A column (ToyoScreen AF-rProtein A HC-650F) was connected to AKTA explorer 100. A 100 mM KH ₂ PO ₄ 150 mM NaCl (pH 7.2) solution was fed to the Protein A column to equilibrate the column, and then the culture supernatant in which the A10-1C04 antibody was expressed was added. After feeding the column with 100 mM KH ₂ PO ₄ 150 mM NaCl (pH 7.2) solution at 6 CV (6 min) and washing the column, the column is washed with 100 mM KH ₂ PO ₄ 150 mM NaCl (pH 7.2) neutral washing solution or various alkalis. The cleaning solution was fed at 6 CV (6 min). The alkaline washing solution used was 100 mM TrisHCl (pH 9.1), 100 mM Gly-NaOH (pH 9.5), 100 mM Na ₂ CO ₃ (pH 10.1), 100 mM Na ₂ CO ₃ (pH 10.6), 100 It is a solution of mM Na ₂ CO ₃ (pH 11.0), 100 mM Na ₂ HPO ₄ (pH 11.5). Then, 100 mM KH ₂ PO ₄ 150 mM NaCl (pH 7.2) solution was sent to the column at 6 CV (6 min), the column was washed, and 100 mM glycine hydrochloride buffer (pH 3.2) was sent at 8 CV (8 min). The antibody bound in the column was eluted, and the target fraction was recovered from the chromatogram. Note that 1.0 M Tris-HCl buffer (pH 8.8) was previously added to the collection tube so that the pH was around neutral. The concentration of the purified antibody was measured with NanoDrop ND-1000 (Thermo Scientific).

FIG. 4 shows the antibody yield when the A10-1C04 antibody was purified from the culture supernatant using the various washing solutions having different pHs. As a result, a yield improving effect was observed when a cleaning solution having a pH of 10.1 or higher was used.

Example 7: pH Dependence of Impurity Removal Effect A Protein A column (MabSelect SuRe) was connected to AKTA explorer 100. PBS (pH 7.2) was fed to the Protein A column to equilibrate the column, and then the culture supernatant expressing the A23-1A05 antibody was added to capture the A23-1A05 antibody. PBS (pH 7.2) was sent to the column at 8 CV (8 min), and the column was washed. Then, 100 mM KH ₂ PO ₄ 150 mM NaCl (pH 7.2) neutral washing solution or 6 types of alkaline washing solutions were used at 6 CV (6 min). Liquid was sent. The alkaline cleaning solution used was 100 mM TrisHCl (pH 9.0), 100 mM Na ₂ CO ₃ (pH 9.5), 100 mM Na ₂ CO ₃ (pH 10.0), 100 mM Na ₂ CO ₃ (pH 10.6), 100 mM Na ₂ It is a solution of CO ₃ (pH 11.0), 100 mM K ₂ HPO ₄ (pH 11.5). Then, PBS (pH 7.2) was sent to the column at 8 CV (8 min) and the column was washed. Then, 100 mM glycine hydrochloride buffer (pH 3.2) was sent at 10 CV (10 min), and the antibody bound to the column was removed. The desired fraction was recovered from the chromatogram after elution. Note that 1.0 M Tris-HCl buffer (pH 8.8) was previously added to the collection tube so that the pH was around neutral.

Fig. 5 shows the result of electrophoresis of SDS-PAGE (Nupage 4-12% Bris-Tris gels (Life Technologies)) according to the manufacturer's package insert for the fraction washed with a neutral washing solution or an alkaline solution. As a result, impurities were observed at pH 9.5 or higher. It was found that the antibody molecule, which is the target protein, was also washed at pH 11.5. FIG. 6 shows the change over time in the absorbance of the washing solution that passed through the column when washed with washing solutions of various pHs. No increase in absorbance was observed in pH 7.2 and 9 cleaning solutions, and no cleaning effect was observed.In pH 9.5 cleaning solutions, an absorbance peak that appears to be an impurity was observed. A high absorbance peak was observed, confirming the cleaning effect.

After washing with each washing solution as described above, the free thiol content of the antibody as the eluted target protein was measured in the same manner as in Example 2. As a result, it was shown that the free thiol content was reduced by performing a washing step with an alkaline washing solution having a pH of 9 or higher (FIG. 7).

Example 8: Influence of washing time with alkaline solution In order to investigate the influence of washing time on the yield improvement effect by the alkali washing step, purification was carried out under conditions of a plurality of liquid feeding times. A Protein A column (ToyoScreen AF-rProtein A HC-650F) was connected to AKTA explorer 100. A 100 mM KH ₂ PO ₄ 150 mM NaCl (pH 7.2) solution was fed to the Protein A column to equilibrate the column, and then the culture supernatant in which the A25-3H04 antibody was expressed was added. A 100 mM KH ₂ PO ₄ 150 mM NaCl (pH 7.2) solution was fed to the column at 6 CV (6 min) to wash the column, and then washed with various alkaline washing solutions. There are 4 types of washing solutions: 100 mM TrisHCl (pH 8.0), 100 mM TrisHCl (pH 9.0), 100 mM Na ₂ CO ₃ (pH 10.0), and 100 mM Na ₂ CO ³ (pH 11.0). , 6,12,18,24,48CV The cleaning process with alkaline cleaning solution was carried out. Thereafter, a 100 mM KH ₂ PO ₄ 150 mM NaCl (pH 7.2) solution was fed to the column at 6 CV (6 min) to wash the column. Next, 100 mM glycine hydrochloride buffer (pH 3.2) was fed at 8 CV (8 min), the antibody bound in the column was eluted, and the desired fraction was recovered from the chromatogram. Note that 1.0 M Tris-HCl buffer (pH 8.8) was previously added to the collection tube so that the pH was around neutral. The concentration of the purified antibody was measured with NanoDrop ND-1000 (Thermo Scientific).

To investigate the effect of alkali cleaning time, 1CV (1 min), 3CV (3 min), 6CV (6 min), 12CV (12 min), 18CV (18 min), 24CV (24 min), 48CV ( The A25-3H04 antibody was eluted after washing with the alkaline washing solution (pH 11) at 48 μmin). As a result of calculating the yield of the antibody, the yield improvement effect by incorporating the alkali washing step was recognized even at 1 CV (1 min).

Example 9: Host cell derived protein Purification 3 (without alkaline washing step):
A Protein A column (ToyoScreen AF-rProtein A-650F, TOSOH) was connected to AKTA explorer 100 (GE Healthcare). 20 mM phosphoric acid, 20 mM citric acid, and 20 mM Tris buffer (PCT buffer) (pH 7.0) were fed to the Protein A column to equilibrate the column, and then the culture supernatant of the A10-1C04 antibody was added. The column was washed with 2 CV of PCT buffer (pH 7.0) and the column was washed, and then washed with 6 CV of PCT buffer (pH 8.5). Subsequently, 9 CV of PCT buffer (pH 5.8) was fed. Finally, PCT buffer (pH 2.7) was fed at 10 CV (10 min), the antibody bound in the column was eluted, and the desired fraction was recovered from the chromatogram.

Purification 4 (with alkali cleaning process):
A Protein A column (ToyoScreen AF-rProtein A-650F, TOSOH) was connected to AKTA explorer 100 (GE Healthcare). PCT buffer (pH 7.0) was fed to the Protein A column to equilibrate the column, and then the culture supernatant of the A10-1C04 antibody was added. The column was washed with 2 CV of PCT buffer (pH 7.0) and washed with an alkaline washing solution of 100 mM Na ₂ CO ₃ buffer (pH 11). Subsequently, 9 CV of PCT buffer (pH 5.8) was fed. Finally, PCT buffer (pH 2.7) was fed at 10 CV (10 min), the antibody bound in the column was eluted, and the desired fraction was recovered from the chromatogram.

The host cell-derived protein contained in the A10-1C04 antibody solution purified by the methods of purification 3 and 4 was measured according to the package insert using an Immunoenzymetric Assay-for-the-Measurement-of-CHO-Host-Cell Protein Protein kit (CYGNUS-TECHNOLOGIES). . Host cell protein, which contained 373443 ppm in the starting material, was reduced to 6978 ppm in the sample purified by purification 3 (without alkali washing step), whereas it was purified by purification 4 (with alkali washing step). In the sample, the concentration was 1691 ppm, and the removal effect of the host cell-derived protein was confirmed by using an alkaline washing step.

Example 10: Fc fusion protein Fc fusion protein ST2-Fc is a fusion protein of the extracellular region of human ST2 protein and the human antibody constant region (Fc). DNA encoding the amino acid sequence of the fusion protein (SEQ ID NO: 52 in the sequence listing) was inserted into pXC-17.4 vector (Lonza) and GS Xceed ™ Gene Expression System (Lonza) using CHOK1SV GS-KO cells. Was used in accordance with the package insert to establish the target CHO stable expression strain. The CHO stable expression strain was cultured from 0.3 × 10 ⁶ cells / mL using an Erlenmeyer flask, and the culture supernatant containing secreted ST2-Fc was recovered. 125 mL of the obtained culture supernatant containing ST2-Fc was purified by the methods of Purification 1 (without alkali washing step) and Purification 2 (with alkali washing step) in Example 1, respectively. The ST2-Fc solution obtained by purification 1 or purification 2 was recovered, and then dialyzed against Slide-A-Lyzer G2 10,000 MWCO (Thermo Scientific). PBS (pH 7.2) was used as the external dialysis solution, and the dialysis external solution was changed halfway. Thereafter, when the concentration was measured, the yield was 46.1 mg in the purification 1 (without the alkali washing step) and 48.4 mg in the purification 2 (with the alkali washing step), and an improvement of about 5% was observed.

The particle size of the purified ST2-Fc solution (1.5 mg / mL) was measured using a dynamic light scattering measurement device (Nanotrac UPA UT151 Nikkiso). As shown in FIG. 8, the sample obtained in purification 2 (with an alkali washing step) contained less aggregate than the sample obtained in purification 1 (without an alkali washing step).

Example 11: Effect of alkaline washing on protein By washing protein under alkaline conditions, there may be concerns such as deactivation of activity and chemical changes. Therefore, for the biological activity of the antibody, the binding to the Fcγ receptor, and the peptide map, the sample purified by the process including the alkaline washing step and the sample purified by the process not including the alkaline washing step were compared.

Example 11-1: Bioactivity of antibodies Human umbilical vein endothelial cells (HUVEC) are suspended in EGM-2 medium (LONZA) and seeded in a 96-well collagen-coated microplate (AGC Technoglass, 4860-010) (1 × 10 ⁴ cells / 100 μL / well). Approximately 24 hours later, the medium was removed by suction, and anti-IL-33 antibody (final concentration) containing polymyxin B (final concentration 10 μg / mL) and hIL-33 (ATGen) (final concentration 0-100 ng / mL) was added. 0-100 μg / mL) was added (150 μL / well) and incubated at 37 ° C. for about 24 hours. Culture supernatants were collected and cytokine levels in the medium were measured using a Human IL-6 ELISA Kit kit (R & D systems). A plurality of antibodies (8 antibodies including A10-1C04, A23-1A05, and A25-3H04) purified by the method of Purification 1 (without alkali washing step) and Purification 2 (with alkali washing step) of Example 1 -6 production inhibitory effect was examined, but there was no difference in the neutralizing ability between the two, and no functional loss was observed due to the alkaline washing.

Example 11-2: Binding to Fcγ receptor Anti-IL-33 antibodies (A23-1A05, A10) purified by the methods of purification 1 (without alkali washing step) and purification 2 (with alkali washing step) of Example 1 -1C04, A26-1F02, A25-3H04) and Fc receptors were analyzed using Biacore T200 (GE healthcare). Fc receptor Recombinant Human FcγRIIIA (R & D Systems, 4325-FC-050) was immobilized on a sensor chip using an anti-His antibody. Anti-His antibody was immobilized according to the manual of His capture kit (GE healthcare, BR-1003-51). A Recombinant Human FcγRIIIA solution prepared to 0.5 μg / mL using HBS-EP + (pH 7.4) solution (GE healthcare) was added at 5 μL / min for 30 sec. Next, various antibody solutions were added in order from a low-concentration sample at 30 μL / min for 60 sec (Single-cycle method). Thereafter, the HBS-EP + (pH 7.4) solution was added at 30 μL / min for 600 sec to dissociate the analyte. The analysis was performed using the two state reaction model with the attached software BIAevaluation software. Dissociation constant (KD), was calculated binding parameters, differences in the dissociation constants K _D and binding parameters for Human FcyRIIIA with and without alkali cleaning treatment is not observed, that binds to Fcγ receptors Fc region by alkali washing performed There was no loss of function.

Example 11-3: Peptide map Antibodies purified by the methods of Purification 1 (without alkali washing step) and Purification 2 (with alkali washing step) of Example 1 (A23-1A05, A10-1C04, A26-1F02, A25) -3H04) were each reduced with DTT, alkylated with iodoacetamide, and then enzymatically digested with trypsin. A C18 column ACQUITY UPLC Peptide BEH C18 Column, 300 mm, 1.7 μm, 2.1 mm × 150 mm (Waters) was connected to a liquid chromatography apparatus Acuity (Waters). Mobile phase A: 0.1% formic acid / water, mobile phase B: 0.1% formic acid / acetonitrile, gradient was sent at a flow rate of 0.2 mL / min so that the concentration of mobile phase B was 1 → 40% / 60 min, and digested with trypsin. Peptide fragments were analyzed on a C18 column. As a result of comparing the obtained UV chromatograms, the peptide map patterns of the samples purified in purification 1 (without alkali washing step) and purification 2 (with alkali washing step) are equivalent, and the chemical changes associated with alkali washing are I was not able to admit.

The method for purifying a protein having an Fc region of the present invention can reduce the free thiol content, self-association property and aggregation property of a protein having an Fc region as compared with conventional methods, and can improve the yield and storage stability. Can be improved. According to the present invention, a protein having an Fc region having excellent physical properties can be produced with high purity at low cost. The human anti-IL-33 monoclonal antibody produced by the present invention can be used as a pharmaceutical composition for diagnosis, treatment, prevention or alleviation of IL-33-related diseases.

Claims (29)

1. A method for purifying a protein having an Fc region,
   (a) contacting a solution containing a protein having an Fc region with an affinity chromatography support, and binding the protein having an Fc region to the affinity chromatography support;
   (b) washing with an alkaline washing liquid having a pH value in the range of 10.1 to 12, and
   (c) elution of a protein having an Fc region,
   A method for purifying a protein having an Fc region, comprising:
2. The method according to claim 1, wherein the alkaline cleaning liquid has a pH value of 10.5 to 11.5.
3. The method according to claim 1 or 2, wherein the alkaline cleaning solution does not contain 0.2 M or more sodium chloride.
4. The method according to any one of claims 1 to 3, wherein the alkaline cleaning solution does not contain arginine.
5. The method according to any one of claims 1 to 4, wherein the carrier for affinity chromatography is a carrier having Protein Pro A, Protein 改 変 G, or a modified form thereof.
6. The method according to any one of claims 1 to 5, wherein the protein having an Fc region is an antibody.
7. The method according to any one of claims 1 to 6, wherein the protein having an Fc region is a human monoclonal antibody, a humanized monoclonal antibody, or a chimeric monoclonal antibody.
8. The method according to any one of claims 1 to 7, wherein the protein having an Fc region is an antibody having a λ chain.
9. A method for producing a protein having an Fc region, comprising the method according to any one of claims 1 to 8.
10. A protein having an Fc region produced by the method according to claim 9.
11. A method for producing a human monoclonal antibody comprising:
    (i) contacting a solution containing a human monoclonal antibody with an affinity chromatography support, and binding the human monoclonal antibody to the affinity chromatography support;
    (ii) washing with an alkaline wash having a pH value in the range of 9 to 12, and
    (iii) a step of eluting the human monoclonal antibody;
    A light chain complementarity determining region 1 (LCDR1), a light chain complementarity determining region 2 (LCDR2), a light chain complementarity determining region 3 (LCDR3), and a heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2) and heavy chain complementarity determining region 3 (HCDR3),
    (a) LCDR1: TGSSSNIGAVYDVH (SEQ ID NO: 1 in the sequence listing), LCDR2: RNNQRPS (SEQ ID NO: 2 in the sequence listing), LCDR3: QTYDSSRWV (SEQ ID NO: 3 in the sequence listing), HCDR1: DYYMN (SEQ ID NO: 4 in the sequence listing) , HCDR2: SISRYSSYIYYADSVKG (SEQ ID NO: 5 in the sequence listing), and HCDR3: DIGGMDV (SEQ ID NO: 6 in the sequence listing)
    (b) LCDR1: SGSSSNIGNNAVS (SEQ ID NO: 7 in the sequence listing), LCDR2: ASNMRVI (SEQ ID NO: 8 in the sequence listing), LCDR3: GAWDDSQKALV (SEQ ID NO: 9 in the sequence listing), HCDR1: NYYMH (SEQ ID NO: 10 in the sequence listing) , HCDR2: SISARSRYHYYADSVKG (SEQ ID NO: 11 in the sequence listing), and HCDR3: LATRHNAFDI (SEQ ID NO: 12 in the sequence listing)
    (c) LCDR1: SGSSSNIGRNAVN (SEQ ID NO: 13 in the sequence listing), LCDR2: ASNMRVS (SEQ ID NO: 14 in the sequence listing), LCDR3: WAWDDSQKVGV (SEQ ID NO: 15 in the sequence listing), HCDR1: NYYMH (SEQ ID NO: 10 in the sequence listing) , HCDR2: SISARSSYIYYADSVKG (SEQ ID NO: 16 in the sequence listing), and HCDR3: LATRNNAFDI (SEQ ID NO: 17 in the sequence listing)
    (d) LCDR1: SGSSSNIGRNAVN (SEQ ID NO: 13 in the sequence listing), LCDR2: ASNMRRS (SEQ ID NO: 18 in the sequence listing), LCDR3: SAWDDSQKVVV (SEQ ID NO: 19 in the sequence listing), HCDR1: RYYMH (SEQ ID NO: 20 in the sequence listing) HCDR2: SISAQSSHIYYADSVEG (SEQ ID NO: 21 of the sequence listing) and HCDR3: LATRQNAFDI (SEQ ID NO: 22 of the sequence listing), or
    (e) LCDR1: SGSSSNIGNNAVN (SEQ ID NO: 23 in the sequence listing), LCDR2: ASNMRRP (SEQ ID NO: 24 in the sequence listing), LCDR3: EAWDDSQKAVV (SEQ ID NO: 25 in the sequence listing), HCDR1: NYYMH (SEQ ID NO: 10 in the sequence listing) , HCDR2: SISARSSYLYYADSVKG (SEQ ID NO: 26 in the sequence listing), and HCDR3: LATRHVAFDI (SEQ ID NO: 27 in the sequence listing)
    A method for producing a human monoclonal antibody, which is a human monoclonal antibody.
12. The combination of the amino acid sequences of the light chain variable region (VL) and heavy chain variable region (VH) of the human monoclonal antibody is as follows:
    (A) VL: QSVLTQPPSASGTPGQRVTISCTGSSSNIGAVYDVHWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCQTYDSSRWVFGGGTKLTVL and VH (SEQ ID NO: 28 of Sequence Listing): EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMNWVRQAPGKGLEWVSSISRYSSYIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDIGGMDVWGQGTLVTVSS (SEQ ID NO: 29 of the Sequence Listing)
    (B) VL: QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNAVSWYQQLPGTAPKLLIYASNMRVIGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGAWDDSQKALVFGGGTKLTVL (SEQ ID NO: of the Sequence Listing 30) and VH: EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYYMHWVRQAPGKGLEWVSSISARSRYHYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLATRHNAFDIWGQGTLVTVSS (SEQ ID NO: 31 of the Sequence Listing)
    (C) VL: QSVLTQPPSASGTPGQRVTISCSGSSSNIGRNAVNWYQQLPGTAPKLLIYASNMRVSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCWAWDDSQKVGVFGGGTKLTVL (SEQ ID NO: 32 of the Sequence Listing) and VH: (SEQ ID NO: 33 of the Sequence Listing) IbuikyuerueruiesujijijierubuikyuPijijiesueruarueruesushieieiesujiefutiefuesuenuwaiwaiemueichidaburyubuiarukyueiPijikeijieruidaburyubuiesuesuaiesueiaruesuesuwaiaiwaiwaieidiesubuikeijiaruefutiaiesuarudienuesukeienutieruwaierukyuemuenuesueruarueiiditieibuiwaiYCARLATRNNAFDIWGQGTLVTVSS
    (D) VL: QSVLTQPPSASGTPGQRVTISCSGSSSNIGRNAVNWYQQLPGTAPKLLIYASNMRRSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCSAWDDSQKVVVFGGGTKLTVL (SEQ ID NO: 34 in Sequence Listing) and VH: (SEQ ID NO: 35 of Sequence Listing) IbuikyuerueruiesujijijierubuikyuPijijiesueruarueruesushieieiesujiefutiefuesuaruwaiwaiemueichidaburyubuiarukyueiPijikeijieruidaburyubuiesuesuaiesueikyuesuesueichiaiwaiwaieidiesubuiijiaruefutiaiesuarudienuesukeienutieruwaierukyuemuenuesueruarueiiditieibuiwaiYCARLATRQNAFDIWGQGTLVTVSS, or
    (E) VL: QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNAVNWYQQLPGTAPKLLIYASNMRRPGVPDRFSGSKSGTSASLAISGLRSEDEADYYCEAWDDSQKAVVFGGGTKLTVL (SEQ ID NO: 36 in Sequence Listing) and VH: (SEQ ID NO: 37 of Sequence Listing) IbuikyuerueruiesujijijierubuikyuPijijiesueruarueruesushieieiesujiefutiefuesuenuwaiwaiemueichidaburyubuiarukyueiPijikeijieruidaburyubuiesuesuaiesueiaruesuesuwaieruwaiwaieidiesubuikeijiaruefutiaiesuarudienuesukeienutieruwaierukyuemuenuesueruarueiiditieibuiwaiYCARLATRHVAFDIWGQGTLVTVSS,
    The manufacturing method according to claim 11.
13. The manufacturing method according to claim 11 or 12, wherein the alkaline cleaning liquid has a pH value of 10.5 to 11.5.
14. The manufacturing method according to any one of claims 11 to 13, wherein the alkaline cleaning liquid does not contain 0.2 M or more sodium chloride.
15. The production method according to any one of claims 11 to 14, wherein the alkaline cleaning liquid does not contain arginine.
16. The production method according to any one of claims 11 to 15, wherein the carrier for affinity chromatography is a carrier having Protein A, Protein G, or a modified form thereof.
17. A human monoclonal antibody produced by the method according to any one of claims 11 to 16.
18. A method for improving the physical properties of a protein having an Fc region contained in a solution,
    (a) contacting a solution containing a protein having an Fc region with an affinity chromatography support, and binding the protein having an Fc region to the affinity chromatography support;
    (b) contacting an alkaline solution having a pH value in the range of 9.1 to 12, and
    (c) elution of a protein having an Fc region,
    Including a method.
19. The method according to claim 18, wherein the method for improving the physical properties of a protein having an Fc region is a method for reducing the free thiol content of a protein having an Fc region.
20. The method according to claim 18, wherein the method for improving the physical properties of a protein having an Fc region is a method for reducing the self-association property of a protein having an Fc region.
21. The method according to claim 18, wherein the method for improving the physical properties of a protein having an Fc region is a method for reducing the aggregation property of a protein having an Fc region.
22. The method according to claim 18, wherein the method for improving the physical properties of a protein having an Fc region is a method for further enhancing the storage stability of a protein having an Fc region.
23. 23. The method according to any one of claims 18 to 22, wherein the alkaline solution has a pH value of 10.6 to 11.5.
24. The method according to any one of claims 18 to 23, wherein the alkaline solution does not contain 0.2 M or more of sodium chloride.
25. The method according to any one of claims 18 to 24, wherein the alkaline solution does not contain arginine.
26. The method according to any one of claims 18 to 25, wherein the carrier for affinity chromatography is a carrier having Protein A, Protein G, or a modified form thereof.
27. The method according to any one of claims 18 to 26, wherein the protein having an Fc region is an antibody.
28. The method according to any one of claims 18 to 27, wherein the protein having an Fc region is a human monoclonal antibody, a humanized monoclonal antibody, or a chimeric monoclonal antibody.
29. The method according to any one of claims 18 to 28, wherein the protein having an Fc region is an antibody having a λ chain.

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