Highly concentrated antibody-containing solution formulation

Abstract

Provided is a stable, highly concentrated antibody-containing formulation suitable for subcutaneous administration, in which dimer formation and deamidation during long-term storage are suppressed. The present invention provides a stable, highly concentrated antibody-containing liquid formulation that contains arginine and methionine, and preferably further contains a histidine buffer.

Description

[Technical field]

[0001] The present invention relates to an antibody-containing formulation, and in particular to a stable, highly concentrated antibody-containing liquid formulation. [Background technology]

[0002] In recent years, various antibody preparations have been developed and put to practical use, but most of them are used as intravenous injection preparations. However, due to needs in the medical field, there is an increasing demand for the development of antibody-containing preparations as self-injectable subcutaneous injection preparations.

[0003] When designing an antibody-containing formulation for subcutaneous injection, the amount of antibody administered per injection is large. However, subcutaneous injections generally have limitations on the amount of fluid injected. It is necessary to increase the concentration of antibodies in the administration solution. Therefore, high-concentration formulations using the so-called freeze-drying concentration technique, in which a freeze-dried formulation is redissolved in a smaller volume of water than before freeze-drying to prepare a high-concentration solution formulation, are often used. However, there is also a great demand for solution formulations that are easy to use and do not require the trouble of redissolving. In addition, in the production of freeze-dried formulations, the viscosity of the formulation increases due to the addition of a cryoprotectant such as sugar, which is not desirable for a formulation for subcutaneous injection, but a solution formulation is thought to be able to avoid this problem.

[0004] A high-concentration antibody-containing solution tends to form a highly viscous solution due to the macromolecular nature of the protein and intermolecular interactions. Furthermore, when storing a protein in a high-concentration solution, degradation phenomena, including the formation of insoluble and / or soluble aggregates, become a problem and must be prevented. In particular, antibody preparations are prone to forming associations during storage in a solution state, leading to the formation of insoluble aggregates. In addition, when a solution preparation is stored for a long period of time, there is a problem that the physiological activity of the antibody molecule is lost due to deamidation of asparagine and other amino acid residues.

[0005] In general, various efforts have been made to stabilize protein formulations with minimal loss of active ingredients even after long-term storage, and these formulations are produced by dissolving the active ingredient and various additives in a buffer solution. However, there are still insufficient techniques to prevent antibody dimerization and deamidation, particularly in high-concentration antibody-containing solution formulations.

[0006] There is a need for stable, highly concentrated antibody-containing formulations suitable for subcutaneous administration that suppress dimer formation and deamidation during long-term storage. Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a stable, high-concentration antibody-containing formulation which is suitable for subcutaneous administration and which is inhibited in dimer formation and deamidation during long-term storage. [Means for solving the problem]

[0008] As a result of intensive research to achieve the above-mentioned object, the present inventors discovered that a highly concentrated, stable antibody-containing liquid formulation can be obtained by adding the amino acid arginine or a salt thereof as a stabilizer, and thus completed the present invention.

[0009] That is, the present invention provides the following. (1) A stable antibody-containing liquid formulation, characterized in that it contains arginine and methionine. (2) The solution formulation according to (1), further comprising a histidine buffer. (3) The solution formulation according to (1) or (2), further comprising a surfactant. (4) The solution formulation according to any one of (1) to (3), wherein the antibody concentration is 50 mg / ml or more. (5) The solution formulation according to any one of (1) to (3), wherein the antibody has a concentration of 100 mg / ml or more. (6) The solution formulation according to any one of (1) to (3), wherein the antibody concentration is 120 mg / ml or more. (7) The solution formulation according to any one of (1) to (6), wherein the antibody is an anti-interleukin-6 receptor antibody. (8) A stable anti-interleukin-6 receptor antibody-containing solution formulation, characterized by containing arginine or methionine. (9) The solution formulation according to any one of (1) to (8), wherein the antibody is a humanized antibody or a human antibody. (10) The solution formulation according to any one of (1) to (9), further comprising tryptophan. (11) The solution formulation according to any one of (1) to (10), having a pH of 4 to 8. (12) The solution formulation according to any one of (1) to (11), wherein the arginine content is 50 to 1500 mM. (13) The solution preparation according to any one of (1) to (12), having a viscosity of 2 to 15 mPa s. (14) The solution formulation according to any one of (1) to (13), which is stable at 22 to 28°C for at least 6 months. (15) The solution formulation according to any one of (1) to (13), wherein formation of an antibody dimer is suppressed. (16) The solution formulation according to any one of (1) to (13), wherein deamidation of an antibody molecule is suppressed. (17) The solution formulation according to any one of (1) to (13) above, which is administered subcutaneously. (18) The solution formulation according to any one of (1) to (13), which is produced without a freeze-drying step in the production process of the solution formulation. (19) A method for suppressing deamidation of antibody molecules in an antibody-containing liquid formulation, comprising adding arginine to the solution. (20) A method for suppressing antibody dimer formation in an antibody-containing liquid formulation, the method comprising adding arginine and methionine to the solution. Effect of the Invention

[0010] The present invention provides a highly concentrated antibody-containing formulation that does not require reconstitution by lyophilization and does not require the trouble of redissolving. The highly concentrated antibody-containing formulation of the present invention can be stably stored in a solution state for a long period of time, and can be produced without a lyophilization step in the production process, eliminating the need to add sugar or other cryoprotectants. [Brief description of the drawings]

[0011] [Figure 1] 1 is a typical chromatograph of Example 1. [Diagram 2] 1 shows the evaluation results of gel permeation chromatography (SEC) in Example 1. [Diagram 3] 1 shows the evaluation results of gel permeation chromatography (SEC) in Example 1. [Figure 4] 1 is a typical chromatograph of Example 2. [Diagram 5] 4 shows the evaluation results of ion exchange chromatography (IEC) of Example 2. [Figure 6] 4 shows the evaluation results of ion exchange chromatography (IEC) of Example 2. [Figure 7] 1 shows the evaluation results of gel permeation chromatography (SEC) in Example 3. [Figure 8] 4 shows the evaluation results of ion exchange chromatography (IEC) in Example 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present invention will be described in detail below. In the present invention, an antibody-containing solution formulation refers to a solution formulation which contains an antibody as an active ingredient and is prepared so as to be able to be administered to animals such as humans, and preferably refers to a solution formulation which is produced without including a lyophilization step in the production process.

[0013] The antibody-containing solution formulation of the present invention is a solution formulation containing a high concentration of an antibody, and the antibody concentration is preferably 50 mg / mL or more, more preferably 100 mg / mL or more, even more preferably 120 mg / mL or more, and even more preferably 150 mg / mL. In particular, there have been no examples of antibody-containing solution formulations of 120 mg / mL or more, preferably 150 mg / mL or more, being put to practical use until now, and the formulation of the present invention has made it possible for the first time to put such a high-concentration antibody-containing solution formulation to practical use. Moreover, from the viewpoint of production, the upper limit of the antibody concentration in the antibody-containing solution formulation of the present invention is generally 300 mg / mL, preferably 250 mg / mL, and more preferably 200 mg / mL. Therefore, the antibody concentration in the high-concentration antibody solution formulation of the present invention is preferably 50 to 300 mg / mL, more preferably 100 to 300 mg / mL, further preferably 120 to 250 mg / mL, and particularly preferably 150 to 200 mg / mL.

[0014] The antibody used in the present invention is not particularly limited as long as it binds to the desired antigen, and may be a polyclonal or monoclonal antibody, although monoclonal antibodies are preferred in that homogeneous antibodies can be stably produced.

[0015] The monoclonal antibodies used in the present invention include not only monoclonal antibodies derived from animals such as humans, mice, rats, hamsters, rabbits, sheep, camels, and monkeys, but also artificially modified recombinant antibodies such as chimeric antibodies, humanized antibodies, and bispecific antibodies. The immunoglobulin class of the antibody is not particularly limited, and may be any class such as IgG, such as IgG1, IgG2, IgG3, and IgG4, IgA, IgD, IgE, and IgM, with IgG and IgM being preferred.

[0016] Furthermore, the antibodies of the present invention include not only whole antibodies, but also antibody fragments such as Fv, Fab, and F(ab)2, and low molecular weight antibodies such as monovalent or divalent or more single-chain Fvs (e.g., diabodies such as scFv, sc(Fv)2, and scFv dimers) in which the variable regions of the antibodies are linked via a linker such as a peptide linker.

[0017] The above-mentioned antibodies of the present invention can be prepared by methods well known to those skilled in the art. Hybridomas producing monoclonal antibodies can be basically prepared using known techniques as follows. That is, a desired antigen or cells expressing the desired antigen are used as a sensitizing antigen, which is immunized according to a conventional immunization method, the resulting immune cells are fused with known parent cells by a conventional cell fusion method, and monoclonal antibody-producing cells (hybridomas) are screened by a conventional screening method. Hybridomas can be prepared, for example, according to the method of Milstein et al. (Kohler, G. and Milstein, C., Methods Enzymol. (1981) 73: 3-46). When the antigen has low immunogenicity, it may be bound to a macromolecule having immunogenicity such as albumin and then immunized.

[0018] Alternatively, a recombinant antibody can be produced by cloning an antibody gene from a hybridoma, incorporating it into a suitable vector, and introducing it into a host using recombinant gene technology (see, for example, Carl, AK Borrebaeck, James, W. Larrick, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990). Specifically, cDNA of the variable region (V region) of an antibody is synthesized from the mRNA of a hybridoma using reverse transcriptase. Once DNA encoding the V region of a target antibody is obtained, it is linked to DNA encoding the desired antibody constant region (C region), and this is incorporated into an expression vector. Alternatively, DNA encoding the V region of an antibody may be incorporated into an expression vector containing DNA of the antibody C region. It is incorporated into the expression vector so that it is expressed under the control of an expression control region, such as an enhancer or promoter. Next, the host cell is transformed with this expression vector to express the antibody.

[0019] In the present invention, recombinant antibodies that have been artificially modified for the purpose of reducing heterologous antigenicity against humans, such as chimeric antibodies and humanized antibodies, are used. These modified antibodies can be produced by known methods. Chimeric antibodies are antibodies consisting of the heavy and light chain variable regions of an antibody from a mammal other than human, for example, a mouse, and the heavy and light chain constant regions of a human antibody, and can be obtained by linking DNA encoding the variable region of a mouse antibody to DNA encoding the constant region of a human antibody, incorporating this into an expression vector, and introducing it into a host for production.

[0020] A humanized antibody is also called a reshaped human antibody, and is a human antibody made by converting the complementarity determining region (CDR) of an antibody from a non-human mammal, such as a mouse, into a human antibody. The human antibody is a mouse antibody that has been transplanted into the CDR of the human antibody, and a general genetic recombination method for this purpose is also known. Specifically, a DNA sequence designed to link the CDR of a mouse antibody and the framework region (FR) of a human antibody is synthesized by PCR from several oligonucleotides that have been prepared so as to have overlapping portions at the ends. The resulting DNA is linked to DNA encoding the constant region of a human antibody, which is then incorporated into an expression vector, which is then introduced into a host to produce the antibody (see European Patent Application Publication No. EP 239400 and International Patent Application Publication No. WO 96 / 02576). The FR of the human antibody that is linked via the CDR is selected from those whose CDRs form a good antigen-binding site. If necessary, amino acids in the framework region of the variable region of the antibody may be replaced so that the CDRs of the reshaped human antibody form an appropriate antigen-binding site (Sato, K. et al., Cancer Res. (1993) 53, 851-856).

[0021] Methods for obtaining human antibodies are also known. For example, human lymphocytes can be sensitized in vitro with a desired antigen or cells expressing the desired antigen, and the sensitized lymphocytes can be fused with human myeloma cells, such as U266, to obtain a desired human antibody having binding activity to the antigen (see JP-B-1-59878). Also, a desired human antibody can be obtained by immunizing a transgenic animal having a full repertoire of human antibody genes with an antigen (see International Patent Application Publication Nos. WO 93 / 12227, WO 92 / 03918, WO 94 / 02602, WO 94 / 25585, WO 96 / 34096, and WO 96 / 33735). Furthermore, a technique for obtaining a human antibody by panning using a human antibody library is also known. For example, the variable region of a human antibody can be expressed on the surface of a phage as a single chain antibody (scFv) by phage display, and a phage that binds to the antigen can be selected. By analyzing the genes of the selected phages, the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined. Once the DNA sequence of the scFv that binds to the antigen is determined, an appropriate expression vector containing the sequence can be constructed to obtain a human antibody. These methods are already well known, and reference can be made to WO 92 / 01047, WO 92 / 20791, WO 93 / 06213, WO 93 / 11236, WO 93 / 19172, WO 95 / 01438, and WO 95 / 15388.

[0022] When the antibody gene is isolated and then introduced into a suitable host to produce the antibody, a suitable combination of host and expression vector can be used. When eukaryotic cells are used as the host, animal cells, plant cells, and fungal cells can be used. Known animal cells include (1) mammalian cells, such as CHO, COS, myeloma, BHK (baby hamster kidney), HeLa, and Vero, (2) amphibian cells, such as Xenopus oocytes, and (3) insect cells, such as sf9, sf21, and Tn5. Known plant cells include cells derived from the genus Nicotiana, such as Nicotiana tabacum, which can be cultured as callus. Known fungal cells include yeasts such as the genus Saccharomyces, e.g., Saccharomyces cerevisiae, and filamentous fungi such as the genus Aspergillus, e.g., Aspergillus niger. When using prokaryotic cells, there are production systems using bacterial cells. Known examples of bacterial cells include Escherichia coli and Bacillus subtilis. Antibodies can be obtained by introducing the desired antibody gene into these cells by transformation and culturing the transformed cells in vitro.

[0023] Furthermore, the antibody of the present invention may be an antibody fragment, a minibody, or a modified antibody. Examples of antibody fragments and minibodies include Fab, F(ab')2, Fv, or monovalent or multivalent single-chain Fv (scFv, sc(Fv)2, etc.) in which the H chain and L chain Fv are linked via an appropriate linker (Huston, JS et al., Proc. Natl. Acad. Sci. USA (1988) 85, 5879-5883). Specifically, antibodies are treated with enzymes such as papain or pepsin to generate antibody fragments, or genes encoding these antibody fragments are constructed and introduced into expression vectors, which are then expressed in appropriate host cells (see, for example, Co, MS et al., J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, AH, Methods Enzymol. (1989) 178, 476-496; Pluckthun, A. and Skerra, A., Methods Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol. (1986) 121, 652-663; Rousseaux, J. et al., Methods Enzymol. (1986) 121, 663-669; Bird, See RE and Walker, BW, Trends Biotechnol. (1991) 9, 132-137).

[0024] As antibody modifications, antibodies bound to various molecules such as polyethylene glycol (PEG) are used. The "antibody" of the present invention also includes these modified antibodies. Such modified antibodies can be obtained by chemically modifying the obtained antibody. These methods have already been established in the field.

[0025] The antibodies contained in the formulation of the present invention include anti-tissue factor antibody, anti-IL-6 receptor antibody, anti-IL-6 antibody, HM1.24 antigen monoclonal antibody, anti-parathyroid hormone-related peptide antibody (anti-PTHrP antibody), anti-glypican-3 antibody, anti-ganglioside GM3 antibody, anti-TPO receptor antibody, agonist antibodies, coagulation factor VIII replacement antibodies, anti-CD3 antibodies, anti-CD20 antibodies, anti-GPIIb / IIIa antibodies body, anti-TNF antibody, anti-CD25 antibody, anti-EGFR antibody, anti-Her2 / neu antibody, anti-RSV antibody, anti-CD33 antibody, anti-CD52 antibody, anti-IgE antibody, anti-CD11a antibody, anti-VEGF antibody, anti-VLA4 antibody, anti-AXL antibody, etc. It can be, but is not limited to, this.

[0026] As reshaped humanized antibodies, preferred antibodies for use in the present invention include humanized anti-interleukin 6 (IL-6) receptor antibody (hPM-1 or MRA) (see International Patent Application Publication No. WO92-19759), humanized anti-HM1.24 antigen monoclonal antibody (see International Patent Application Publication No. WO98-14580), humanized anti-parathyroid hormone-related peptide antibody (anti-PTHrP antibody) (see International Patent Application Publication No. WO98-13388), humanized anti-tissue factor antibody (see International Patent Application Publication No. WO99-51743), and anti-glypican-3 humanized IgG1κ antibody (see International Patent Application No. PCT / JP05 / 013103). Particularly preferred as a humanized antibody for use in the present invention is a humanized anti-IL-6 receptor antibody.

[0027] As for the human IgM antibody, we have an anti-ganglioside GM3 recombinant human IgM antibody (international patent application pending). See Publication No. WO05-05636) and the like are preferred. Preferred examples of minibodies include anti-TPO receptor agonist diabodies (see International Patent Application Publication No. WO02-33072) and anti-CD47 agonist diabodies (see International Patent Application Publication No. WO01-66737).

[0028] In order to evaluate the storage stability of a sample containing a high concentration of an antibody, the present inventors investigated the effects of various additives by thermal acceleration tests and light acceleration tests. As a result, a solution in which a high concentration of an antibody was dissolved in a buffer containing the amino acid arginine showed a lower amount of dimer formation than a solution without arginine, and it was found that arginine is effective as a stabilizer that suppresses dimer formation. Furthermore, in a buffer containing methionine in addition to arginine, In a solution containing a high concentration of antibody, a similar effect of inhibiting dimer formation was observed at a lower total concentration of arginine and methionine than that of arginine alone, and thus it was found that a synergistic effect was exhibited by the combined use of arginine and methionine. It was also found that the addition of arginine inhibited deamidation of antibody molecules. These results are exemplified in the examples described later in this specification as test results using a sample containing 180 mg / ml of humanized anti-IL-6 receptor antibody.

[0029] That is, by containing arginine as a stabilizer, it is possible to obtain a stable antibody formulation in which the formation of antibody dimers is reduced and deamidation is prevented. Therefore, the first aspect of the present invention is characterized by adding arginine to the solution, thereby suppressing the formation of dimers or deamidation of antibody molecules in the antibody-containing solution formulation. And, the aspect of the stable antibody-containing solution formulation is characterized by containing an antibody and arginine in a buffer. Also, as described above, the antibody-containing solution formulation of the present invention further contains methionine, thereby exerting a synergistic effect by the combined use of arginine and methionine. Therefore, the second aspect of the present invention is characterized by adding arginine and methionine to the solution, and in particular, it relates to suppressing the formation of antibody dimers in the antibody-containing solution formulation. And, the aspect of the stable antibody-containing solution formulation is characterized by containing an antibody, arginine, and methionine in a buffer.

[0030] The arginine used in the present invention may be any of its derivatives and salts, and L-arginine and its salts are particularly preferred. The methionine used in the present invention may be any of its derivatives and salts, and L-methionine and its salts are particularly preferred.

[0031] When the antibody-containing liquid formulation of the present invention contains only arginine without adding methionine, the amount of arginine is preferably 50 to 1500 mM, more preferably 100 to 1000 mM, and even more preferably 200 to 700 mM. When the antibody-containing liquid formulation of the present invention contains arginine and methionine, the total concentration of arginine and methionine is 50 to 1200 mM, for example, the amount of arginine is preferably 40 to 1000 mM and the amount of methionine is 10 to 200 mM, more preferably the amount of arginine is 50 to 700 mM and the amount of methionine is 10 to 100 mM, and even more preferably the amount of arginine is 100 to 300 mM and the amount of methionine is 10 to 50 mM.

[0032] The buffer solution is prepared using a buffering agent, which is a substance for maintaining the pH of the solution. In the high-concentration antibody-containing solution formulation of the present invention, the pH of the solution is preferably 4 to 8, more preferably 5.0 to 7.5, even more preferably 5.5 to 7.2, and even more preferably 6.0 to 6.5. The buffering agent usable in the present invention is capable of adjusting the pH within this range and is medicamentally acceptable. Such buffering agents are known to those skilled in the art of solution formulations, and examples of such buffering agents include inorganic salts such as phosphate (sodium or potassium) and sodium bicarbonate; organic acid salts such as citrate (sodium or potassium), sodium acetate, and sodium succinate; and acids such as phosphoric acid, carbonic acid, citric acid, succinic acid, malic acid, and gluconic acid. Furthermore, Tris and Good's buffers such as MES, MOPS, and HEPES, histidine (e.g., histidine hydrochloride), and glycine may be used. In the high-concentration antibody-containing solution formulation of the present invention, the buffer solution is preferably a histidine buffer solution or a glycine buffer solution, and particularly preferably a histidine buffer solution. The concentration of the buffer solution is generally 1 to 500 mM, preferably 5 to 100 mM, and more preferably 10 to 20 mM. When a histidine buffer solution is used, the buffer solution preferably contains 5 to 25 mM histidine, and more preferably 10 to 20 mM histidine.

[0033] In the "stable" high-concentration antibody-containing solution formulation of the present invention, no significant change is observed for at least 12 months, preferably 2 years, and more preferably 3 years, at refrigerated temperatures (2-8°C); or for at least 3 months, preferably 6 months, and more preferably 1 year, at room temperature (22-28°C). For example, the total amount of dimer and decomposition product after storage for 2 years at 22°C is 5.0% or less, preferably 1.5% or less. or less than 2%, more preferably less than 1.5%, or the total amount of dimers and decomposition products after storage at 25°C for 6 months is less than 5.0%, preferably less than 2%, more preferably less than 1.5%. The following is the result.

[0034] The formulation of the present invention may further contain a surfactant. Examples of the surfactant include nonionic surfactants, such as sorbitan fatty acid esters such as sorbitan monocaprylate, sorbitan monolaurate, and sorbitan monopalmitate; glycerin fatty acid esters such as glycerin monocaprylate, glycerin monomyriate, and glycerin monostearate; polyglycerin fatty acid esters such as decaglyceryl monostearate, decaglyceryl distearate, and decaglyceryl monolinoleate; polyoxyethylene sorbitan fatty acid esters such as polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate, and polyoxyethylene sorbitan tristearate; polyoxyethylene sorbitan fatty acid esters such as polyoxyethylene sorbitan tetrastearate and polyoxyethylene sorbit tetraoleate; polyoxyethylene glycerin fatty acid esters such as polyoxyethylene glyceryl monostearate; polyethylene glycol distearate. polyethylene glycol fatty acid esters such as tearates; polyoxyethylene alkyl ethers such as polyoxyethylene lauryl ether; polyoxyethylene polyoxypropylene glycol ether, polyoxyethylene polyoxypropylene propyl ether, polyoxyethylene polyoxypropylene cetyl ether, and other polyoxyethylene polyoxypropylene alkyl ethers; polyoxyethylene alkyl phenyl ethers such as polyoxyethylene nonylphenyl ether; polyoxyethylene hydrogenated castor oils such as polyoxyethylene castor oil and polyoxyethylene hydrogenated castor oil (polyoxyethylene hydrogen castor oil); polyoxyethylene beeswax derivatives such as polyoxyethylene sorbitol beeswax; polyoxyethylene lanolin derivatives such as polyoxyethylene lanolin; polyoxyethylene fatty acid amides such as polyoxyethylene stearic acid amide, and other surfactants having an HLB of 6 to 18; anionic surfactants, for example, alkyl sulfates having an alkyl group having 10 to 18 carbon atoms, such as sodium cetyl sulfate, sodium lauryl sulfate, sodium oleyl sulfate, and other surfactants;Typical examples include polyoxyethylene alkyl ether sulfates having an average added mole number of ethylene oxide of 2 to 4 and an alkyl group having 10 to 18 carbon atoms, such as sodium polyoxyethylene lauryl sulfate; alkyl sulfosuccinate salts having an alkyl group having 8 to 18 carbon atoms, such as sodium lauryl sulfosuccinate; natural surfactants, such as lecithin, glycerophospholipids; phingophospholipids such as sphingomyelin; and sucrose fatty acid esters of fatty acids having 12 to 18 carbon atoms. One or more of these surfactants can be added in combination to the formulation of the present invention.

[0035] Preferred surfactants are polyoxyethylene sorbitan fatty acid esters and polyoxyethylene polyoxypropylene alkyl ethers, particularly preferred are polysorbates 20, 21, 40, 60, 65, 80, 81, 85 and Pluronic® type surfactants, most preferred are polysorbate 20, 80 and Pluronic F-68 (Poloxamer 188).

[0036] The amount of surfactant added to the antibody formulation of the present invention is generally 0.0001 to 10% ( The concentration is preferably 0.001 to 5%, and more preferably 0.005 to 3%.

[0037] In another embodiment of the invention, the formulation of the invention preferably comprises the following components: A) Anti-IL-6 receptor antibody B) arginine and / or methionine, and optionally further amino acids (e.g. tryptophan) as additional components. C) a buffering agent, and D) Surfactants The present invention is essentially composed of:

[0038] The term "substantially composed" means that it does not contain any components other than those typically added to formulations, such as the optional added components described below, such as suspending agents, solubilizing agents, isotonicity agents, preservatives, adsorption inhibitors, diluents, excipients, pH adjusters, soothing agents, sulfur-containing reducing agents, and antioxidants.

[0039] The above (B) "arginine and / or methionine, and further another amino acid (e.g., tryptophan) as an optional additional component" means that the types of amino acids that can be contained as additives in the formulation include (b-1) arginine; (b-2) arginine and methionine; (b-3) methionine, and may further contain another amino acid. A preferred example of the other amino acid is tryptophan, and as tryptophan, any of its derivatives and its salts may be used, and in particular, L-tryptophan or its salts are preferable.

[0040] If necessary, suspending agents, solubilizing agents, isotonicity agents, preservatives, adsorption inhibitors, diluents, excipients, pH adjusters, soothing agents, sulfur-containing reducing agents, antioxidants and the like may be appropriately added to the formulations of the present invention.

[0041] Examples of suspending agents include methylcellulose, polysorbate 80, hydroxyethylcellulose, gum arabic, powdered tragacanth, sodium carboxymethylcellulose, polyoxyethylene sorbitan monolaurate, and the like.

[0042] Examples of solution adjuvants include polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinamide, polyoxyethylene sorbitan monolaurate, macrogol, and castor oil fatty acid ethyl ester.

[0043] Examples of the isotonic agent include sodium chloride, potassium chloride, calcium chloride, and the like. Examples of preservatives include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, sorbic acid, phenol, cresol, and chlorocresol.

[0044] Examples of anti-adsorption agents include human serum albumin, lecithin, dextran, ethylene oxide-propylene oxide copolymer, hydroxypropyl cellulose, methyl cellulose, polyoxyethylene hydrogenated castor oil, and polyethylene glycol.

[0045] Examples of sulfur-containing reducing agents include those having a sulfhydryl group, such as N-acetylcysteine, N-acetylhomocysteine, thioctic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and its salts, sodium thiosulfate, glutathione, and thioalkanoic acids having 1 to 7 carbon atoms.

[0046] Examples of the antioxidant include erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, α-tocopherol, tocopherol acetate, L-ascorbic acid and its salts, L-ascorbyl palmitate, L-ascorbyl stearate, sodium hydrogen sulfite, sodium sulfite, triamyl gallate, propyl gallate, and chelating agents such as disodium ethylenediaminetetraacetate (EDTA), sodium pyrophosphate, and sodium metaphosphate.

[0047] The antibody-containing liquid formulation of the present invention is usually administered parenterally, for example, by injection (subcutaneous injection, intravenous injection, intramuscular injection, etc.), transdermal, transmucosal, nasal, pulmonary, etc., but can also be administered orally. For subcutaneous injection, the antibody dose per administration is large (about 100 to 200 mg), but the injection volume is limited, so the formulation of the present invention is particularly suitable for subcutaneous injection.

[0048] The osmotic pressure ratio of the antibody-containing liquid formulation of the present invention is preferably about 0.5-4, more preferably about 0.7-2, and even more preferably about 1. The viscosity of the antibody-containing liquid formulation of the present invention is preferably about 2 to 15 mPa s, and more preferably about 4 to 10 mPa s, provided that the viscosity in the present invention is measured by a rotational viscometer method using a cone-plate type viscometer (Japanese Pharmacopoeia, 15th Edition, General Test Methods, 2.53 Viscosity Measurement Method).

[0049] In the present invention, based on the results of the Examples described below, the addition of arginine alone, arginine and methionine, or methionine alone makes it possible to obtain a stable solution formulation with little antibody dimerization or deamidation even during long-term storage.

[0050] In yet another embodiment, the present invention provides a method for suppressing deamidation of an antibody-containing liquid formulation, which comprises adding arginine or a salt thereof to the solution. In yet another aspect, there is provided a method for suppressing antibody dimer formation in an antibody-containing liquid formulation, comprising adding arginine and methionine to the solution.

[0051] In the above two methods, the antibody is an anti-interleukin-6 receptor antibody, which is preferably a humanized or human antibody. The present invention will be described in more detail by the following examples, but the scope of the present invention is not limited to these examples.

[0052] [Example] Antibody samples The anti-IL-6 receptor humanized antibody is a humanized antibody produced according to the method described in Reference Example 2 of JP-A-8-99902, utilizing the human elongation factor Iα promoter described in Example 10 of International Patent Application Publication No. WO92 / 19759. Note that in the tables of the Examples, this antibody is sometimes referred to as MRA. EXAMPLES

[0053] Stabilizing effect of the combination of arginine and methionine The effect of a combination of arginine and methionine on the stabilization of a liquid formulation containing an anti-IL-6 receptor humanized antibody was evaluated.

[0054] In this study, samples A1 to A9 were used to evaluate the combined effect of arginine and methionine. The formulation of each evaluation sample is as follows:

[0055] [Table 1-1]

[0056] To evaluate the stability of the solution formulation, a thermal acceleration test was performed on each sample (storage at 40°C for 3 months and at 25°C for 6 months). The purity of the antibody before and after thermal acceleration was evaluated by gel permeation chromatography (SEC). The analytical conditions were as follows: [Gel filtration chromatography] The sample is used as the measurement solution as is.

[0057] For 1 μL of the measurement solution, perform the test using liquid chromatography under the following conditions, measure the peak areas of dimers, monomers, and low molecular weight decomposition products (LMW) using an automatic analysis method, and calculate their amounts (%).

[0058] [Table 1-2]

[0059]

number

[0060] A typical chromatograph is shown in FIG. The evaluation results of gel permeation chromatography (SEC) obtained in this example are shown in Table 1 and FIG. 3. Thus, for the samples to which arginine was added (samples No. A2 to A6), the amount of dimers after accelerated incubation at 40°C for 3 months and 25°C for 6 months was lower than that of the sample to which arginine was not added (sample No. A1), and the inhibitory effect of arginine on dimer formation was confirmed. It was also confirmed that the amount of dimers decreased in proportion to the amount of arginine added. On the other hand, for the samples to which methionine was added to arginine (100 mM) (samples No. A7 to A9), the amount of dimers after accelerated incubation at 40°C for 3 months and 25°C for 6 months was lower than that of the samples with an arginine concentration of 150 mM (samples No. A3 and A4), which were almost the same total stabilizer concentration, and was equivalent to that of the sample with an arginine concentration of 300 mM (sample No. A6). This result is considered to indicate a synergistic inhibitory effect on dimer formation due to the combination of arginine and methionine.

[0061] Furthermore, no effect of arginine or methionine was observed on the amount of low molecular weight decomposition products.

[0062] [Table 1-3] EXAMPLES

[0063] Inhibitory effect of arginine on deamidation The inhibitory effect of arginine-induced deamidation on a liquid formulation containing an anti-IL-6 receptor humanized antibody was evaluated.

[0064] In this study, samples No. A10 to A15 and No. A16 with different amounts of arginine and methionine were used. The formulations of the evaluation samples were as follows:

[0065] [Table 2-1]

[0066] To evaluate the stability of the solution formulation, a thermal acceleration test was performed on each sample (storage at 40°C for 3 months and 25°C for 6 months). The purity of the antibody before and after thermal acceleration was evaluated by ion exchange chromatography (IEC). The analytical conditions were as follows:

[0067] [Ion exchange chromatography] Add purified water to the sample to prepare a solution containing approximately 1 mg of anti-IL-6 receptor humanized antibody per mL. The resulting solution is used as the measurement solution for each sample.

[0068] Test 30μL of the test solution using liquid chromatography under the following conditions. The peak areas are measured by an automated analysis method, and the amounts (%) of MRA Pre, MRA Main, MRA Sub-1, MRA Sub-2, MRA R-1, 1Q(H)-MRA, 2Q(H)-MRA, and other related substances (Others) are calculated by the area percentage method.

[0069] MRA Pre is the sum of peaks eluted at shorter retention times than the main component, and is the anti-IL-6 receptor. The peak contains multiple degradation products, mainly deamidated forms of the target humanized antibody. A small amount of product means that deamidation of the antibody is suppressed.

[0070] [Table 2-2]

[0071]

number

[0072] A typical chromatogram is shown in Figure 4. MRA Pre is a chromatogram of all peaks that appear before MRA Main. It is the sum of the lines. The evaluation results of the ion exchange chromatography (IEC) in this example are shown in Table 2 and in Figs. 5 and 6. As shown in Fig. 1, for the samples with added arginine (samples No. A11 to A15), the amount of the pre-peak due to acceleration at 40°C for 3 months and at 25°C for 6 months was significantly greater than that of the samples without added arginine (samples No. The amount of the Pre peak was lower than that of the supplement No. A10), confirming the inhibitory effect of arginine on the generation of the Pre peak. It was also confirmed that the amount of the Pre peak decreased in proportion to the amount of arginine added. On the other hand, the samples to which methionine was added (samples A16 to A18) were incubated at 40°C for 3 weeks. The amount of Pre peak after accelerated incubation at 25°C for 1 month and 6 months was equivalent to that of the sample without arginine (sample No. A10), and no effect of methionine addition was observed.

[0073] [Table 2-3] EXAMPLES

[0074] Stabilizing effect of the combination of arginine and methionine (2) As in Example 1, the effect of a combination of arginine and methionine on the stabilization of a liquid formulation containing an anti-IL-6 receptor humanized antibody was evaluated.

[0075] In this study, in order to evaluate the combined effect of arginine and methionine, evaluation samples No. A19 to A27 were prepared. The formulation of each evaluation sample is as follows:

[0076] [Table 3-1]

[0077] To evaluate the stability of the solution formulation, each sample was subjected to a photoaccelerated test (total illumination of 1.2 million lux and total illumination of 1.5 million lux). UV radiation energy: 200 W·h / m 2The purity of the antibody before and after photoacceleration was evaluated by gel permeation chromatography (SEC) and ion exchange chromatography (IEC) in the same manner as in Examples 1 and 2.

[0078] The evaluation results of gel permeation chromatography (SEC) in this example are shown in Table 3 and FIG. As described above, the amount of dimers due to light acceleration was lower in the samples with added arginine (samples No. A20 to A24) than in the sample without added arginine (sample No. A19). It was possible to confirm the inhibitory effect on dimer formation. It was also possible to confirm that the amount of dimers decreased in proportion to the amount of arginine added. On the other hand, for the samples (samples A25 to A27) in which methionine was added to arginine (100 mM), the amount of dimers due to light acceleration was lower than that of the sample (sample A22) in which the arginine concentration was 150 mM, which was almost the same as the total stabilizer concentration. However, the arginine concentration was lower than that of the samples with arginine concentrations of 200 mM and 300 mM (samples No. A23 and A24). The results are believed to indicate a synergistic inhibitory effect on dimer formation by the combination of arginine and methionine.

[0079] No effect of arginine or methionine was observed on the amount of low molecular weight decomposition products.

[0080] [Table 3-2]

[0081] Next, the evaluation results of ion exchange chromatography (IEC) are shown in Table 4 and FIG. Thus, for the samples to which arginine was added (samples No. A20 to A24), the amount of Pre peak due to photoacceleration was lower than that of the sample to which arginine was not added (sample No. A19), and the inhibitory effect of arginine on the generation of Pre peaks was confirmed. It was also confirmed that the amount of Pre peaks decreased in proportion to the amount of arginine added. On the other hand, for the samples to which methionine was added to arginine (100 mM) (samples No. A25 to A27), the amount of dimers due to photoacceleration was lower than that of the sample with an arginine concentration of 150 mM (sample No. A22), which was almost the same as the total stabilizer concentration, and was also lower than that of the samples with arginine concentrations of 200 mM and 300 mM (samples No. A23 and A24). This result is considered to indicate the inhibitory effect of Pre peak generation as a synergistic effect of the combination of arginine and methionine.

[0082] [Table 4]

Claims

1. A method for suppressing deamidation of asparagine residues in a solution preparation containing a monoclonal IgG antibody at 50 mg / ml or more, the method comprising adding components such that the solution preparation has an arginine concentration of 50 to 300 mM, a nonionic surfactant concentration of 0.005 to 3% (w / v), a histidine concentration of 5 to 100 mM, and a pH of 5.5 to 7.2, the antibody being an anti-tissue factor antibody, an anti-IL-6 receptor antibody, an anti-IL-6 antibody, an HM1.24 antigen monoclonal antibody, an anti-parathyroid hormone-related peptide antibody (anti-PTHrP antibody), an anti-glypican-3 antibody, an anti-IL-6 ... The method of the present invention relates to a method for treating cancer, comprising administering to the patient a therapeutically effective amount of an antibody selected from the group consisting of an anti-CD3 antibody, an anti-ganglioside GM3 antibody, a coagulation factor VIII surrogate antibody, an anti-CD3 antibody, an anti-CD20 antibody, an anti-GPIIb / IIIa antibody, an anti-TNF antibody, an anti-CD25 antibody, an anti-EGFR antibody, an anti-Her2 / neu antibody, an anti-RSV antibody, an anti-CD33 antibody, an anti-CD52 antibody, an anti-CD11a antibody, an anti-VEGF antibody, an anti-VLA4 antibody, and an anti-AXL antibody.

2. The method described in claim 1, which suppresses deamidation of antibody molecules caused by heat or light during storage of the formulation.

3. The method of claim 1, wherein the nonionic surfactant is polysorbate 80 or 20.

4. The method according to any one of claims 1 to 3, wherein the antibody is an anti-IL-6 receptor antibody or a coagulation factor VIII surrogate antibody.

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