Highly concentrated antibody-containing solution formulation

Abstract

To provide a stable antibody-containing preparation suitable for subcutaneous administration in which dimer formation and deamidation during long-term storage are suppressed.SOLUTION: There is provided a stable antibody-containing solution formulation containing arginine and methionine.SELECTED DRAWING: None

Description

[Technical Field]

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

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

[0003] When designing an antibody-containing formulation for subcutaneous injection, it is important to consider the large amount of antibody administered per dose. However, subcutaneous injections generally have limitations on the amount of fluid injected (approximately 100-200 mg). High concentrations of antibodies in administration solutions are required. Therefore, high-concentration formulations are often prepared by reconstituting a lyophilized formulation using a smaller volume of water than before lyophilization, a technique known as lyophilization concentration. However, there is also a great demand for easy-to-use solution formulations that do not require the labor of reconstitution. Furthermore, in the production of lyophilized formulations, the addition of cryoprotectants such as sugars increases the viscosity of the formulation, which is undesirable for formulations intended for subcutaneous injection. However, solution formulations are thought to avoid this problem.

[0004] High-concentration antibody-containing solutions tend to form highly viscous solutions due to the macromolecular properties of proteins and intermolecular interactions. Furthermore, when proteins are stored in high-concentration solutions, degradation phenomena, including the formation of insoluble and / or soluble aggregates, become a problem, and these must be prevented. Antibody formulations, in particular, are prone to form aggregates and insoluble aggregates during storage in solution. Furthermore, when solution formulations are stored for long periods of time, there is a problem of loss of physiological activity of antibody molecules 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, technologies for preventing antibody dimerization and deamidation remain insufficient, particularly for 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 suitable for subcutaneous administration, in which dimer formation and deamidation during long-term storage are suppressed. [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 by containing 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 concentration is 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 liquid 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 liquid 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), which has 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 the formation of antibody dimers 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), 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. [Effects of the Invention]

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

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

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

[0013] The antibody-containing liquid formulations of the present invention are solution formulations containing high concentrations of antibodies, with antibody concentrations of preferably 50 mg / mL or higher, more preferably 100 mg / mL or higher, even more preferably 120 mg / mL or higher, and even more preferably 150 mg / mL. In particular, there have been no examples of antibody-containing liquid formulations with concentrations of 120 mg / mL or higher, preferably 150 mg / mL or higher, being put into practical use until now, and the formulations of the present invention have made it possible for the first time to put such high-concentration antibody-containing liquid formulations into practical use. Furthermore, from the viewpoint of production, the upper limit of the antibody concentration in the antibody-containing liquid 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, even more 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, but a monoclonal antibody is preferred in that it allows stable production of a homogeneous antibody.

[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. Furthermore, the immunoglobulin class of the antibody is not particularly limited, and may be any class, such as IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA, IgD, IgE, or IgM, although IgG and IgM are 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 minibodies such as monovalent or divalent or higher single-chain Fvs (e.g., scFv, sc(Fv)2, and diabodies such as scFv dimers) in which the variable regions of antibodies are linked via a linker such as a peptide linker.

[0017] The above-mentioned antibodies of the present invention can be produced by methods well known to those skilled in the art. Hybridomas producing monoclonal antibodies can basically be produced using known techniques as follows: Specifically, a desired antigen or cells expressing the desired antigen are used as a sensitizing antigen, and immunization is carried out using a conventional immunization method. The resulting immune cells are fused with known parent cells using a conventional cell fusion method, and monoclonal antibody-producing cells (hybridomas) are screened using a conventional screening method. Hybridomas can be produced, 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 can be conjugated to an immunogenic macromolecule such as albumin and then immunized.

[0018] Alternatively, an antibody gene can be cloned from a hybridoma, inserted into an appropriate vector, and then introduced into a host to produce a recombinant antibody using genetic engineering (see, for example, Carl, A.K. Borrebaeck, James, W. Larrick, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990). Specifically, cDNA encoding the variable region (V region) of the antibody is synthesized from the mRNA of the hybridoma using reverse transcriptase. Once DNA encoding the V region of the desired antibody is obtained, For example, this may be ligated to DNA encoding the desired antibody constant region (C region), and the resulting product may be used as an expression vector. Alternatively, DNA encoding the antibody V region may be incorporated into an expression vector containing DNA encoding the antibody C region. The DNA 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, host cells can be 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 to humans, such as chimeric antibodies and humanized antibodies, are used. These modified antibodies can be produced using known methods. Chimeric antibodies are antibodies consisting of the heavy and light chain variable regions of an antibody from a non-human mammal, such as 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] Humanized antibodies are also called reshaped human antibodies, and are made by converting the complementarity determining regions (CDRs) of a non-human mammalian antibody, such as a mouse antibody, into human antibodies. This method involves transplanting the CDRs of a mouse antibody into the framework regions (FRs) of a human antibody, and common genetic recombination techniques are known. Specifically, a DNA sequence designed to link the CDRs of a mouse antibody to the framework regions (FRs) of a human antibody is synthesized by PCR from several oligonucleotides engineered to have overlapping ends. The resulting DNA is ligated to DNA encoding the constant regions of a human antibody, then incorporated into an expression vector, which is then introduced into a host for production (see European Patent Application Publication No. EP 239400 and International Patent Application Publication No. WO 96 / 02576). The FRs of the human antibody linked via the CDRs are selected so that the CDRs form a good antigen-binding site. If necessary, amino acids in the framework regions of the antibody variable region can be substituted 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 with antigen-binding activity (see Japanese Patent Publication No. 1-59878). Alternatively, a desired human antibody can be obtained by immunizing a transgenic animal carrying a full repertoire of human antibody genes with the 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 human antibodies by panning using a human antibody library is also known. For example, the variable regions of human antibodies can be expressed on the surface of phages as single-chain fragments (scFvs) using phage display, and phages that bind 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 known, an appropriate expression vector containing the sequence can be constructed to obtain a human antibody. These methods are already well known, and are described in WO 92 / 01047, WO 92 / 20791, WO 93 / 06213, Reference can be made to WO 93 / 11236, WO 93 / 19172, WO 95 / 01438, and WO 95 / 15388.

[0022] When antibody genes are isolated and then introduced into a suitable host to produce antibodies, a suitable combination of host and expression vector can be used. When eukaryotic cells are used as hosts, 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 from the genus Nicotiana, such as Nicotiana tabacum, which can be cultured as callus. Known fungal cells include yeasts such as the Saccharomyces genus, e.g., Saccharomyces cerevisiae, and filamentous fungi such as the Aspergillus genus, e.g., Aspergillus niger. When using prokaryotic cells, there are production systems that use bacterial cells. Known bacterial cells include E. coli and Bacillus subtilis. These cells The desired antibody gene is then introduced into the cells by transformation, and the transformed cells are cultured in vitro to obtain the antibody.

[0023] Furthermore, the antibodies of the present invention may be antibody fragments, minibodies, or modified antibodies. Examples of antibody fragments and minibodies include Fab, F(ab'), Fv, or monovalent or multivalent single-chain Fvs (e.g., scFv, sc(Fv)) in which the Fvs of the H chain and L chain are linked via an appropriate linker (Huston, JS et al., Proc. Natl. Acad. Sci. USA (1988) 85, 5879-5883). can be done. 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 (e.g., 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, 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 encompasses these modified antibodies. Such modified antibodies can be obtained by chemically modifying the antibody obtained. These methods have already been established in this field.

[0025] 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,

[0026] Examples of reshaped humanized antibodies 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 humanized anti-glypican-3 IgG1κ antibody (see International Patent Application Publication No. WO99-51743). (See application number PCT / JP05 / 013103) are preferred antibodies for use in the present invention. A particularly preferred humanized antibody for use in the present invention is a humanized anti-IL-6 receptor antibody.

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

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

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

[0030] The arginine used in the present invention may be any of a single compound, a derivative thereof, or a salt thereof, with L-arginine or a salt thereof being particularly preferred.The methionine used in the present invention may be any of a single compound, a derivative thereof, or a salt thereof, with L-methionine or a salt thereof being 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, preferably 40 to 1000 mM arginine and 10 to 200 mM methionine, more preferably 50 to 700 mM arginine and 10 to 100 mM methionine, and even more preferably 100 to 300 mM arginine and 10 to 50 mM methionine.

[0032] The buffer solution is prepared using a buffering agent, which is a substance that maintains 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. Buffers that can be used in the present invention are those that can adjust the pH within this range and are pharmaceutically acceptable. Such buffers are well known to those skilled in the art of solution formulations, and examples 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, Good's buffers such as Tris, MES, MOPS, and HEPES, histidine (e.g., histidine hydrochloride), and glycine may also be used. In the high-concentration antibody-containing solution formulation of the present invention, the buffer solution is preferably a histidine buffer or glycine buffer, with a histidine buffer being particularly preferred. 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] The "stable" high-concentration antibody-containing solution formulations of the present invention show no significant changes 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 dimers and decomposition products after storage at 2°C for 2 years is 5.0% or less, preferably 1.0% 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 surfactants 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 monomyliate, 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 sorbit 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 stearates; 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 sorbitan 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, and sodium oleyl sulfate;Typical examples include polyoxyethylene alkyl ether sulfates, such as sodium polyoxyethylene lauryl sulfate, in which the average number of moles of ethylene oxide added is 2 to 4 and the alkyl group has 10 to 18 carbon atoms; alkyl sulfosuccinates, such as sodium lauryl sulfosuccinate, in which the alkyl group has 8 to 18 carbon atoms; 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 to the formulations 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 polysorbates 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% ( w / v), preferably 0.001 to 5%, and more preferably 0.005 to 3%.

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

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

[0039] The above (B) "arginine and / or methionine, and optionally another amino acid (e.g., tryptophan)" 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; and (b-3) methionine, and may further contain another amino acid. A preferred example of the other amino acid is tryptophan, and tryptophan may be used alone, as a derivative, or as a salt thereof, with L-tryptophan or a salt thereof being particularly desirable.

[0040] If necessary, suspending agents, solubilizing agents, isotonicity agents, preservatives, anti-adsorption agents, diluents, excipients, pH adjusters, soothing agents, sulfur-containing reducing agents, antioxidants, etc. 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, and polyoxyethylene sorbitan monolaurate.

[0042] Examples of the solution adjuvant include polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinamide, polyoxyethylene sorbitan monolaurate, magrogol, and castor oil fatty acid ethyl esters.

[0043] Examples of isotonic agents 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 antioxidants 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 solution formulations of the present invention are usually administered parenterally, for example, by injection (subcutaneous, intravenous, intramuscular, etc.), transdermal, transmucosal, nasal, or pulmonary route, but can also be administered orally. For subcutaneous injection, the antibody dose per administration is large (approximately 100 to 200 mg), but the injection volume is limited, so the formulations of the present invention are 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, as measured by a rotational viscometer using a cone-and-plate 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, it is possible to obtain a stable solution formulation with little antibody dimer formation or deamidation even during long-term storage by adding arginine alone, arginine and methionine, or methionine alone.

[0050] In yet another embodiment, the present invention provides a method for suppressing deamidation in an antibody-containing liquid formulation, which comprises adding arginine or a salt thereof to the solution. In yet another embodiment, 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 sample The humanized anti-IL-6 receptor antibody was produced using the human elongation factor Iα promoter described in Example 10 of International Patent Application Publication No. WO92 / 19759, according to the method described in Reference Example 2 of Japanese Patent Laid-Open Publication No. 8-99902. Note that this antibody is sometimes referred to as MRA in the tables of the Examples. [Example]

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

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

[0055] [Table 1-1]

[0056] To evaluate the stability of the solution formulations, 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] Test 1 μL of the test solution using liquid chromatography under the following conditions, and measure the peak areas of dimer, monomer, and low molecular weight decomposition products (LMW) using an automated analysis method. Measure and calculate the amount (%).

[0058] [Table 1-2]

[0059]

number

[0060] A typical chromatogram is shown in Figure 1. The evaluation results of gel permeation chromatography (SEC) obtained in this example are shown in Table 1 and Figure 2. The results are shown in Figure 3. As shown above, for the samples containing arginine (samples A2 to A6), the dimer amounts after accelerated incubation at 40°C for 3 months and at 25°C for 6 months were lower than those for the sample without arginine (sample A1), confirming the inhibitory effect of arginine on dimer formation. It was also confirmed that the dimer amount decreased in proportion to the amount of arginine added. On the other hand, for the samples containing arginine (100 mM) and methionine (samples A7 to A9), the dimer amounts after accelerated incubation at 40°C for 3 months and at 25°C for 6 months were lower than those for the samples with an arginine concentration of 150 mM (samples A3 and A4), which had approximately the same total stabilizer concentration, but were comparable to those for the sample with an arginine concentration of 300 mM (sample 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 degradation products.

[0062] [Table 1-3] [Example]

[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 A10 to A15 and A16 with different amounts of arginine and methionine were used. The formulation of each evaluation sample is as follows:

[0065] [Table 2-1]

[0066] To evaluate the stability of the solution formulations, 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 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 prepared solution is used as the measurement solution for each sample.

[0068] Test 30 μL of the test solution using liquid chromatography under the following conditions. The peak areas are measured by automated analysis, 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. This peak contains multiple degradation products, primarily 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 works. The evaluation results of 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 after 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. A11 to A15). The amount of the Pre-peak was lower than that of 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 heated at 40°C for 3 months. The amount of the Pre peak after accelerated incubation at 25°C for 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] [Example]

[0074] Stabilizing effect of the combination of arginine and methionine (2) As in Example 1, the effect of the combination of arginine and methionine on the stability 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 Nos. A19 to A27 were prepared. The formulation of each evaluation sample was as follows:

[0076] [Table 3-1]

[0077] To evaluate the stability of the solution formulation, each sample was subjected to accelerated light testing (total illuminance of 1.2 million lux and total approx. UV irradiation energy 200 W·h / m 2The purity of the antibody before and after photoacceleration was determined by gel permeation chromatography (SEC) and ion exchange chromatography in the same manner as in Examples 1 and 2. The results were evaluated using the IEC method.

[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 photoacceleration was lower in the samples with added arginine (samples No. A20 to A24) than in the sample without added arginine (sample No. A19). The inhibitory effect on dimer formation was confirmed. It was also confirmed that the amount of dimer 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 dimer due to photoacceleration 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. This was lower than 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 degradation products.

[0080] [Table 3-2]

[0081] Next, the evaluation results of ion exchange chromatography (IEC) are shown in Table 4 and FIG. As described above, the amount of Pre peak due to photoacceleration 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 also confirmed that the amount of pre-peak decreased in proportion to the amount of arginine added. For the samples with added arginine (samples No. A25 to A27), the amount of dimers due to photoacceleration was lower than that of the sample with 150 mM arginine (sample No. A22), which had almost the same total stabilizer concentration. In addition, the arginine concentration was lower than that of the samples with 200 mM and 300 mM (samples No. A23 and A24). These results suggest that the combination of arginine and methionine has a synergistic effect in inhibiting the production of pre-peaks.

[0082] [Table 4]

Claims

1. A method for inhibiting deamidation of asparagine residues in a solution formulation containing 120 to 250 mg / mL of a monoclonal IgG1 or IgG4 antibody, the method comprising adding components such that the solution formulation has an arginine concentration of 50 to 300 mM, a nonionic surfactant concentration of 0.005 to 3% (w / v), and a histidine concentration of 5 to 25 mM, and a pH of 5.5 to 7.

2.

2. The method according to claim 1, wherein deamidation of antibody molecules due to heat or light during storage of the formulation is suppressed.

3. A method for producing a solution formulation containing 120 to 250 mg / mL of a monoclonal IgG1 or IgG4 antibody, in which deamidation of asparagine residues in the antibody is inhibited, the method comprising adding each component so that the solution formulation has an arginine concentration of 50 to 300 mM, a nonionic surfactant concentration of 0.005 to 3% (w / v), and a histidine concentration of 5 to 25 mM, and a pH of 5.5 to 7.

2.

4. The method according to claim 3, which is a method for producing a formulation in which deamidation of antibody molecules due to heat or light during storage of the formulation is suppressed.

5. A method for storing a solution formulation containing 120 to 250 mg / mL of a monoclonal IgG1 or IgG4 antibody, in which deamidation of asparagine residues in the antibody is inhibited, the method comprising adding each component to the solution formulation so that the concentration of arginine in the solution formulation is 50 to 300 mM, the concentration of polysorbate 80 or 20 is 0.005 to 3% (w / v), the concentration of histidine is 5 to 25 mM, and the pH is 5.5 to 7.

2.

6. The method according to claim 5, which is a method for preserving a formulation, wherein deamidation of antibody molecules due to heat or light during storage of the formulation is suppressed.

7. The method according to any one of claims 1 to 6, wherein the nonionic surfactant is polysorbate 80 or 20.

8. The method according to any one of claims 1 to 6, wherein the liquid formulation is an injection.

9. The method according to any one of claims 1 to 6, wherein the antibody is a humanized antibody or a human antibody.

10. The method according to any one of claims 1 to 6, wherein the histidine concentration is about 20 mM.

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