HK1231734B - Solution preparation containing antibody at high concentration - Google Patents

Description

Solution preparations containing high concentrations of antibodies

This application is a divisional application of the invention patent application 200880119066.5 of the same name filed on December 26, 2008.

Technical Field

The present invention relates to a preparation containing an antibody, and in particular to a stable solution preparation containing a high concentration of an antibody.

Background Art

In recent years, various antibody preparations have been developed for practical use, but many are administered intravenously. Meanwhile, due to practical needs in the medical field, there is a growing demand for the development of antibody-containing preparations for subcutaneous self-administration.

On the one hand, the single antibody administration dose is large (approximately 100-200 mg), and on the other hand, the subcutaneous injection solution volume is generally limited. Therefore, when designing antibody-containing formulations for subcutaneous injection, it is necessary to highly concentrate the antibody in the administration solution. To this end, the lyophilized formulation is reconstituted in a smaller volume of water than before lyophilization to produce a high-concentration solution formulation. High-concentration formulations prepared using so-called lyophilization and concentration techniques are now widely used. However, there is also a great demand for convenient solution formulations that can be used without the need for a reconstitution step. In addition, when preparing lyophilized formulations, the addition of cryoprotectants such as sugars increases the viscosity of the formulation, making it generally unsuitable for subcutaneous injection. If a solution formulation is used, this problem can be avoided.

Solutions containing high concentrations of antibodies tend to form solutions with relatively high intrinsic viscosity due to the macromolecular properties of proteins and intermolecular interactions. Furthermore, when proteins are stored in highly concentrated solutions, there is a problem of degradation, primarily the formation of insoluble and/or soluble aggregates, which should be prevented. Antibody preparations, in particular, are prone to forming polymers and insoluble aggregates when stored in solution. Furthermore, when solution preparations are stored for extended periods, there is also the problem of antibody molecules losing their physiological activity due to deamidation of amino acid residues other than asparagine.

Typically, to minimize loss of active ingredients in protein formulations even after long-term storage, various methods are employed to stabilize the formulation by dissolving the active ingredient and various additives in a buffer. However, technologies for preventing the formation of antibody dimers and deamidation, particularly in solution formulations containing high antibody concentrations, remain incomplete.

Therefore, there is a need for a formulation containing a high concentration of antibodies, which has the characteristics of being able to inhibit dimer formation and deamidation during long-term storage, being stable, and being suitable for subcutaneous administration.

Summary of the Invention

Technical issues that the invention intends to solve

The object of the present invention is to provide a preparation containing a high concentration of an antibody, which is characterized in that the formation of dimers and the occurrence of deamidation can be suppressed during long-term storage, and the preparation is stable and suitable for subcutaneous administration.

Technical solutions to problems

To achieve the above objectives, after diligent research, the inventors of the present invention discovered that by adding arginine, an amino acid, and its salts as stabilizers, a stable solution formulation containing high-concentration antibodies can be obtained, thereby completing the present invention.

That is, the present invention provides the following contents.

(1) A stable antibody-containing solution formulation characterized by containing arginine and methionine.

(2) A solution preparation of (1), further comprising a histidine buffer.

(3) The solution preparation of (1) or (2), further comprising a surfactant.

(4) The solution preparation of (1) to (3), wherein the antibody concentration is 50 mg/mL or higher.

(5) The solution preparation of (1) to (3), wherein the antibody concentration is 100 mg/mL or higher.

(6) The solution preparation of (1) to (3), wherein the antibody concentration is 120 mg/mL or higher.

(7) The solution preparation of (1) to (6), wherein the antibody is an anti-interleukin-6 receptor antibody.

(8) A stable solution preparation containing an anti-interleukin-6 receptor antibody, characterized by containing arginine or methionine.

(9) The solution preparation of (1) to (8), wherein the antibody is a humanized antibody or a human antibody.

(10) The solution preparation of (1) to (9), further comprising tryptophan.

(11) The solution preparation of (1) to (10), wherein the pH of the solution preparation is 4 to 8.

(12) The solution preparation of (1) to (11), wherein the content of arginine is 50 to 1500 mM.

(13) The solution preparation of (1) to (12) has a viscosity of 2 to 15 mPa·s.

(14) The solution preparation of (1) to (13), wherein the solution preparation is stable at 22 to 28°C for at least 6 months.

(15) The solution preparation of (1) to (13), characterized in that the formation of antibody dimers is suppressed.

(16) The solution preparation of (1) to (13), characterized in that deamidation of the antibody molecule is suppressed.

(17) A solution preparation of (1) to (13), which is administered subcutaneously.

(18) The solution preparation of (1) to (13), wherein the solution preparation is prepared without a freeze-drying step.

(19) A method for inhibiting deamidation of an antibody molecule in an antibody-containing solution formulation, comprising: adding arginine to the solution.

(20) A method for inhibiting the formation of antibody dimers in a solution preparation containing an antibody, comprising: adding arginine and methionine to the solution.

Effects of the Invention

A solution formulation containing a high concentration of an antibody can be provided without the need for freeze-drying, concentration, reconstitution, or reconstitution steps. The high-concentration antibody solution formulation of the present invention can be stored stably in a solution state for a long period of time. Because the preparation process does not include a freeze-drying step, there is no need to add sugars or other lyoprotectants.

BRIEF DESCRIPTION OF THE DRAWINGS

[Figure 1] shows a typical chromatogram of Example 1.

[ Figure 2 ] shows the evaluation results of gel filtration chromatography (SEC) in Example 1.

[ Figure 3 ] shows the evaluation results of gel filtration chromatography (SEC) in Example 1.

[Figure 4] shows a typical chromatogram of Example 2.

[ Fig. 5 ] shows the evaluation results of ion exchange chromatography (IEC) in Example 2.

[ Fig. 6 ] shows the evaluation results of ion exchange chromatography (IEC) in Example 2.

[ Figure 7 ] shows the evaluation results of gel filtration chromatography (SEC) in Example 3.

[ Fig. 8 ] shows the evaluation results of ion exchange chromatography (IEC) in Example 3.

Implementation Method

Hereinafter, the present invention will be described in detail.

In the present invention, an antibody-containing solution preparation refers to a solution preparation containing an antibody as an active ingredient and prepared in a manner that can be administered to humans and other animals, particularly a solution preparation prepared without a lyophilization step during the preparation process.

The antibody-containing solution formulation of the present invention refers to a solution formulation containing a high concentration of an antibody, preferably having an antibody concentration of 50 mg/mL or greater, more preferably 100 mg/mL or greater, more preferably 120 mg/mL or greater, and even more preferably 150 mg/mL. In particular, to date, there have been no practical examples of solution formulations containing an antibody at a concentration of 120 mg/mL or greater, preferably 150 mg/mL or greater. Therefore, the formulation of the present invention makes such a high-concentration antibody-containing solution formulation practical for the first time.

Furthermore, from a production perspective, 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-containing solution formulation of the present invention is preferably 50-300 mg/mL, more preferably 100-300 mg/mL, even more preferably 120-250 mg/mL, and particularly preferably 150-200 mg/mL.

The antibodies used in the present invention are not particularly limited as long as they can bind to the desired antigen and may be polyclonal or monoclonal antibodies. However, monoclonal antibodies are preferred for stable production of homogeneous antibodies.

The monoclonal antibodies used in the present invention include not only monoclonal antibodies derived from humans, mice, rats, hamsters, rabbits, sheep, camels, monkeys, and the like, but also artificially engineered recombinant antibodies such as chimeric antibodies, humanized antibodies, and bispecific antibodies. Furthermore, the immunoglobulin class of the antibodies is not particularly limited and may be any of the following: IgG (IgG1, IgG2, IgG3, IgG4, etc.), IgA, IgD, IgE, and IgM, with IgG and IgM being preferred.

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 higher-valent single-chain Fvs (diabodies such as scFv, sc(Fv) ₂ , or scFv dimers) in which antibody variable regions are linked by linkers such as polypeptide linkers.

The antibodies of the present invention can be prepared according to methods known to those skilled in the art.

Hybridomas that produce monoclonal antibodies can basically be prepared using known methods as follows. That is, the desired antigen or cells expressing the desired antigen are used as sensitizing antigens, which are immunized using conventional immunization methods, and then the resulting immune cells are fused with known mother cells using conventional cell fusion methods. Monoclonal antibody-producing cells (hybridomas) are screened using conventional screening methods to prepare them. Hybridomas can be prepared by referring to the method used by Milstein et al. (Kohler. G. and Milstein, C., Methods Enzymol. (1981) 73: 3-46). When the immunogenicity of the antigen is low, immunization can be performed by binding to immunogenic macromolecules such as albumin.

In addition, antibody genes can be cloned from hybridomas, integrated into suitable vectors, and then introduced into hosts. Genetic recombinant antibodies can be produced using genetic recombination technology (e.g., see Carl, A.K. Borrebaeck, James, W. Larrick, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990). Specifically, cDNA of the antibody variable region (V region) is synthesized from hybridoma mRNA using reverse transcriptase. If DNA encoding the target antibody V region is available, it is ligated with DNA encoding the desired antibody constant region (C region) and integrated into an expression vector. Alternatively, the DNA encoding the antibody V region can be integrated into an expression vector containing the DNA of the antibody C region. The antibody can be integrated into an expression vector to be expressed in a manner that can be regulated by expression regulatory regions (e.g., enhancers, promoters), and then the antibody can be expressed by transforming host cells with the expression vector.

In the present invention, in order to achieve the purpose of reducing heterologous antigenicity to humans, artificially modified genetically recombinant antibodies, such as chimeric antibodies and humanized antibodies, can be used. These modified antibodies can be prepared by known methods. Chimeric antibodies are antibodies composed of the variable regions of the antibody heavy and light chains of mammals other than humans (such as mice) and the constant regions of the antibody heavy and light chains of humans. They can be obtained by connecting DNA encoding the variable regions of mouse antibodies with DNA encoding the constant regions of human antibodies, integrating them into expression vectors, and then introducing them into host cells for production.

Humanized antibodies, also known as reshaped antibodies, are antibodies obtained by transplanting the complementary determining regions (CDRs) of mammalian antibodies (such as mice) other than humans into the complementary determining regions of human antibodies. Conventional gene recombination techniques are known. Specifically, a DNA sequence designed to connect the CDRs of mouse antibodies and the framework regions (FRs) of human antibodies is synthesized by PCR using several oligonucleotides having overlapping ends. The obtained DNA is connected to the DNA encoding the constant region of a human antibody, which is then integrated into an expression vector and then introduced into a host to produce the resulting DNA (see European Patent Application Publication EP 239400, International Patent Application Publication WO 96/02576). The human antibody FR connected by the CDR can be selected to form a good antigen binding site in the complementary determining region. As needed, in order to form a suitable antigen binding site in the complementary determining region of the reconstructed human antibody, the amino acids in the variable region framework region of the antibody can also be replaced (Sato, K. et al., Cancer Res. (1993) 53, 851-856).

In addition, methods for obtaining human antibodies are known. For example, human lymphocytes are sensitized in vitro with a desired antigen or cells expressing a desired antigen, and the sensitized lymphocytes are fused with human myeloma cells (such as U266) to obtain the desired human antibodies with antigen-binding activity (reference to Japanese Patent Publication No. 1-59878). In addition, the desired human antibodies can be obtained by immunizing transgenic animals with a full set of human antibody genes with antigens (reference to International Patent Application Publications WO93/12227, WO92/03918, WO94/02602, WO94/25585, WO96/34096, WO96/33735). Furthermore, the technology of using human antibody libraries to obtain human antibodies by panning is also known. For example, phages that bind to antigens can be screened by expressing the human antibody variable region as a single-chain antibody (scFv) on the phage surface through phage display. If the genes of the screened phage are analyzed, the DNA sequence encoding the human antibody variable region that binds to the antigen can be determined. Once the DNA sequence of the antigen-binding scFv is known, a suitable expression vector containing that sequence can be prepared, thereby obtaining a human antibody. Such methods are also well known, and reference can be made to WO92/01047, WO92/20791, WO93/06213, WO93/11236, WO93/19172, WO95/01438, and WO95/15388.

When the antibody gene is isolated and introduced into a suitable host to prepare the antibody, a suitable host can be used in combination with an expression vector. When eukaryotic cells are used as hosts, animal cells, plant cells, and fungal cells can be used. As animal cells, (1) mammalian cells (such as CHO, COS, myeloma, BHK (baby hamster kidney), HeLa, Vero), (2) amphibian cells (such as African clawed frog oocytes), or (3) insect cells (such as Sf9, Sf21, Tn5) are known. As plant cells, for example, Nicotiana (such as cells derived from Nicotiana tabacum) are known, and callus culture can be performed on them. As fungal cells, for example, yeast (such as Saccharomyces (such as Saccharomyces cerevisiae)) and mold (such as Aspergillus (such as Aspergillus niger)) are known. When prokaryotic cells are used, there is a production system using bacterial cells. Examples of bacterial cells include Escherichia coli (E. coli) and Bacillus subtilis. Antibodies can be obtained by transforming these cells with the target gene and culturing the transformed cells in vitro.

Furthermore, the antibodies of the present invention may be antibody fragments, low-molecular-weight antibodies, and modified antibodies. For example, antibody fragments or low-molecular-weight antibodies include Fab, F(ab')2, Fv, or monovalent or divalent or higher-valent single-chain Fvs (scFv, sc(Fv) ₂ , etc.) obtained by linking H and L chains of Fv via an appropriate linker (Huston, J. S. et al., Proc. Natl. Acad. Sci. USA (1988) 85, 5879-5883). Specifically, the antibody fragments can be obtained by treating the antibody with an enzyme (e.g., papain or pepsin), or by constructing genes encoding the antibody fragments, introducing the genes into expression vectors, and expressing the genes in appropriate host cells (e.g., see Co, M S et al., J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, A H, 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, R E and Walker, BW, Trends Biotechnol. (1991) 9, 132-137).

As modified antibodies, antibodies conjugated with various molecules such as polyethylene glycol (PEG) can be used. The "antibody" of the present invention includes these modified antibodies. To obtain such modified antibodies, the obtained antibodies can be chemically modified. These methods have been established in the art.

Examples of the antibodies contained in the preparation of the present invention include, but are not limited to, anti-tissue factor antibodies, anti-IL-6 receptor antibodies, anti-IL-6 antibodies, HM1.24 antigen monoclonal antibodies, anti-parathyroid hormone-related peptide antibodies (anti-PTHrP antibodies), anti-glypican-3 antibodies, anti-ganglioside GM3 antibodies, anti-TPO receptor agonist antibodies, coagulation factor VIII replacement antibodies, anti-CD3 antibodies, anti-CD20 antibodies, anti-GPIIb/IIIa antibodies, anti-TNF antibodies, anti-CD25 antibodies, anti-EGFR antibodies, anti-Her2/neu antibodies, anti-RSV antibodies, anti-CD33 antibodies, anti-CD52 antibodies, anti-IgE antibodies, anti-CD11a antibodies, anti-VEGF antibodies, anti-VLA4 antibodies, and anti-AXL antibodies.

Preferred reshaped humanized antibodies used in the present invention include humanized anti-interleukin-6 (IL-6) receptor antibodies (hPM-1 or MRA) (see International Patent Application Publication WO92-19759), humanized anti-HM1.24 antigen monoclonal antibodies (see International Patent Application Publication WO98-14580), humanized anti-parathyroid hormone-related peptide antibodies (anti-PTHrP antibodies) (see International Patent Application Publication WO98-13388), humanized anti-tissue factor antibodies (see International Patent Application Publication WO99-51743), and anti-glypican-3 humanized IgG1κ antibodies (see International Patent Application PCT/JP05/013103). Humanized anti-IL-6 receptor antibodies are particularly preferred as humanized antibodies used in the present invention.

As the human IgM antibody, a recombinant human IgM antibody against ganglioside GM3 is preferred (see International Patent Application Publication WO05-05636).

As low-molecular-weight antibodies, anti-TPO receptor agonist diabodies (see International Patent Application Publication WO02-33072), anti-CD47 agonist diabodies (see International Patent Application Publication WO01-66737), and the like are preferred.

To evaluate the storage stability of samples containing high-concentration antibodies, the present inventors used thermal acceleration tests and light acceleration tests to investigate the effects of various additives. The results revealed that solutions prepared by dissolving high-concentration antibodies in a buffer containing the amino acid arginine resulted in lower levels of dimer formation compared to solutions without arginine, suggesting that arginine is effective as a stabilizer for inhibiting dimer formation. Furthermore, solutions prepared by dissolving high-concentration antibodies in a buffer containing both arginine and methionine showed the same dimer inhibition effect, even at a lower combined concentration of arginine and methionine than when arginine alone was used. This suggests that the combined use of arginine and methionine produces a synergistic effect. Furthermore, the addition of arginine was found to inhibit deamidation of antibody molecules. These findings are exemplified in the Examples described later in this specification using samples containing 180 mg/mL of humanized anti-IL-6 receptor antibody.

That is, by including arginine as a stabilizer, a stable antibody preparation can be produced that reduces the formation of antibody dimers and prevents deamidation. Therefore, a first embodiment of the present invention is characterized in that arginine is added to the solution, thereby inhibiting the formation of antibody dimers or deamidation in an antibody-containing solution preparation. Furthermore, as an embodiment of a stable antibody-containing solution preparation, it is characterized in that the buffer contains an antibody and arginine. Moreover, as described above, the antibody-containing solution preparation of the present invention further contains methionine, and the combination of arginine and methionine exerts a synergistic effect. Therefore, a second embodiment of the present invention is characterized in that arginine and methionine are added to the solution, particularly inhibiting the formation of antibody dimers in an antibody-containing solution preparation. Furthermore, as an embodiment of a stable antibody-containing solution preparation, it is characterized in that the buffer contains an antibody, arginine, and methionine.

As the arginine used in the present invention, any one of a monomer, a derivative thereof, or a salt thereof can be used, and L-arginine or a salt thereof is particularly preferred. As the methionine used in the present invention, any one of a monomer, a derivative thereof, or a salt thereof can be used, and L-methionine or a salt thereof is particularly preferred.

In the antibody-containing solution formulation of the present invention, when methionine is not added and only arginine is contained, the amount of arginine is preferably 50-1500 mM, more preferably 100-1000 mM, and even more preferably 200-700 mM. When the antibody-containing solution formulation of the present invention contains both arginine and methionine, the total concentration of arginine and methionine is 50-1200 mM. For example, the amount of arginine is preferably 40-1000 mM and the amount of methionine is 10-200 mM; more preferably, the amount of arginine is 50-700 mM and the amount of methionine is 10-100 mM, and even more preferably, the amount of arginine is 100-300 mM and the amount of methionine is 10-50 mM.

The buffer is prepared using a buffering agent, which is a substance used to maintain the pH of the solution. In the solution formulation containing a high concentration of the antibody of the present invention, the pH of the solution is preferably pH 4-8, more preferably 5.0-7.5, more preferably 5.5-7.2, and even more preferably 6.0-6.5. The buffer that can be used in the present invention is a pharmaceutically acceptable buffer that can adjust the pH within this range. Such buffers are well known to those skilled in the art in the field of solution formulations, for example, 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; in addition, acids such as phosphoric acid, carbonic acid, citric acid, succinic acid, malic acid, and gluconic acid can also be used. Tris-based and excellent buffers such as MES, MOPS, and HEPES, histidine (such as histidine hydrochloride), and glycine can also be used. In the solution formulation containing a high concentration of the antibody of the present invention, the buffer is preferably a histidine buffer or a glycine buffer, with histidine buffer being particularly preferred. The concentration of the buffer is generally 1 to 500 mM, preferably 5 to 100 mM, and more preferably 10 to 20 mM. When a histidine buffer is used, the buffer preferably contains 5 to 25 mM histidine, and more preferably 10 to 20 mM histidine.

The "stable" solution formulation containing a high concentration of an antibody of the present invention should exhibit no significant changes when stored at refrigerated temperatures (2-8°C) for at least 12 months, preferably 2 years, and more preferably 3 years, or at room temperature (22-28°C) for at least 3 months, preferably 6 months, and more preferably 1 year. For example, after 2 years of storage at 5°C, the sum of the amount of dimers and degradation products should be less than 5.0%, preferably less than 2%, and more preferably less than 1.5%. After 6 months of storage at 25°C, the sum of the amount of dimers and degradation products should be less than 5.0%, preferably less than 2%, and more preferably less than 1.5%.

The formulations of the present invention may also contain a surfactant.

Examples of surfactants include:

Nonionic surfactants, such as:

Sorbitan fatty acid esters (sorbitan monooctanoate, sorbitan monolaurate, sorbitan monopalmitate, etc.);

Glyceryl fatty acid esters (glyceryl monocaprylate, glyceryl monomyristate, glyceryl monostearate, etc.);

Polyglycerol fatty acid esters (decaglyceryl monostearate, decaglyceryl distearate, decaglyceryl linoleate, etc.);

Polyoxyethylene sorbitan fatty acid esters (polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate, polyoxyethylene sorbitan tristearate, etc.);

Polyoxyethylene sorbitan fatty acid esters (polyoxyethylene sorbitan tetrastearate, polyoxyethylene sorbitan tetraoleate, etc.);

Polyoxyethylene glycerol fatty acid esters (polyoxyethylene glycerol monostearate, etc.);

Polyethylene glycol fatty acid esters (polyethylene glycol distearate, etc.);

Polyoxyethylene alkyl ether (polyoxyethylene lauryl ether, etc.);

Polyoxyethylene polyoxypropylene alkyl ether (polyoxyethylene polyoxypropylene glycol ether, polyoxyethylene polyoxypropylene propyl ether, polyoxyethylene polyoxypropylene hexadecyl ether, etc.);

Polyoxyethylene alkylphenyl ether (polyoxyethylene nonylphenyl ether, etc.);

Polyoxyethylene hydrogenated castor oil (polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, etc.);

Polyoxyethylene beeswax derivatives (polyoxyethylene sorbitol beeswax, etc.);

Polyoxyethylene lanolin derivatives (polyoxyethylene lanolin, etc.);

Polyoxyethylene fatty acid amides (polyoxyethylene stearic acid amide, etc.) with HLB 6 to 18;

Anionic surfactants, such as:

Alkyl sulfates having an alkyl group having 10 to 18 carbon atoms (sodium hexadecyl sulfate, sodium lauryl sulfate, sodium oleyl sulfate, etc.);

Polyoxyethylene alkyl ether sulfates (such as polyoxyethylene sodium lauryl sulfate) with an average ethylene oxide addition mole number of 2 to 4 and an alkyl carbon atom number of 10 to 18;

Alkyl sulfosuccinate salts having an alkyl group carbon number of 8 to 18 (such as sodium dodecyl sulfosuccinate);

Natural surfactants, such as:

Lecithin, glycerophospholipids;

Sphingomyelins (sphingomyelin, etc.);

Sucrose fatty acid esters of fatty acids having 12 to 18 carbon atoms, etc.

The preparation of the present invention may contain one of these surfactants or a combination of two or more of these surfactants.

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

The amount of surfactant added to the antibody preparation of the present invention is generally 0.0001 to 10% (w/v), preferably 0.001 to 5%, and more preferably 0.005 to 3%.

As another aspect of the present invention, the preparation of the present invention is preferably actually composed of the following ingredients:

A) Anti-IL-6 receptor antibody

B) Arginine and/or methionine, and other amino acids (e.g., tryptophan) may be added as optional additional components.

C) buffer, and

D) surfactant.

The so-called "actual composition" means that it does not contain any ingredients other than the optional additives described later (such as suspending agents, solubilizers, isotonic agents, preservatives, adsorption inhibitors, diluents, excipients, pH adjusters, analgesics, sulfur-containing reducing agents, antioxidants, etc. that are added to conventional preparations).

The phrase "arginine and/or methionine, and other amino acids (e.g., tryptophan) may be added as optional additional components" in (B) above means that the types of amino acids that may be added as additives to the formulation include: (b-1) arginine; (b-2) arginine and methionine; and (b-3) methionine. Other amino acids may also be included. The other amino acids are preferably tryptophan, for example. Tryptophan may be used as a monomer, a derivative thereof, or a salt thereof. L-tryptophan or a salt thereof is particularly preferred.

The preparation of the present invention may be supplemented with suspending agents, solubilizing agents, isotonic agents, preservatives, adsorption inhibitors, diluents, excipients, pH regulators, analgesics, sulfur-containing reducing agents, antioxidants, etc. as needed.

Examples of the suspending agent include methylcellulose, polysorbate 80, hydroxyethylcellulose, gum arabic, tragacanth powder, sodium carboxymethylcellulose, and polyoxyethylene sorbitan monolaurate.

Examples of the solubilizing agent include polyoxyethylene hydrogenated castor oil, polysorbate 80, niacinamide, polyoxyethylene sorbitan monolaurate, polyethylene glycol, and castor oil fatty acid ethyl ester.

Examples of isotonic agents include sodium chloride, potassium chloride, and calcium chloride.

Examples of the preservative include methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, sorbic acid, phenol, cresol, and chlorocresol.

Examples of the adsorption inhibitor include human serum albumin, lecithin, dextran, a copolymer of ethylene oxide and propylene oxide, hydroxypropyl cellulose, methyl cellulose, polyoxyethylene hydrogenated castor oil, and polyethylene glycol.

Examples of the sulfur-containing reducing agent include N-acetylcysteine, N-acetylhomocysteine, lipoic acid, thiodiethanol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and its salts, sodium thiosulfate, glutathione, and substances having a mercapto group such as thioalkanoic acids having 1 to 7 carbon atoms.

Examples of the antioxidant include erythorbic acid, butylated hydroxytoluene, butylated hydroxyanisole, α-tocopherol, tocopheryl acetate, L-ascorbic acid and its salts, L-ascorbyl palmitate, L-ascorbyl stearate, sodium bisulfite, sodium sulfite, tripentyl gallate, propyl gallate, or chelating agents such as sodium ethylenediaminetetraacetate (EDTA), sodium pyrophosphate, and sodium metaphosphate.

The antibody-containing solution formulations of the present invention are typically administered non-orally, for example, by injection (subcutaneous, intravenous, intramuscular, etc.), transdermal, transmucosal, nasal, or transpulmonary administration, but oral administration is also possible. Subcutaneous injections allow for a large dosage of antibody per dose (approximately 100-200 mg), but this also limits the amount of injection solution required. Therefore, the formulations of the present invention are particularly suitable for subcutaneous injection.

The osmotic pressure ratio of the antibody-containing solution preparation 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 solution preparation of the present invention is preferably about 2 to 15 mPa·s, more preferably about 4 to 10 mPa·s, but the viscosity of the present invention is measured by a rotational viscometer method using a cone-plate viscometer (15th revised Japanese Pharmacopoeia General Test Methods 2.53 Viscosity Measurement Method).

In the present invention, the results of the Examples described below indicate that the addition of arginine alone, arginine and methionine, or methionine alone can provide stable solution preparations with low levels of antibody dimer formation and deamidation even during long-term storage.

As another embodiment of the present invention, a method for inhibiting deamidation of a solution formulation containing an antibody is provided, comprising: adding arginine or a salt thereof to the solution.

Furthermore, as another embodiment, a method for inhibiting the formation of antibody dimers in a solution formulation containing an antibody is provided, comprising: adding arginine and methionine to the solution.

In the above two methods, the antibody is preferably an anti-interleukin-6 receptor antibody that is a humanized antibody or a human antibody.

The present invention will be described in more detail below by way of examples. However, these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention.

Example

Antibody samples

The humanized anti-IL-6 receptor antibody was prepared by 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 Application Laid-Open No. 8-99902. In the tables of the examples, it may be referred to as MRA.

Example 1:

Stabilizing effect due to the combination of arginine and methionine

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

In this study, to evaluate the effects of the combination of arginine and methionine, evaluation samples No. A1 to A9 were prepared. The formulations of each evaluation sample are as follows.

【Table 1-1】

【formula】

To evaluate the stability of the solution formulation, each sample was subjected to a heat acceleration test (stored at 40°C for 3 months and 25°C for 6 months). The purity of the antibody before and after heat acceleration was evaluated using gel filtration chromatography (SEC). The analytical conditions are as follows.

Gel filtration chromatography

The sample was directly used as the assay solution.

1 μL of the test solution was taken and measured by liquid chromatography under the following conditions. The peak areas of dimer, monomer, and low molecular weight degradation product (LMW) were measured by automatic analysis, and their amounts (%) were determined.

【Table 1-2】

Analysis conditions

Chromatography column: TSKgel G3000SW x 1 7.8mm ID x 30cm (TOSOH)

Mobile phase: pH 7.0 phosphate buffer (50 mmol/L phosphate buffer, pH 7.0, containing 300 mmol/L sodium chloride and 0.05% sodium azide)

Sample injection volume: about 180 μg of anti-IL-6 receptor humanized antibody

Flow rate: 1mL/min

Detection wavelength: 280nm

【Number 1】

Calculation formula

Total peak area = monomer peak area + dimer peak area + low molecular weight degradation product (LMW) peak area

Dimer amount (%) = (dimer peak area/total area of each peak) × 100

Low molecular weight degradation product (LMW) (%) = (peak area of low molecular weight degradation product/total area of each peak) × 100

A typical chromatogram is shown in Figure 1.

The gel filtration chromatography (SEC) evaluation results obtained in this example are shown in Table 1 and Figures 2 and 3. As can be seen, the samples supplemented with arginine (Sample Nos. A2 to A6) exhibited lower dimer levels during accelerated tests at 40°C for 3 months and 25°C for 6 months than the sample without arginine (Sample No. A1), demonstrating that arginine inhibits dimer formation. Furthermore, the reduction in dimer levels was proportional to the amount of arginine added. Meanwhile, the samples supplemented with methionine (Sample Nos. A7 to A9) exhibited lower dimer levels during accelerated tests at 40°C for 3 months and 25°C for 6 months than the samples supplemented with 150 mM arginine (Sample Nos. A3 and A4), which had similar concentrations of the full stabilizer, and comparable dimer levels to the sample supplemented with 300 mM arginine (Sample No. A6). These results suggest that the synergistic effect of the combination of arginine and methionine contributes to the inhibition of dimer formation.

Furthermore, no effect of arginine and methionine on the amount of low-molecular-weight degradation products was detected.

【Table 1-3】

Table 1

Example 2:

Inhibitory effect of deamidation caused by arginine

The effect of arginine on the deamidation inhibition of a solution formulation containing an anti-IL-6 receptor humanized antibody was evaluated.

In this study, we prepared evaluation samples Nos. A10 to A15 and A16 to A18, each containing varying amounts of arginine and methionine. The formulations for each evaluation sample are as follows.

【Table 2-1】

【formula】

To evaluate the stability of the solution formulation, each sample was subjected to a heat acceleration test (stored at 40°C for 3 months and 25°C for 6 months). The purity of the antibody before and after heat acceleration was evaluated using ion exchange chromatography (IEC). The analytical conditions are as follows.

【Ion exchange chromatography】

Purified water was added to the sample to prepare a solution containing approximately 1 mg of the anti-IL-6 receptor humanized antibody in 1 mL as the measurement solution for each sample.

30 μL of the assay solution was taken and subjected to liquid chromatography under the following conditions. The peak areas were measured by automated analysis, and the amounts (%) of pre-MRA, main MRA, sub-1 MRA, sub-2 MRA, R-1 MRA, 1Q(H)-MRA, 2Q(H)-MRA, and other substances (Others) were determined by the area percentage method.

The pre-MRA peak is the sum of peaks eluting at a shorter retention time than the main component and contains various degradation products, primarily the deamidated form of the anti-IL-6 receptor humanized antibody. Low amounts of this pre-MRA peak indicate that deamidation of the antibody is suppressed.

【Table 2-2】

Analysis conditions

Chromatography column: ProPac WCX-10 4×250mm (DIONEX)

Mobile phase: Liquid A: 25mmol/L MES buffer at pH 6.1

Solution B: 25 mmol/L MES buffer (containing 250 mmol/L sodium chloride) at pH 6.1

Sample injection volume: about 30 μg of anti-IL-6 receptor humanized antibody

Flow rate: 0.5mL/min

Detection wavelength: 280nm

【Number 2】

Calculation formula

Total peak area = total peak area before MRA + MRA main peak area + MRA sub-1 peak area + MRA sub-2 peak area + MRA sub-3 peak area + MRA R-1 peak area + total peak area of 1Q(H)-MRA + total peak area of 2Q(H)-MRA + sum of peak areas of other substances (Others)

Amount before MRA (%) = (total peak area before MRA/total area of each peak) × 100

A typical chromatogram is shown in Figure 4. Pre-MRA refers to the sum of all peaks that appear before the main MRA.

The results of ion exchange chromatography (IEC) evaluation obtained in this example are shown in Table 2 and Figures 5 and 6. As can be seen, the amount of pre-peak in samples supplemented with arginine (Sample Nos. A11 to A15) was lower than that in the sample without arginine (Sample No. A10) during the accelerated test at 40°C for 3 months and 25°C for 6 months, indicating that arginine inhibits pre-peak formation. Furthermore, the reduction in pre-peak amount was proportional to the amount of arginine added. On the other hand, the amount of pre-peak in samples supplemented with methionine (Sample Nos. A16 to A18) was equivalent to that in the sample without arginine (Sample No. A10) after the accelerated test at 40°C for 3 months and 25°C for 6 months, indicating that no effect of methionine addition was detected.

【Table 2-3】

Table 2

Example 3:

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 solution formulation containing an anti-IL-6 receptor humanized antibody was evaluated.

In this study, to evaluate the effects of the combination of arginine and methionine, evaluation samples No. A19 to A27 were prepared. The formulations of each evaluation sample are as follows.

【Table 3-1】

【formula】

To evaluate the stability of the solution formulation, each sample was subjected to a light acceleration test (total illumination 1.2 million lux and total near-UV irradiation energy 200 W·h/m ² ). As in Examples 1 and 2, the purity of the antibody before and after light acceleration was evaluated using gel filtration chromatography (SEC) and ion exchange chromatography (IEC).

The gel filtration chromatography (SEC) evaluation results obtained in this example are shown in Table 3 and Figure 7 . As can be seen, the samples containing arginine (Sample Nos. A20 to A24) produced less dimer in the light-accelerated test than the sample without arginine (Sample No. A19), indicating that arginine inhibits dimer formation. Furthermore, the decrease in dimer formation was proportional to the amount of arginine added. Meanwhile, the samples containing methionine added to 100 mM arginine (Sample Nos. A25 to A27) produced less dimer in the light-accelerated test than the sample containing 150 mM arginine (Sample No. A22), which had a similar concentration of the full stabilizer, and also less than the samples containing 200 mM and 300 mM arginine (Sample Nos. A23 and A24). These results suggest that the synergistic effect of the combination of arginine and methionine can inhibit dimer formation.

No effect of arginine and methionine on the amount of low molecular weight degradation products was detected.

【Table 3-2】

Table 3

Next, the evaluation results of ion exchange chromatography (IEC) are shown in Table 4 and FIG8 .

As can be seen, the amount of pre-peak in the light-accelerated test for samples containing arginine (Sample Nos. A20-A24) was lower than that in the sample without arginine (Sample No. A19), indicating that arginine inhibits pre-peak formation. Furthermore, the reduction in pre-peak amount was proportional to the amount of arginine added. Meanwhile, the amount of dimer formation in samples containing methionine added to 100 mM arginine (Sample Nos. A25-A27) was lower than that in the sample containing 150 mM arginine (Sample No. A22), which had a similar total stabilizer concentration, and also lower than that in samples containing 200 mM and 300 mM arginine (Sample Nos. A23 and A24). These results suggest that the synergistic effect of the combination of arginine and methionine can suppress pre-peak formation.

【Table 4】

This manual also includes the following:

A stable antibody-containing solution formulation characterized by containing arginine and methionine.

2. The solution preparation of embodiment 1, further comprising a histidine buffer.

3. The solution preparation according to embodiment 1 or 2, further comprising a surfactant.

4. The solution preparation according to any one of embodiments 1 to 3, wherein the antibody concentration is 50 mg/mL or higher.

5. The solution preparation according to any one of embodiments 1 to 3, wherein the antibody concentration is 100 mg/mL or higher.

6. The solution preparation according to any one of embodiments 1 to 3, wherein the antibody concentration is 120 mg/mL or higher.

7. The solution preparation according to any one of embodiments 1 to 6, wherein the antibody is an anti-interleukin-6 receptor antibody.

8. A stable solution preparation containing an anti-interleukin-6 receptor antibody, characterized by containing arginine or methionine.

9. The solution formulation according to any one of embodiments 1 to 8, wherein the antibody is a humanized antibody or a human antibody.

10. The solution preparation according to any one of embodiments 1 to 9, further comprising tryptophan.

11. The solution preparation according to any one of embodiments 1 to 10, wherein the pH of the solution preparation is 4 to 8.

12. The solution preparation according to any one of embodiments 1 to 11, wherein the content of arginine is 50 to 1500 mM.

13. The solution preparation according to any one of embodiments 1 to 12, wherein the viscosity is 2 to 15 mPa·s.

14. The solution formulation of any one of embodiments 1 to 13, wherein the solution formulation is stable at 22-28°C for at least 6 months.

15. The solution preparation according to any one of embodiments 1 to 13, wherein the formation of antibody dimers is suppressed.

16. The solution preparation according to any one of embodiments 1 to 13, wherein deamidation of the antibody molecule is suppressed.

17. The solution preparation according to any one of embodiments 1 to 13, which is administered subcutaneously.

18. The solution preparation according to any one of embodiments 1 to 13, wherein the solution preparation is prepared without a freeze-drying step.

19. A method for inhibiting deamidation of an antibody molecule in an antibody-containing solution formulation, comprising: adding arginine to the solution.

20. A method for inhibiting antibody dimer formation in an antibody-containing solution formulation, comprising: adding arginine and methionine to the solution.

Claims (6)

1. A method for inhibiting deamidation of a humanized anti-IL-6 receptor antibody MRA in a liquid preparation, comprising: adding arginine to the liquid preparation, wherein the formulation comprises at least 150 mg/ml of humanized anti-IL-6 receptor antibody MRA, wherein arginine is added to the preparation in an amount of 100 to 300 mM, and The preparation was not freeze-dried. 2. The method of claim 1, wherein the formulation Include: 10-20 mM histidine buffer, and 0.005-3w/v% polysorbate 80, and It has a pH of 6.0 to 6.5. 3. The method of claim 1, wherein arginine is added to the preparation in an amount of 150 to 300 mM. 4. The method of claim 3, wherein arginine is added to the preparation in an amount of 200 to 300 mM. 5. The method of claim 1, wherein the formulation comprises 180 mg/ml of humanized anti-IL-6 receptor antibody MRA. 6. The method of claim 1, wherein the formulation is for subcutaneous administration.