Antibody-Including Preparation

Abstract

The present invention relates to stable antibody-containing solution formulations in which the formation of agglomeration of emicizumab (ACE910), a dual antibody, which is functionally substituted with FVIII, is prevented. Specifically, the present invention relates to a solution formulation containing the above-mentioned antibody at a pH of 4.5 to 6.5, comprising the aforementioned dual antibody at 20 to 180 mg / mL, 10 mM to 40 mM histidine-aspartate buffer, ploxamer 188 at 0.2 to 1 mg / mL, and 100 mM to 300 mM arginine.

Description

Description Preparation containing antibody Technical background The present invention relates to formulations comprising a bivalent antibody that is functionally substituted with its own coagulation factor VIII (FVIII) that binds to blood coagulation factor IX (FIX) and / or activated blood coagulation factor IX (FIXa) and blood coagulation factor X (FX). Prior knowledge Functionally substituted FVIII dual antibodies that bind to coagulation factor IX (FIX) and / or coagulation factor IX (FIXa) and coagulation factor X (FX) have been discovered (Non-inventive Documents 1 and 2; Inventive Documents 1 to 3). The dual antibody emicizumab (ACE910) improves the reduction in coagulation response due to FVIII deficiency and poor function by functionally replacing FVIII; therefore, clinical trials have been conducted on hemophilia A patients. Most of the antibody aqueous formulations have been developed, and the high-concentration antibody aqueous formulations reported so far are formulations using histidine and arginine (Patent Document 4) and formulations using histidine / aspartate buffer (Patent Document 5). In addition, stable liquid pharmaceutical antibody formulations containing amyloid β (Aβ) using histidine / histidine-HCl as buffer (Patent Document 6) have been reported. However, stable solution formulations in which the formation of aggregates and / or components is prevented by charge heterogeneity have not been reported for formulations containing the previously mentioned dual antibodies. Reference list Patent document [Invention Document 1]: WO2005 / 035756 [Invention Document]2: WO2006 / 109592 [Patent Document [3: WO2012 / 067176] [Patent Document [4: WO2002 / 030463] [Patent Document [5: WO2011 / 090088] [Patent Document [6: WO2013 / 131866] [Non-inventory document] [Non-patented document [1: Nat Med. 2012; 18(10):1570-74 [Non-inventory document [2: PLoS One. 2013; 8(2):e57479 [Summary of the invention] [Problem to be solved] The present invention aims to provide stable solution formulations comprising emicizumab (ACE910), which is a dual antibody functionally substituted with FVIII that binds to FIX and / or FIXa and FX. [Tools for solving problems] As a result of the research conducted to achieve the above-mentioned object, the present inventors have discovered that a pH 4.5 to 6.5 solution formulation comprising the aforementioned dual antibody at 20 to 180 mg / mL, 10 mM to 40 mM histidine / aspartate buffer, ploxamer 188 at 0.2 to 1 mg / mL, and 100 mM to 300 mM arginine, can be a stable antibody-containing solution formulation in which the formation of a mass and / or components with charge heterogeneity is prevented, thereby completing the present invention. Specifically, the present invention provides the following: [1] Antibody solution formulation pH 4.5 to 6.5, which includes: A bivalent antibody at 20 to 180 mg / mL, wherein a primary polypeptide and a third polypeptide form a pair, and a secondary polypeptide and a fourth polypeptide form a pair, wherein the primary polypeptide comprises an H chain comprising the amino acid sequences of H-chain CDRs 1, 2, and 3 of SEQ ID NOs: 1, 2, and 3 (H-chain CDRs Q499), respectively; the secondary polypeptide comprises an H chain comprising the amino acid sequences of H-chain CDRs 1, 2, and 3 of SEQ ID NOs: 4, 5, and 6 (H-chain CDRs J327), respectively; and the third polypeptide and the fourth polypeptide comprise a common L chain comprising the amino acid sequences of L-chain CDRs 1, 2, and 3 of SEQ ID NOS: 7, 8, and 9 (L-chain CDRs L404), respectively; 10 mM to 40 mM histidine / aspartate buffer; 0.2 to 1 mg / mL of ploxamer 188; and 100 mM to 300 mM arginine. [2] A solution formulation of the antibody [1], wherein in the dual antibody, the primary polypeptide and the tertiary polypeptide form a pair, and the secondary polypeptide and the fourth polypeptide form a pair, wherein the primary polypeptide comprises an H chain comprising the amino acid sequence of SEQ ID NO: 10; the secondary polypeptide comprises an H chain comprising the amino acid sequence of SEQ ID NO: 11, and the primary polypeptide and the fourth polypeptide comprise a common L chain of SEQ ID NO: 12. [3] A solution formulation of the antibody [1] or [2], wherein the concentration of ploxamer 188 is 0.5 mg / mL. [4] The antibody solution formulation of any one of [1] to [3], wherein said pH is 6.0. [5] The antibody solution formulation of any of [1] to [4], wherein the concentration of the histidine / aspartate buffer is 20 mM. [6] The antibody solution formulation of any of [1] to [5], wherein the arginine concentration is 150 mM. [7] The antibody solution formulation of any one of [1] to [6], wherein it does not substantially contain chloride ion or acetate ion. [8] Antibody solution formulation pH 6, which includes: A dual antibody at 20 to 180 mg / mL, wherein the primary polypeptide and the third polypeptide form a pair, and a secondary polypeptide and a fourth polypeptide form a pair, wherein the primary antibody comprises an H chain comprising the amino acid sequence of SEQ ID NO: 10; the secondary polypeptide comprises an H chain comprising the amino acid sequence of SEQ ID NO: 11; and the third polypeptide and the fourth polypeptide comprise a common L chain of SEQ ID NO: 12; 20 mM L-histidine / aspartate buffer; 0.5 mg / mL of ploxamer 188; and 150 mM of L-arginine. [9] A solution formulation of the antibody of any of [1] to [8], for use in subcutaneous administration.

[10] A solution formulation of the antibody of any of [1] to [9], for use in the treatment of hemophilia A.

[11] A method for stabilizing an antibody in a solution formulation containing the antibody, comprising adding histidine / aspartate buffer, ploxamer 188, and arginine to the solution, whereby the histidine / aspartate buffer concentration is 10 mM to 40 mM, the ploxamer 188 concentration is 0.2 to 1 mg / mL, and the arginine concentration is 100 mM to 300 mM.

[12] A method for preventing aggregation (cluster formation) of antibody in a solution formulation containing antibody, comprising adding histidine / aspartate buffer, ploxamer 188, and arginine to the solution, whereby the concentration of histidine / aspartate buffer is 10 mM to 40 mM, the concentration of ploxamer 188 is 0.2 to 1 mg / mL, and the concentration of arginine is 100 mM to 300 mM.

[13] A method for preventing a component with charge heterogeneity in an antibody-containing formulation, which comprises adding a histidine / aspartate buffer to the solution, wherein the concentration of the histidine / aspartate buffer is 10 mM to 40 mM. [Invention Impact] The present invention provides antibody-containing formulations that exhibit excellent stability. Furthermore, the provision of antibody-containing formulations in which the formation of aggregates and / or components with charge heterogeneity is prevented in the solution state itself is also made possible by the present invention. [Summary explanation of maps] Figure 1 shows photographs showing the insoluble foreign materials present after the shaking tests of Example 8 (a: 0 mg / mL of ploxamer 188; b: 0.5 mg / mL of ploxamer 188). Figure 2 shows images showing the number of insoluble microparticles (microparticles / mL) present after the shaking and water and freeze tests of Example 8. [Methods for implementing the invention] The present invention will be explained in detail below. The present invention provides a pH 4.5 to 7.5 solution formulation comprising: emicizumab (ACE910) at 20 to 180 mg / mL, which is a dual antibody functionally substituted for FVIII that binds to FIX and / or FIXa and FX; 10 mM to 40 mM histidine / aspartate buffer; ploxamer 188 at 0.2 to 1 mg / mL; and 100 mM to 300 mM arginine. Emicizumab (ACE910), which is the previously mentioned dual antibody, is described below. A dual antibody (Q499-z121 / J327-z119 / L404-k) in which the primary polypeptide and the third polypeptide form a pair, and the secondary polypeptide and the fourth polypeptide form a pair; wherein the primary polypeptide comprises the H chain comprising the amino acid sequences of the H-chain CDRs 1, 2, and 3 of SEQ ID NOs: 1, 2, and 3, respectively (H-chain CDRs Q499); the secondary polypeptide comprises the H chain comprising the amino acid sequences of the H-chain CDRs 1, 2, and 3 of SEQ ID NOs: 4, 5, and 6, respectively (H-chain CDRs J327); and the third polypeptide and the fourth polypeptide comprising the L chain comprise the amino acid sequences of L-chain CDRs 1, 2, and 3 of SEQ ID NOS: 7, 8, and 9 (L-chain CDRs L404), respectively. More specifically, the aforementioned dual antibody is a dual antibody in which the primary polypeptide and the third polypeptide form a pair; and the secondary polypeptide and the fourth polypeptide form a pair; wherein the primary polypeptide comprises an H chain comprising the amino acid sequence of the H-chain variable region of SEQ ID NO: 13, the secondary polypeptide comprises an H chain comprising the amino acid sequence of the H-chain variable region of SEQ ID NO: 14, and the primary polypeptide and the fourth polypeptide comprise a common L chain comprising the amino acid sequence of the L-chain variable region of SEQ ID NO: 15. More specifically, the aforementioned dual antibody is a dual antibody (Q499-z121 / J327-z119 / L404-k) in which the primary polypeptide and the serum polypeptide form a pair, and the secondary polypeptide and the fourth polypeptide form a pair; wherein the primary polypeptide comprises the H chain comprising the amino acid sequence of SEQ ID NO: 10, the secondary polypeptide comprises the H chain comprising the amino acid sequence of SEQ ID NO: 11, and the third polypeptide and the fourth polypeptide comprise the L chain comprising the sequence of SEQ ID NO: 12. Such antibodies can be obtained by the methods described in WO2005 / 035756, WO2006 / 109592, WO2012 / 067176 and the like. The concentration of the antibody in the formulation of the present invention is not particularly limited, but is preferably 20 mg / mL to 180 mg / mL. Examples include 20 mg / mL, 30 mg / mL, 40 mg / mL, 120 mg / mL, 150 mg / mL, and 180 mg / mL. The upper limit of the concentration of the antibody in the formulation of the present invention is not particularly limited, but is typically 250 mg / mL. The antibodies used in the present invention are not particularly limited as long as they bind to a desired antigen, and they may be polyclonal or monoclonal antibodies. Monoclonal antibodies are preferred in this case as homogeneous antibodies can be stably produced. Amino acids in the amino acid sequences of the present invention may be post-translationally modified (for example, modification of an N-terminal glutamine to a pyroglutamic acid by pyroglutamylation is known to one skilled in the art). Naturally, such post-translationally modified amino acids are included in the antibodies used in the present invention. In the present invention, the term "functionally replacing FVII" means identifying FIX or FIXa, and FX, and promoting FX activity by FIXa (promoting FXa product by FIXa). The promoting activity of FXa product can be assessed using, for example, a measurement system comprising FXIa, FX, the synthetic substrate S-2222 (synthetic substrate for FXa), and phospholipid. Such a measurement system shows a correlation with the severity of the disease and clinical symptoms in cases of hemophilia A (Rosen S, Anderson M, Blumba KM et al. Clinical applications of the chromogenic substrate method for the determination of FVIII activity, Thromb Haemost 1985; 54: 811-23). In the present invention, the term "common L chain" refers to an L chain that can form pairs with two or more different H chains, and shows the ability to bind to any antigen. Here, the term "different H chains" preferably refers to H chains of antibodies against different antigens, but is not limited thereto; it refers to H chains whose amino acid sequences are different from each other. Common L chains can be obtained, for example, according to the method described in WO 2006 / 109592. In the present invention, the term "stable antibody-containing formulation" refers to a formulation in which aggregates and / or charge-inhomogeneous components of proteins such as antibodies are difficult to form, i.e., formulations in which detrimental reactions, including the formation of insoluble aggregates, soluble aggregates, charge-inhomogeneous components, are difficult to form in solution. "Charge-inhomogeneous components" refer to components with protein surface charges that differ from those of the parent component due to deamidation, oxidation, hydrolysis, and the like. In the present invention, "polypeptide" generally refers to polypeptides and proteins having a length of about ten amino acids or more. Typically, they are biologically derived polypeptides, but are not specifically limited thereto, and may be, for example, polypeptides having an artificially designed sequence. In addition, they may be any naturally occurring polypeptide, synthetic polypeptides, recombinant polypeptides, and the like. In addition, fragments of the above-mentioned polypeptides are also included in the polypeptides of the present invention. The term "antibody" is used generically, and includes single-stranded antibodies, multi-stranded antibodies, dimers, multimers, multiple antibodies (e.g., bivalent antibodies), antibody derivatives, and modified antibodies (Miller K et al. J Immunol. 2003, 170(9), 4854-61) to the extent that they exhibit a desired biological activity. Antibodies may be murine antibodies, human antibodies, humanized antibodies, mock antibodies, and those derived from other species, or artificially synthesized antibodies. The antibodies described herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), or class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) or subclass of immunoglobulin molecules. Immunoglobulins can be derived from any species (for example, human, mouse, or rabbit). The terms "antibody", "immunoglobulin", and "immunoglobulin" are used interchangeably in general. A "dual antibody" refers to an antibody with two variable regions, each recognizing different epitopes, where the variable regions are present on the same antibody molecule. Dual antibodies may be antibodies that recognize two or more different antigens, or antibodies that recognize two or more different epitopes on the same antigen. Dual antibodies may include not only whole antibodies but also antibody derivatives. Recombinant antibodies produced by using genetic engineering techniques can be used as antibodies. Recombinant antibodies can be obtained by cloning a DNA encoding the antibody from hybridomas or antibody-producing cells such as sensitized lymphocytes that produce antibodies; inserting this into a vector, and then introducing it into hosts (host cells) to produce the antibody. Dual antibodies are not limited to those of the IgG type; for example, dual IgG-type antibodies can be secreted from a hybrid hybridoma (quadroma) produced by fusing two types of hybridomas that produce IgG antibodies (Milstein C et al. Nature 1983, 305: 537-540). They can also be secreted by introducing L-chain and H-chain genes into cells that replace the two desired IgG types, i.e., a total of four types of genes, for co-expression of the genes. The antibodies of the present invention can be produced by methods known to those skilled in the art. Typically, DNA encoding the desired antibody is inserted into an expression vector. Insertion into the expression vector is carried out in such a way that expression is under the control of expression regulatory regions, such as enhancers or promoters. Host cells are then transformed using this expression vector to express the antibody. Suitable combinations of host and expression vector can be used for this purpose. The antibodies of the present invention obtained can be isolated from inside the host cells or outside the cells (medium, etc.), and purified to obtain a substantially pure and homogeneous antibody. These antibodies can be isolated and purified by methods commonly used for the isolation and purification of antibodies, and the methods are not limited in any way. For example, these antibodies can be isolated and purified by appropriate selection and combination of column chromatography, filtration, ultrafiltration, salting out or precipitation, solvent precipitation, distillation, immunoprecipitation, polyacrylamide-SDS gene electrophoresis, isoelectrofocusing, dialysis, recrystallization, and the like. In a preferred aspect, the histidine / aspartate buffer in the formulation of the present invention is a buffer prepared by titrating a solution, such as an aqueous solution supplemented with histidine as a free amino acid, with a liquid, such as an aqueous solution containing aspartic acid as a free amino acid. Alternatively, the buffer can be prepared by adding the amino acids in reverse order, or by direct titration with powders. The present inventors conducted freeze-thaw tests, thermal acceleration tests, long-term storage tests, and cryopreservation tests to evaluate the effects of various additives on the stability of the samples containing the above-mentioned dual antibodies during their storage. As a result, the present inventors have found that the formation of aggregates and / or components with charge heterogeneity is prevented by using a histidine buffer, compared to phosphate buffer, citrate buffer, and acetate buffer. Furthermore, the present inventors have found that the formation of aggregates and / or components with heterogeneous charge is prevented by using aspartic acid, which is an acidic amino acid, as a counterion species for the buffer, i.e., by using a histidine / aspartate buffer as the buffer. The concentration (amount) of the histidine / aspartate buffer in the formulations of the present invention is preferably 10 to 100 mM, and most preferably 10 to 40 mM. In addition, examples of the concentration (amount) of the histidine / aspartate buffer are 10 mM, 20 mM, and 40 mM. Furthermore, compared to sodium chloride, which has been reported to be a stabilizer for antibody-containing formulations, the addition of arginine has been found to exhibit superior stabilizing effects (i.e., effects of preventing clump formation and effects of preventing components with charge heterogeneity). The concentration (amount) of arginine in the formulations of the present invention is preferably 100 mM and 300 mM. Examples of arginine concentrations (amount) include 100 mM, 150 mM, 200 mM and 300 mM. The pH of the solution of the formulation of the present invention is preferably 4.5 to 6.5, more preferably 5.5 to 6.5, and even more preferably 5.5 to 6. Examples of pH include 5.5 and 6. Surfactants present in the formulations of the present invention include, for example, polysorbate 20 (PS20), and Pluronic F-68 (Ploxamer 188; polyethylene (160) polyoxypropylene (30) glycol), with Ploxamer 188 being particularly preferred. The amount of Ploxamer 188 (or PX188) added to the formulation of the present invention is preferably 0.2 mg / mL to 1 mg / mL. Examples of the amount of Ploxamer 188 added to the formulation include 0.2 mg / mL, 0.5 mg / mL, 0.8 mg / mL, and 1 mg / mL. The histidine used in the present invention may be histidine itself or a derivative thereof, and L-histidine is preferably preferred. The arginine used in the present invention may be arginine itself, a derivative thereof, or a salt thereof. And L-arginine or a salt thereof is preferably preferred. Preferred salts of arginine include aspartate salt and glutamate salt. The formulations of the present invention may additionally comprise amino acids. Preferred amino acids for use in the present invention are natural amino acids or amino acid derivatives, and preferred amino acids are particularly L-methionine and L-proline. The formulations of the present invention may additionally comprise sugars. Preferred sugars used in the present invention are sucrose, trehalose, meglumine, and sorbitol. The amount of amino acid or sugar added to the formulations of the present invention is typically 1 mM to 1000 mM, preferably 5 mM to 500 mM, and most preferably 10 mM to 300 mM. The formulations of the present invention may additionally comprise inorganic salts. Preferred inorganic salts used in the present invention are magnesium salts and calcium salts. Furthermore, it is preferred that the formulation of the present invention does not contain anions other than aspartic acid as a counterion for the buffer (buffering agent) or stabilizer. In one aspect, examples of such formulations include formulations that do not substantially contain chloride ion or acetate ion. "Does not substantially contain chloride ion or acetate ion" means that the concentrations of chloride ion and acetate ion are, for example, 5 mM or less, preferably 2 mM or less, and most preferably 1 mM or less. Highly stable antibody-containing formulations can be produced without increasing osmotic pressure by using aspartic acid, which has a high stabilizing effect as a counterion, and does not substantially contain chloride ion or acetate ion with a stabilizing effect. If necessary, the formulations of the present invention may additionally include suitable cryoprotectants, suspending agents, solubilizing agents, isotonizing agents, preservatives, absorption inhibitors, diluents, excipients, pH adjusters, analgesics, sulfur-containing reducing agents, antioxidants, and the like. Antifreezes include, for example, sugars such as trehalose, sucrose, and sorbitol. Stabilizing agents, for example, include hardened polyoxyethylene castor oil, polysorbate 80, nicotinamide, polyoxyethylene sorbitan monolaurate, macrogol, and castor oil fatty acid ethyl ester. Isotonic agents include, for example, sodium chloride, potassium chloride, and calcium chloride. Preservatives, for example, include methyl-p-hydroxybenzoate, ethyl-p-hydroxybenzoate, sorbic acid, phenol, cresol, and chlorocresol. Absorption inhibitors include, for example, human serum albumin, lecithin, dextran, ethyleneoxy / propylene oxide copolymer, hydroxypropyl glucose, methyl glucose, polyoxyethylene hardened castor oil, and polyethylene glycol. Sulfur-containing reducing agents include, for example, those containing sulfhydryl groups such as N-acetylcysteine, N-acetylhomocysteine, thioctic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbital, thioglycolic acid and its salts, sodium thiosulfate, glutathione, and thioalkanoic acids having one or more carbon atoms. Antioxidants, for example, include erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, α-tocopherol, tocopherol acetate, L-ascorbic acid and its salts, L-ascorbic acid palmitate, L-ascorbic acid stearate, sodium hydrogen sulfite, sodium sulfite, triamyl gallate, propyl gallate, and chelating agents such as disodium ethylenediaminetetraacetate (EDTA), sodium pyrophosphate, and sodium metaphosphate. In one example, the formulation of the present invention is as follows: Antibody solution formulation pH 6, which includes: A dual antibody at 20 to 180 mg / mL, wherein the primary polypeptide and the third polypeptide form a pair, and a secondary polypeptide and the fourth polypeptide form a pair, wherein the primary polypeptide comprises an H chain comprising the amino acid sequence of SEQ ID NO: 10, the secondary polypeptide comprises an H chain comprising the amino acid sequence of SEQ ID NO: 11, and the third polypeptide and the fourth polypeptide comprise a common L chain of SEQ ID NO: 12; 20 mM L-histidine / aspartate buffer; 0.5 mg / mL of ploxamer 188; and 150 mM L-arginine; Or Antibody solution formulation pH 6, which includes: Emicizumab dual antibody (ACE910) at 20 to 180 mg / mL, 20 mM L-histidine / aspartate buffer; 0.5 mg / mL of ploxamer 188; and 150 mM L-arginine. In another example, the formulation of the present invention is as follows: Antibody solution formulation pH 6, which includes: A dual antibody at 20 to 180 mg / mL, wherein the primary antibody and the tertiary antibody form a pair, and the secondary polypeptide and the fourth polypeptide form a pair, wherein the primary polypeptide comprises an H chain comprising the amino acid sequence of SEQ ID NO: 10, the secondary polypeptide comprises an H chain comprising the amino acid sequence of SEQ ID NO: 11, and the tertiary polypeptide and the fourth polypeptide comprise a common L chain of SEQ ID NO: 12; 20 mM L-histidine / aspartate buffer; 0.05 mg / mL of PS20; and 150 mM L-arginine; Or Antibody solution formulation pH 6, which includes: Emicizumab dual antibody (ACE910) at 20 to 180 mg / mL, 20 mM L-histidine / aspartate buffer; 0.05 mg / mL of PS20; and 150 mM L-arginine. The antibody-containing formulations of the present invention can be administered to a patient by any suitable route, for example, by bolus injection or continuous infusion over a period of time, intravenously, intramuscularly, or subcutaneously. Intravenous administration or subcutaneous administration is preferred. The dose of emicizumab (ACE910), for example, is 0.001 to 1000 mg / kg, and the administration interval is at least one day or more. More specifically, for example, after administering emicizumab (ACE910) at an initial dose of 1 mg / kg, emicizumab (ACE910) can be administered at a continuous dose of 0.3 mg / kg once weekly. Alternatively, for example, after administering emicizumab (ACE910) at an initial dose of 3 mg / kg, emicizumab (ACE910) can be administered at a continuous dose of 1 mg / kg once weekly. In another example, after administering emicizumab (ACE910) at an initial dose of 3 mg / kg, emicizumab (ACE910) can be administered at a continuous dose of 3 mg / kg once weekly. The antibody-containing formulations of the present invention can be used for diseases that are caused and / or progress due to a reduction or deficiency in the activity of FVIII and / or coagulation factor VIII (FVIIIa). For example, they can be used for hemophilia A, hemophilia A in which inhibitors to FVIII / FVIIIa have appeared, acquired hemophilia A, von Willebrand's disease, without being limited thereto. Another embodiment of the present invention is a method for stabilizing an antibody in a solution formulation containing the antibody. Preferably, the method for stabilizing an antibody in a solution formulation containing the antibody comprises adding a histidine / aspartate buffer, ploxamer 188, and arginine to the solution. Another embodiment of the present invention is a method for reducing the aggregation (cluster formation) of antibodies in a solution formulation containing antibodies. Preferably, the method for reducing the aggregation (cluster formation) of antibodies in a solution formulation containing antibodies comprises adding a histidine / aspartate buffer, ploxamer 188, and arginine to the solution. In addition, the above-mentioned method for stabilizing the antibody and a method for reducing aggregation (cluster formation) of the antibody comprises adding histidine / aspartate buffer, ploxamer 188, and arginine to the solution, and preferably the antibody concentration is 20 to 180 mg / mL, the histidine / aspartate buffer concentration is 10 mM to 40 mM, the ploxamer 188 concentration is 0.2 to 1 mg / mL, the arginine concentration is 100 mM to 300 mM, and the pH is 4.5 to 6.5; or most preferably the antibody concentration is 20 to 180 mg / mL, the histidine / aspartate buffer concentration is 20 mM, the ploxamer 188 concentration is 0.5 mg / mL, the arginine concentration is 150 mM, and the pH is 6. Another example of the present invention is a method for reducing a component with charge heterogeneity in an antibody-containing formulation. Preferably, the method for reducing a component with charge heterogeneity in an antibody-containing formulation comprises adding a histidine / aspartate buffer to the solution. Most preferably, the method for reducing a component with charge heterogeneity in an antibody-containing formulation comprises adding a histidine / aspartate buffer to the solution, wherein the concentration of the histidine / aspartate buffer is 10 mM to 40 mM, or at 20 mM. In another embodiment of the present invention, a method for reducing the charge heterogeneity component in an antibody-containing formulation comprises adding a histidine / aspartate buffer, ploxamer 188, and arginine to the solution. Most preferably, a method for reducing a component with charge heterogeneity in an antibody-containing formulation comprises adding a histidine / aspartate buffer, ploxamer 188, and arginine to the solution, and preferably the antibody concentration is 20 to 180 mg / mL, the histidine / aspartate buffer concentration is 10 mM to 40 mM, the ploxamer 188 concentration is 0.2 to 1 mg / mL, the arginine concentration is 100 mM to 300 mM, and the pH is 4.5 to 6.5, or most preferably the antibody concentration is 20 to 180 mg / mL, the histidine / aspartate buffer concentration is 20 mM, the ploxamer 188 concentration is 0.5 mg / mL, the arginine concentration is 150 mM, and the pH is 6. In the above-mentioned method for stabilizing an antibody, a method for reducing aggregation (cluster formation) of the antibody, and a method for reducing a component with charge heterogeneity, the antibody is preferably a bivalent antibody and most preferably emicizumab (ACE910). As used herein, aspects referred to by the phrase "comprising" include those referred to by the phrase "essentially comprising", and those referred to by the phrase "consisting of". The numerical values ​​referred to herein may vary within certain limits, for example, depending on the means or equipment, the measurement conditions, and the process used by the person skilled in the art, and to the extent that they are within the limits that achieve the purpose of the invention, they may include a deviation of approximately, for example, 10%. All inventions and references expressly cited herein are hereby incorporated by reference into this specification in their entirety. The present invention will be further illustrated by the following examples, but should not be construed as being limited thereto. [Examples] [Example 1] Effects of preventing histidine aggregation during thermally accelerated storage of humanized IgG4 antibody ACE910 (1)Materials ACE910 is a dual humanized IgG4 antibody that recognizes both coagulation factor IX and coagulation factor X, which is expected to prevent bleeding in hemophilia A by functionally replacing activated coagulation factor VIII. (2)Test samples Liquid formulations of pH 6.0 containing ACE910 at 100 mg / mL, NaCl at 150 mmol / L, and each of the following buffers were prepared: phosphate buffer at 20 mmol / L; citrate buffer at 20 mmol / L; acetate buffer at 20 mmol / L; and histidine buffer at 20 mmol / L. Glass vials were filled with 5 to 15 L of the compounds, respectively. The thus prepared humanized antibody-containing solution formulations were left in a temperature-regulated bath at 25°C for eight weeks, and then used as test samples. (3)Methods for measuring and calculating the amount of ACE910 accumulations The concentrations in the samples were measured by size exclusion chromatography (SEC) using a G3000SWXL (Tosoh) column with phosphate buffer (50 mmol / L, pH7.0) containing 300 mmol / L sodium chloride for the mobile phase at a flow rate of 0.5 mL / min. Of the identified peaks, the peak with the largest area and height was determined to be the monomer, and the peaks identified before the monomer were collectively referred to as the aggregation peaks (high molecular weight species, HMWS). Peak areas were calculated for all peaks, and the peak area ratio of the desired peak was calculated using the following equation: Peak level ratio of desired peak (%) = 100x(peak level of desired peak) divided by (peak level of desired peak + total peak level of other peaks) (4)Results The results obtained are shown in Table 1. [Table [1] As is clear from Table 1, when supplemented with histidine at 20 mmol / L, the sample showed an aggregation-inhibitory effect after thermal acceleration at 25°C for eight weeks. [Example 2] Inhibition-aggregation effects of salt and arginine concentration during thermally accelerated storage and freeze-thaw of humanized IgG4 antibody ACE910 (1)Materials The antibody described in Example 1 was used. (2)Test samples Liquid formulations of pH 6.0 containing ACE910 at 100 mg / mL, histidine at 20 mmol / L, each of the following additives: NaCl at 50 mmol / L; NaCl at 75 mmol / L; NaCl at 150 mmol / L; and arginine at 150 mmol / L were prepared. Glass vials were filled with 5 to 15 L of the formulations, respectively. The humanized antibody-containing solution formulations prepared in this manner were left in a temperature-regulated bath at 25°C for eight weeks, or were subjected to freeze-thaw (F / T) cycles (5°C / -20°C), and then tested as samples. (3)Methods for measuring and calculating the amount of ACE910 accumulations The methods described in Example 1 were implemented. (4) Results The results obtained are shown in Table 2. [Table 2] As is clear from Table 2, when supplemented with arginine at 150 mmol / L, the samples showed a high aggregation-inhibitory effect after thermal acceleration testing at 25°C for eight weeks and freezing and thawing. [Example 3] Inhibitory effects of aspartic acid during freezing and thawing of humanized IgG4 antibody ACE910. (1)Materials The antibody described in Example 1 was used. (2)Test samples Liquid formulations of pH 6.0 containing ACE910 at 100 mg / mL, histidine at 20 mmol / L, and NaCl at 150 mmol / L or sodium L-aspartic acid at 150 mmol / L as counterion were prepared. Glass vials were filled with 5 to 15 L of the formulations, respectively. The thus prepared humanized antibody-containing solution formulations were subjected to ten freeze-thaw cycles (5°C / -20°C), and then used as test samples. (3)Methods for measuring and calculating the amount of ACE910 accumulation The methods described in Example 1 were implemented. (4)Results The results are shown in Table 3. [Table 3] As is clear from Table 3, when supplemented with aspartic acid, the samples show a high resuscitation-retention effect after freezing and thawing. [Example 4] Effects of preventing aggregation and charge-inhomogeneous components by pH during thermally accelerated storage of humanized IgG4 antibody ACE910 (1)Materials The antibody described in Example 1 was used. (2)Test samples Liquid formulations containing ACE910 at 100 mg / mL, histidine-aspartic acid at 20 mmol / L, and arginine-aspartic acid at 150 mmol / L, and having pH values ​​of 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, or 7.5 were prepared. Glass vials were filled with 5 to 15 L of the formulations, respectively. The thus prepared humanized antibody-containing solution formulations were left in a temperature-regulated bath at 25°C for eight weeks, and then used as test samples. (3)Methods for measuring and calculating the amount of ACE910 aggregates The methods described in Example 1 were implemented. (4)Measurement and calculation methods of ACE910 components with charge heterogeneity The amount of components with charge heterogeneity in the sample was measured by ion exchange chromatography (IEC) through a BioPro QA-F column (YMC) using Tris-HCl buffer (20 mmol / L, pH 7.8) as mobile phase A and Tris-HCl buffer (20 mmol / L, pH 7.8) containing sodium chloride (500 mmol / L) as mobile phase B, at a flow rate of 0.5 mL / min. Of the identified peaks, the peak with the largest area and height was determined as the main peak, and the peaks identified after the main peak were collectively referred to as acidic peaks. The peak area was calculated for all peaks, and the peak area ratio of the desired peak was calculated using the following equation: Peak level ratio of desired peak (%): 100x(peak level of desired peak) divided by (peak level of desired peak + total peak level of other peaks) (5)Results The results are shown in Table 4. [Table 4] As is clear from Table 4, samples at pH 4.5 to 6.5, and especially at pH 5.5 and pH 6.0, showed a high influence of aggregation and inhibitory components with charge heterogeneity after storage at 25°C. [Example 5] Effects of preventing aggregation and charge-inhomogeneous components by histidine concentration during thermally accelerated storage of humanized IgG4 antibody ACE910 (1)Cases The antibody described in Example 1 was used. (2)Test samples Liquid pH 6.0 formulations containing ACE910 at 100 mg / mL, arginine at 150 mmol / L, and histidine-aspartic acid at 5 mmol / L, 10 mmol / L, 20 mmol / L, or 40 mmol / L were prepared. Glass vials were filled with 5 to 15 L of the formulations, respectively. The thus prepared humanized antibody-containing solution formulations were left in a temperature-regulated bath at 25°C for eight weeks, and then used as test samples. (3) Methods for determining and calculating the amount of ACE910 accumulations The methods described in Example 1 were implemented. (4)Measurement and calculation methods of ACE910 components with charge heterogeneity The methods described in Example 4 were implemented. (5)Results The results are shown in Table 5. [Table 5] As is clear from Table 5, samples containing 10 mmol / L or more of histidine-aspartic acid showed an effect of preventing aggregation and components with charge heterogeneity after storage at 25°C. [Example 6] Inhibition-accumulation effects of arginine concentration during freezing and thawing, thermally accelerated storage, and cryopreservation of the humanized IgG4 antibody ACE910 (1)Materials The antibody described in Example 1 was used. (2)Test samples Liquid pH 6.0 formulations containing ACE910 at 100 mg / mL, histidine-aspartic acid at 20 mmol / L, and arginine at 75 mmol / L, 100 mmol / L, 150 mmol / L, 200 mmol / L, or 300 mmol / L were prepared. Glass vials were filled with 5 to 15 L of the formulations, respectively. The thus prepared humanized antibody-containing solution formulations were subjected to ten freeze-thaw cycles (5°C / -20°C), or placed in a temperature-regulated bath at 25°C for eight weeks or at -20°C for six months, and then used as test samples. (3)Methods for measuring and calculating the amount of ACE910 accumulations The methods described in Example 1 were implemented. (4)Results The results are shown in Table 6. [Table 6] As is clear from Table 6, samples containing arginine at 100 mmol / L or more showed an inhibitory effect on aggregation after freezing and thawing, after storage at 25°C, and after storage at 20°C. [Example 7] Effects of Insoluble Foreign Substances and Insoluble Microparticles Inhibition by Ploxamer 188 During Storage at 5°C of Humanized IgG4 Antibody ACE910 (1)Materials The antibodies described in 1 were used. (2)Test samples Liquid formulations at pH 6.0 containing ACE910 at 80 mg / mL, histidine-aspartic acid at 20 mmol / L, arginine at 150 mmol / L, and each of the following additives were prepared: ploxamer 188 at 0 mg / mL; ploxamer 188 at 0.2 mg / mL; ploxamer 188 at 0.5 mg / mL; ploxamer 188 at 1.0 mg / mL; polysorbate 20 at 0.05 mg / mL; and polysorbate 20 at 1.0 mg / mL. Glass vials were filled with 1.0 mL of the formulations, respectively. The thus prepared humanized antibody-containing solution formulations were left in a refrigerator at 5°C for five months, and then used as test samples. (3) Methods for examining insoluble foreign substances The presence of insoluble foreign matter was assessed by placing the sample on a sample platform (a visual test stand for vials), rotating the sample, and observing the vial. (4)Method for measuring insoluble microparticles The number of insoluble microparticles in the solution was determined using a microparticle counter (Hach Ultra Analytics, Model 9703). (5)Results The results are shown in Table 7. [Table 7] As is clear from Table 7, samples containing PS20 at 0.05 mg / mL and samples containing ploxamer 188 at 0.2 mg / mL or more showed a high inhibitory effect on the formation of insoluble microparticles and insoluble foreign materials after storage at 5°C. [Example 8] Effects of Insoluble Foreign Substances and Insoluble Microparticles Inhibition by Ploxamer 188 During a Vibrational Stress and Ice-Water Storage of Humanized IgG4 Antibody ACE910 (1)Cases The antibody described in Example 1 was used. (2)Test samples Liquid formulations of pH 6.0 containing ACE910 at 150 mg / mL, histidine-aspartic acid at 20 mmol / L, arginine-aspartic acid at 150 mmol / L, and each of the following additives were prepared: ploxamer 188 at 0 mg / mL; ploxamer 188 at 0.2 mg / mL; ploxamer 188 at 0.5 mg / mL; and ploxamer 188 at 0.8 mg / mL. Glass vials were filled with 0.9 mL of the formulations, respectively. The thus prepared humanized antibody-containing solution formulations were subjected to vibration for 24 hours at a speed of 200 strokes / min using a shaker at room temperature, or ten freeze-thaw cycles (5°C / -20°C), and then used as samples. (3) Method for examining insoluble substances The method was performed as described in Example 7. (4)Method for measuring insoluble microparticles The method was performed as described in Example 7. (5)Results The results obtained are shown in Table 8 and Figures 1 and 2. [Table 8] As is clear from Table 8 and Figures 1 and 2, samples containing ploxamer 188 at 0.2 mg / mL or more showed a high inhibitory effect on the formation of insoluble microparticles and insoluble foreign materials after exposure to vibrational stress and ice-water storage. [Example 9] Effects of ACE910 humanized IgG4 antibody concentration on stability during thermally accelerated storage and ice-water storage (1)Materials The antibody was used as described in Example 1. (2)Test samples Liquid formulations of pH 6.0 containing histidine-aspartic acid at 20 mmol / L, arginine-aspartic acid at 150 mmol / L, ploxamer 188 at 0.5 mg / mL, and ACE910 at 20 mg / mL, 30 mg / mL, 40 mg / mL, 120 mg / mL, 150 mg / mL, or 180 mg / mL were prepared. Glass vials were filled with 0.65 mL of the formulations, respectively. The thus prepared humanized antibody-containing solution formulations were kept in a temperature-regulated bath at 40°C for eight weeks, and subjected to five or ten freeze-thaw cycles (25°C / -20°C), and then used as test samples. (3)Methods for measuring and calculating the amount of ACE910 accumulations The methods described in Example 1 were implemented. (4)Measurement and calculation methods of ACE910 components with charge heterogeneity The amount of charge-inhomogeneous components in the sample was measured by anion exchange chromatography (AIEC) through a TSKgel Q-STAT column (Waters) using Tris-HCl buffer (50 mmol / L, pH 8.0) as mobile phase A and Tris-HCl buffer (50 mmol / L, pH 8.0) containing sodium chloride (200 mmol / L) as mobile phase B at a flow rate of 0.5 mL / min. Of the identified peaks, the peak with the largest area and height was determined as the main peak, and the peaks identified before the main peak were collectively referred to as basic peaks, and the peaks identified after the main peak were collectively referred to as acidic peaks. In addition, the amount of components with charge heterogeneity was measured by cation ion chromatography (CIEC) through a ProPac WCX-10G column (Thermo Scientific) using a buffer containing Tris at 9.6 mmol / L, piperazine at 6.0 mmol / L, and imidazole at 11.0 mmol / L (pH 6.0) as mobile phase A and a buffer containing Tris at 9.6 mmol / L, piperazine at 6.0 mmol / L, imidazole at 11.0 mmol / L, and NaCl at 100 mmol / L (pH 10.1) as mobile phase B at a flow rate of 0.5 mL / min. Of the identified peaks, the peak with the largest area and height was determined to be the BiAb peak. The peaks identified before the BiAb peak were collectively referred to as pre-peaks, and the peaks identified after the BiAb peak were collectively referred to as post-peaks. The calculated peak area for all peaks, and the peak area ratio of the desired peak were calculated using the following equation: Peak level ratio of desired peak (%) = 100x(peak level of desired peak) divided by (peak level of desired peak + total peak level of other peaks) (5)Results The results are shown in Table 9. “SE”, “AE”, and “CE” indicate the results from size exclusion chromatography, anion exchange chromatography, and cation exchange chromatography, respectively. [Table 9] As can be seen from Table 9, by comparing the samples containing ACE910 at 20 mg / mL to 180 mg / mL, it was shown that the samples have an equal and sufficient stability after storage at 40°C and after freeze-thaw. [Industrial applicability] Compared with conventional formulations, the antibody soluble formulations of the present invention have excellent stability in solution, and exhibit inhibited aggregation of proteins such as antibody molecules after storage at low, ambient, and high temperatures, and after freeze-thaw. The antibody soluble formulations of the present invention in which degradation reactions are difficult to occur can be used, for example, for the treatment of hemophilia A by subcutaneous administration.

Claims

CLAIMS 1. An antibody solution formulation of pH 4.5 to 6.5, which comprises: a bispecific antibody at 20 to 180 mg / mL, wherein a first polypeptide and a third polypeptide form a pair, and a second polypeptide and a fourth polypeptide form a pair, wherein the first polypeptide comprises an H chain comprising the amino acid sequences of H - chain CDR s 1, 2, and 3 of SEQ ID NOs: 1, 2, and 3 (H - chain CDRs of Q499), respectively; the second polypeptide comprises an H chain comprising the amino acid sequences of H - chain CDR s 1, 2, and 3 of SEQ ID NOs: 4, 5, and 6 (H - chain CDRs of J327), respectively; and the third polypeptide and the fourth polypeptide comprise a common L chain comprising the amino acid sequences of L - chain CDR s 1, 2, and 3 of SEQ ID NOs: 7, 8, and 9 (L - chain CDRs of L404), respectively; 10 mM to 40 mM histidine / aspartate buffer; 0.2 to 1 mg / mL Poloxamer 188; and 100 mM to 300 mM arginine.

2. The antibody solution formulation of claim 1, wherein in the bispecific antibody , the first polypeptide and the third polypeptide form a pair, and the second polypeptide and the fourth polypeptide form a pair , wherein the first polypeptide comprises an H chain comprising the amino acid sequence of SEQ ID NO: 10 ; the second polypeptide comprises an H chain comprising the amino acid sequence of SEQ ID NO: 11, and the third polypeptide and the fourth polypeptide comprise a common L chain of SEQ ID NO: 12.

3. The antibody solution formulation of claim 1 or 2, wherein the concentration of Poloxamer 188 is 0.5 mg / mL.

4. The antibody solution formulation of any one of claims 1 to 3, wherein said pH is 6.

5. The antibody solution formulation of any one of claims 1 to 4, wherein the concentration of histidine / aspartate buffer is 20 mM.

6. The antibody solution formulation of any one of claims 1 to 5, wherein the concentration of arginine is 150 mM.

7. The antibody solution formulation of any one of claims 1 to 6, which does not substantially contain a chloride ion or an acetate ion.

8. An antibody solution formulation of pH 6, which comprises: a bispecific antibody at 20 to 180 mg / mL, wherein a first polypeptide and a third polypeptide form a pair, and a second polypeptide and a fourth polypeptide form a pair , wherein the first polypeptide comprises an H chain comprising the amino acid sequence of SEQ ID NO: 10 ; the second polypeptide comprises an H chain comprising the amino acid sequence of SEQ ID NO: 11 ; and the third polypeptide and the fourth polypeptide comprise a common L chain of SEQ ID NO: 12; 2 0 mM L- histidine / aspartate buffer; 0. 5 mg / mL Poloxamer 188; and 1 50 mM L-arginine.

9. The antibody solution formulation of any one of claims 1 to 8, for use in subcutaneous administration.

10. The antibody solution formulation of any one of claims 1 to 9, for use in the treatment of hemophilia A.

11. A method for stabilizing an antibody in an antibody-containing solution formulation, which comprises adding a histidine / aspartate buffer, Poloxamer 188, and arginine to the solution, whereby the concentration of the histidine / aspartate buffer is 10 mM to 40 mM, the concentration of Poloxamer 188 is 0.2 to 1 mg / mL, and the concentration of arginine is 100 mM to 300 mM.

12. A method for suppressing the association (aggregate formation) of an antibod y in an antibody-containing solution formulation, which comprises adding a histidine / aspartate buffer, Poloxamer 188, and arginine to the solution, whereby the concentration of the histidine / aspartate buffer is 10 mM to 40 mM, the concentration of Poloxamer 188 is 0.2 to 1 mg / mL, and the concentration of arginine is 100 mM to 300 mM.

13. A method for suppressing a component with charge heterogeneity in an antibody-containing formulation, which comprises adding a histidine / aspartate buffer to the solution, wherein the concentration of histidine / aspartate buffer is 10 mM to 40 mM.

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