Antibody-containing preparations

Abstract

This invention relates to a solution formulation of a stable antibody containing Emicizumab (ACE910), a dual-specific antibody that replaces the function of FVIII, capable of inhibiting aggregate formation. Specifically, it relates to the above-mentioned antibody-containing solution formulation containing 20–180 mg / mL of the aforementioned dual-specific antibody, 10 mM–40 mM histidine-aspartate buffer, 0.2–1 mg / mL Poloxamer 188, and 100 mM–300 mM arginine, with a pH of 4.5–6.5.

Description

Technical Field

[0001] This invention relates to a formulation containing a dual-specific antibody that binds to both factor IX (FIX) and / or activates factor IX (FIXa) and factor X (FX) to replace the function of factor VIII (FVIII). Prior Technology

[0002] A dual-specific antibody system that binds to both factor IX (FIX) and / or activated factor IX (FIXa) and factor X (FX) to replace the function of factor VIII (FVIII) has been disclosed (Non-Patent Literature 1, 2 and Patent Literature 1, 2, 3). Clinical trials were conducted on patients with hemophilia A using the dual-specific antibody Emicizumab (ACE910) to replace the function of FVIII in order to improve insufficient coagulation response caused by FVIII deficiency and dysfunction. To date, numerous solution formulations of antibodies have been continuously developed, including high-concentration solution formulations. Formulations using histidine or arginine have been reported (Patent Document 4), and formulations using histidine-aspartate buffer have been reported (Patent Document 5). On the other hand, antibody formulations containing sedative liquid pharmaceuticals using histidine / histidine-HCl as a buffer for amyloid protein β (Aβ) have been reported (Patent Document 6). However, there are no reports of stable solution formulations containing the aforementioned dual-specific antibodies that exhibit aggregate formation and / or inhibition of charge-isotropic components. [Previous Technical Documents] [Patent Literature]

[0003] Patent Document 1: WO2005 / 035756 Patent 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-patent literature]

[0004] Non-patent literature 1: Nat Med. 2012; 18(10): 1570-74 Non-patent literature 2: PLoS One. 2013; 8(2): e57479 Summary of the Invention

[0005] [The problem the invention aims to solve]

[0006] The purpose of this invention is to provide a stable solution formulation containing Emicizumab (ACE910), a dual-specific antibody that binds to both FIX and / or FIXa and FX and can replace the function of FVIII. [Technical means to solve the problem]

[0007] As a result of extensive research to achieve the above objectives, the inventors discovered that a solution containing 20-180 mg / mL of the aforementioned dual-specific antibody, 10 mM-40 mM histidine-aspartate buffer, 0.2-1 mg / mL Poloxamer 188, 100 mM-300 mM arginine, and a pH of 4.5-6.5 is a solution containing a stable antibody that can inhibit aggregate formation, thus completing the present invention.

[0008] In other words, the present invention provides the following content. [1] An antibody solution formulation, comprising A dual-specific antibody with a concentration of 20-180 mg / mL, consisting of a first and third polypeptide forming a pair, and a second and fourth polypeptide forming a pair. The antibody is formed by the first polypeptide being an H-chain containing the amino acid sequences 1, 2, and 3 (H-chain CDR of Q499), the second polypeptide being an H-chain containing the amino acid sequences 1, 2, and 3 (H-chain CDR of J327), and the third and fourth polypeptides being a common L-chain containing the amino acid sequences 1, 2, and 3 (L-chain CDR of L404). 10 mM~40 mM histidine-aspartate buffer, 0.2~1 mg / mL of Poloxamer 188, and Arginine at 100 mM to 300 mM, And its pH value is 4.5~6.5. [2] The antibody solution preparation described in [1] is a dual-specific antibody formed by pairing the first polypeptide with the third polypeptide, and pairing the second polypeptide with the fourth polypeptide. The dual-specific antibody is formed by the first polypeptide being an H chain formed by the amino acid sequence of sequence identification number 10, the second polypeptide being an H chain formed by the amino acid sequence of sequence identification number 11, and the third polypeptide and the fourth polypeptide being a common L chain of sequence identification number 12. [3] The antibody solution preparation of [1] or [2], wherein the concentration of Poloxamer 188 is 0.5 mg / mL. [4] The antibody solution preparation of any of [1] to [3], wherein the pH value is 6.0. [5] The antibody solution preparation of any of [1] to [4] wherein the concentration of the histidine-aspartate buffer solution is 20 mM. [6] The antibody solution preparation of any of [1] to [5] wherein the concentration of arginine is 150 mM. [7] The antibody solution preparations of any of the items [1] to [6] do not actually contain chloride ions and acetate ions. [8] An antibody solution formulation, comprising: A dual-specific antibody with a concentration of 20-180 mg / mL is formed by pairing the first and third polypeptides, and the second and fourth polypeptides. The antibody is composed of an H-chain formed by the amino acid sequence of sequence identification number 10 for the first polypeptide, an H-chain formed by the amino acid sequence of sequence identification number 11 for the second polypeptide, and a common L-chain formed by the third and fourth polypeptides of sequence identification number 12. 20 mM L-histidine-aspartate buffer, 0.5 mg / mL of Poloxamer 188, and 150 mM L-arginine, And its pH value is 6. [9] The antibody solution preparations of any of [1] to [8] are intended for subcutaneous administration.

[10] An antibody solution preparation such as any of [1] to [9] is used to treat hemophilia A.

[11] A method for stabilizing antibodies in an antibody-containing solution preparation, comprising adding histidine-aspartate buffer, Poloxamer 188 and arginine to the solution, wherein the concentration of 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 inhibiting antibody aggregation (aggregation) in an antibody-containing solution preparation, comprising adding histidine-aspartate buffer, Poloxamer 188 and arginine to the solution, wherein the concentration of 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 reducing the charge-opposite components in an antibody-containing solution preparation includes adding histidine-aspartate buffer to the solution, wherein the concentration of histidine-aspartate buffer is 10 mM to 40 mM. [Invention Effects]

[0009] This invention provides antibody-containing formulations with good stability. Furthermore, this invention provides antibody-containing formulations that inhibit aggregate formation and / or charge-opposites in solution. Simple Explanation of the Diagram

[0010] Figure 1 shows a photograph of the insoluble impurities after the shaking test in Example 8 (a: 0 mg / mL Poloxamer 188, b: 0.5 mg / mL Poloxamer 188). Figure 2 shows the number of insoluble microparticles (microparticles / mL) after the shaking test and freeze-thaw cycle in Example 8. Implementation

[0011] The present invention will now be described in detail.

[0012] This invention provides a solution containing 20-180 mg / mL of Emicizumab (ACE910), a dual-specific antibody that binds to both FIX and / or FIXa and FX to replace the function of FVIII, 10 mM-40 mM histidine-aspartate buffer, 0.2-1 mg / mL Poloxamer 188, and 100 mM-300 mM arginine, with a pH of 4.5-6.5.

[0013] Emicizumab (ACE910), the dual specific antibody mentioned above, is described below.

[0014] The dual-specific antibody, which forms a pair between the first and third polypeptides and a pair between the second and fourth polypeptides, is formed by the first polypeptide being an H chain containing the amino acid sequences 1, 2, and 3 of the H chain CDR of Q499 (the H chain CDR of Q499), the second polypeptide being an H chain containing the amino acid sequences 1, 2, and 3 of the H chain CDR of J327 (the H chain CDR of J327), and the third and fourth polypeptides being L chains containing the amino acid sequences 1, 2, and 3 of the L chain CDR of L404 (the L chain CDR of L404).

[0015] More specifically, the aforementioned dual-specific antibody, which is a pairing of the first and third polypeptides and the second and fourth polypeptides, is a dual-specific antibody formed by the first polypeptide being an H chain containing the amino acid sequence of the H chain variable region of sequence identification number 13, the second polypeptide being an H chain containing the amino acid sequence of the H chain variable region of sequence identification number 14, and the third and fourth polypeptides being a common L chain containing the amino acid sequence of the L chain variable region of sequence identification number 15.

[0016] More specifically, the aforementioned dual-specific antibody is a dual-specific antibody formed by pairing the first and third polypeptides, and the second and fourth polypeptides. It is a dual-specific antibody (Q499-z121 / J327-z119 / L404-k) formed by pairing the first polypeptide with the amino acid sequence of sequence identification number 10, the second polypeptide with the amino acid sequence of sequence identification number 11, and the third and fourth polypeptides with the common L chain of sequence identification number 12. This antibody can be obtained using methods described in WO2005 / 035756, WO2006 / 109592, WO2012 / 067176, etc.

[0017] The concentration of antibodies contained in the formulations of this invention is not particularly limited, but the preferred antibody concentration is 20 mg / mL to 180 mg / mL. For example, the antibody concentrations contained in the formulations of this invention are 20 mg / mL, 30 mg / mL, 40 mg / mL, 120 mg / mL, 150 mg / mL, and 180 mg / mL. The upper limit of the antibody concentration contained in the formulations of this invention is not particularly limited, but is typically 250 mg / mL.

[0018] The antibodies used in this invention can be any antibodies that bind to the desired antigen, and there are no particular limitations. They can be multi-strain antibodies or monoclonal antibodies. From the viewpoint that the same antibody can be stably produced, monoclonal antibodies are preferred.

[0019] Furthermore, the amino acids contained in the amino acid sequence described in this invention may also be subject to post-translational modifications (for example, modifying N-terminal glutamic acid to pyroglutamic acid by pyroglutamylation is a well-known modification in the industry), and even if such amino acids are subject to post-translational modifications, they are still included in the antibodies used in this invention.

[0020] In this invention, "replacing the function of FVIII" means identifying FIX or FIXa, and FX, and promoting the activation of FX via FIXa (promoting the production of FX via FIXa). The FXa-promoting activity can be assessed, for example, by assays formed from FIXa, FX, the synthetic matrix S-2222 (the synthetic matrix of FXa), and phospholipids. These assays show correlation with the severity and clinical symptoms of hemophilia A cases (Rosen S, Andersson M, BLombäck M et al. Clinical applications of a chromogenic substrate method for determination of FVIII activity. Thromb Haemost 1985; 54: 811-23).

[0021] In this invention, the term "common L-chain" refers to an L-chain that pairs with two or more different H-chains and acquires binding energies against different antigens. Here, "different H-chains" preferably refers to antibody H-chains targeting different antigens, but is not limited to this; it can mean H-chains with different amino acid sequences. The common L-chain can, for example, be obtained according to the method described in WO2006 / 109592.

[0022] Furthermore, the "stable antibody-containing preparation" in this invention refers to a preparation that does not easily generate aggregates of proteins such as antibodies and / or components with opposite charges, that is, a preparation that does not easily cause deterioration reactions, primarily the formation of insoluble aggregates, soluble aggregates, components with opposite charges, etc., in solution.

[0023] The component with the opposite charge refers to the component whose surface charge differs from that of the main component through processes such as deacetylation, oxidation, and hydrolysis.

[0024] In this invention, "polypeptide" generally refers to peptides and proteins having a length of 10 or more amino acids. Furthermore, it is generally a biologically derived polypeptide, but there are no particular limitations; for example, it can also be a polypeptide formed by artificially designed sequences. Additionally, it can be any of the following: natural polypeptides, synthetic polypeptides, recombinant polypeptides, etc. Moreover, fragments of the aforementioned polypeptides are also included in the polypeptides of this invention.

[0025] The term "antibody" is used in its broadest sense, referring to any substance that possesses the desired biological activity. It can be a monoclonal antibody, a polyclonal antibody, a dimer, a multimer, a multispecific antibody (e.g., a bispecific antibody), an antibody derivative, or an antibody modification (Miller K et al. J Immunol. 2003, 170(9), 4854-61). Antibodies can be mouse, human, humanized, chimeric, or from other sources, and can also be synthetically produced. The antibodies described in this invention can be any type (e.g., IgG, IgE, IgM, IgD, and IgA), type (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subtype of immunoglobulin. Immunoglobulins can be obtained from any source (e.g., human, mouse, chimeric). Furthermore, the terms "antibody," "immunoglobulin," and "immunoglobulin" are used interchangeably in a broad sense.

[0026] A "dual-specific" antibody system refers to an antibody molecule containing two variable regions that recognize different antigenic determinants. A dual-specific antibody can recognize two or more different antigens, or it can recognize two or more different antigenic determinants on the same antigen. Dual-specific antibodies can be complete antibodies or may include antibody derivatives.

[0027] Recombinant antibodies, which can be used as antibodies, are produced using gene recombination technology. Recombinant antibodies can be obtained by selecting the DNA encoding the antibody from antibody-producing cells such as fusion tumors or sensitive lymphocytes that produce antibodies, incorporating it into a vector, and then introducing it into a host (host cell) for production.

[0028] Bispecific antibodies are not limited to IgG. For example, IgG bispecific antibodies can be secreted by a chimeric fusion tumor (quadroma) formed by fusing two fusion tumors that produce IgG antibodies (Milstein C et al. Nature 1983, 305: 537-540). In addition, a total of four genes, namely the genes for the two IgG L chains and H chains that constitute the target, can be introduced into cells to be expressed and secreted together.

[0029] The antibodies of this invention can be manufactured using methods known to those skilled in the art. Specifically, DNA encoding the target antibody is constructed into an expression vector. At this time, under the control of expression control regions, such as enhancers or promoters, the antibodies are constructed into the expression vector in an expressive manner. Then, host cells are transposed using this expression vector to express the antibody. Suitable combinations of hosts and expression vectors can be used.

[0030] The antibodies obtained thereby are isolated from host cells or extracellularly (culture medium, etc.) to purify substantially high-purity and homogeneous antibodies. The separation and purification of antibodies can be performed using methods commonly used in antibody purification, without particular limitations. For example, antibodies can be separated and purified by appropriately selecting and combining chromatography columns, filter membranes, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-PAGE electrophoresis, isoelectric point electrophoresis, dialysis, recrystallization, etc.

[0031] The preferred form of the histidine-aspartate buffer in the formulation of this invention is a buffer solution containing aspartate in its free amino acid state, added to a solution such as an aqueous solution, and adjusted by liquid titration with an aqueous solution or similar liquid. Alternatively, the order of addition can be reversed for adjustment, or the buffer solution can be directly titrated with powder.

[0032] To evaluate the stability of samples containing the aforementioned dual-specific antibodies during storage, the inventors assessed the effects of various additives through freeze-thaw tests, accelerated heat treatment tests, long-term storage tests, and cryopreservation tests. The results showed that, compared to phosphate buffer, citrate buffer, and acetate buffer, histidine buffer could inhibit aggregate formation and / or charge-averse components.

[0033] Furthermore, by using aspartic acid, an acidic amino acid with a relative ion type, that is, by using histidine-aspartate buffer as a buffer, aggregate formation and / or components with opposite charges can be suppressed.

[0034] The concentration (amount) of histidine-aspartate buffer in the formulation of the present invention is preferably 10-100 mM, more preferably 10-40 mM. Furthermore, the concentration (amount) of histidine-aspartate buffer is, for example, 10 mM, 20 mM, or 40 mM.

[0035] Furthermore, compared with sodium chloride, which has been reported as a stabilizer in antibody-containing preparations, the addition of arginine showed a higher stabilizing effect (inhibition of aggregate formation and inhibition of charge-dissimilar components).

[0036] The concentration (amount) of arginine in the formulation of the present invention is preferably 100 mM to 300 mM. In addition, the concentration (amount) of arginine is, for example, 100 mM, 150 mM, 200 mM, or 300 mM.

[0037] The pH value of the solution of the formulation of the present invention is preferably 4.5-6.5, more preferably 5.5-6.5, and even more preferably 5.5-6. Furthermore, the pH value of the solution is, for example, 5.5 or 6.

[0038] The surfactants contained in the formulations of this invention are, for example, polysorbate 20 (PS20) and Pluronic F-68 (Poloxamer-188: polyethylene oxide (160) polyethylene oxide (30) glycol), with Poloxamer-188 being particularly preferred. For the formulations of this invention, the amount of Poloxamer-188 (Poloxamer 188 or PX188) added is preferably 0.2 mg / mL to 1 mg / mL. Furthermore, the amount of Poloxamer-188 added to the formulations of this invention is, for example, 0.2 mg / mL, 0.5 mg / mL, 0.8 mg / mL, or 1 mg / mL.

[0039] The histidine used in this invention can be any single product or its derivative, with L-histidine being particularly preferred. The arginine used in this invention can be any single product, its derivative, or its salt, with L-arginine or its salt being preferred. Furthermore, the salts of arginine are preferably aspartate or glutamate.

[0040] The formulations of this invention may include other amino acids. Preferably, the amino acids used in this invention are natural amino acids or amino acid derivatives, particularly L-methionine and L-proline.

[0041] The formulation of this invention may contain other sugars. Preferred sugars used in this invention are sucrose, trehalose, meglumine, and sorbitol.

[0042] For the formulation of the present invention, the amount of added amino acids or sugars is generally 1 mM to 1000 mM, preferably 5 mM to 500 mM, and even more preferably 10 mM to 300 mM.

[0043] The formulation of this invention may contain other inorganic salts. The preferred inorganic salts used in this invention are magnesium salts and calcium salts.

[0044] Furthermore, the relative ion of the formulation of the present invention, which serves as a buffer or stabilizer, is preferably free of anions other than aspartic acid. Examples of such formulations include those substantially free of chloride and acetate ions. "Substantially free of chloride and acetate ions" means, for example, that the chloride and acetate ion concentrations are 5 mM or less, more preferably 2 mM or less, and even more preferably 1 mM or less. By substantially eliminating chloride and acetate ions with low stabilizing effects and using aspartic acid relative ions with high stabilizing effects, a highly stable antibody-containing formulation can be manufactured without increasing osmotic pressure.

[0045] In addition, if necessary, appropriate cryoprotectants, suspending agents, solubilizers, isotonics, preservatives, anti-adsorption agents, diluents, excipients, pH adjusters, analgesics, sulfur-containing reducing agents, antioxidants, etc., may be added to the formulation of the present invention.

[0046] Cryoprotectants include, for example, sugars such as trehalose, sucrose, and sorbitol.

[0047] Co-solvents include, for example, polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinic acid amine, polyoxyethylene dehydrated sorbitan monolaurate, polyethylene glycol, ethyl castor oil, etc.

[0048] Isotropic agents include, for example, sodium chloride, potassium chloride, calcium chloride, etc.

[0049] Preservatives include, for example, methylparaben, ethylparaben, sorbic acid, phenol, cresol, chlorocresol, etc.

[0050] Anti-adhesion agents include, for example, human serum albumin, lecithin, glycosaminoglycan, ethylene oxide-propylene oxide copolymer, hydroxypropyl cellulose, methylcellulose, polyoxyethylene hydrogenated castor oil, polyethylene glycol, etc.

[0051] Sulfur-containing reducing agents include, for example, N-acetycysteine, N-acetylosine, lipoic acid, thiodiethylene glycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and its salts, sodium thiosulfate, glutathione, and thioalkyl acids with carbon numbers 1 to 7, etc., which contain thiol groups.

[0052] Antioxidants include, for example, isoascorbic acid, butylated hydroxytoluene, butylated hydroxyanisole, α-tocopherol, tocopheryl acetate, L-ascorbic acid and its salts, L-ascorbate palmitate, L-ascorbate stearate, sodium bisulfite, sodium sulfite, triamterene gallate, propyl gallate or disodium ethylenediaminetetraacetate (EDTA), sodium pyrophosphate, sodium metaphosphate, and other chelating agents.

[0053] The first embodiment of the formulation of the present invention is described below. An antibody solution formulation, comprising A dual-specific antibody with a concentration of 20-180 mg / mL, consisting of a first and third polypeptide pairing, and a second and fourth polypeptide pairing. The first polypeptide is an H-chain formed by the amino acid sequence at sequence identification number 10; the second polypeptide is an H-chain formed by the amino acid sequence at sequence identification number 11; and the third and fourth polypeptides are a common L-chain formed by sequence identification number 12. 20 mM histidine-aspartate buffer, 0.5 mg / mL of PoLoxamer 188, and 150 mM L-arginine, Its pH value is 6. or An antibody solution formulation, comprising: Emicizumab (ACE910), a dual-specific antibody, is available in concentrations of 20-180 mg / mL. 20 mM L-histidine-aspartate buffer, 0.5 mg / mL of Poloxamer 188 150 mM L-arginine, Its pH value is 6. Another embodiment of the formulation of the present invention is as follows. An antibody solution formulation, comprising: A dual-specific antibody with a concentration of 20-180 mg / mL, consisting of a pairing of the first and third polypeptides, and a pairing of the second and fourth polypeptides, is formed by the first polypeptide being an H-chain formed by the amino acid sequence of sequence identification number 10, the second polypeptide being an H-chain formed by the amino acid sequence of sequence identification number 11, and the third and fourth polypeptides being a common L-chain formed by the sequence identification number 12. 20 mM L-histidine-aspartate buffer 0.05 mg / mL of PS20, 150 mM L-arginine, Its pH value is 6. or An antibody solution formulation, comprising: Emicizumab (ACE910), a dual-specific antibody, is available in concentrations of 20-180 mg / mL. 20 mM L-histidine-aspartate buffer, 0.05 mg / mL PS20, 150 mM L-arginine, Its pH value is 6.

[0054] The present invention relates to the administration of antibody preparations, which can be delivered to patients via any suitable route. For example, via pills or over a period of time via intravenous, intramuscular, or subcutaneous injection. Intravenous or subcutaneous administration is preferred.

[0055] The dosage of Emicizumab (ACE910) is, for example, 0.001~1000 mg / kg, with an interval of at least 1 day. More specifically, for example, after an initial dose of 1 mg / kg Emicizumab (ACE910), subsequent doses of 0.3 mg / kg Emicizumab (ACE910) can be administered weekly. Alternatively, for example, after an initial dose of 3 mg / kg Emicizumab (ACE910), subsequent doses of 1 mg / kg Emicizumab (ACE910) can be administered weekly. Alternatively, for example, after an initial dose of 3 mg / kg Emicizumab (ACE910), subsequent doses of 3 mg / kg Emicizumab (ACE910) can be administered weekly.

[0056] The antibody preparations of this invention can be used for patients with disease onset and / or disease progression due to low or absent activity of FVIII and / or activated coagulation factor VIII (FVIIIa), such as hemophilia A, hemophilia A with FVIII / FVIIIa inhibitors, acquired hemophilia A, von Willebrand disease, etc., but are not limited to these patients.

[0057] Another embodiment of the present invention is a method for stabilizing antibodies in an antibody-containing solution formulation. Preferably, the method for stabilizing antibodies in an antibody-containing solution formulation includes the above-described method of adding histidine-aspartate buffer, Poloxamer 188, and arginine to the solution.

[0058] Furthermore, another embodiment of the present invention is a method for inhibiting antibody aggregation (aggregate formation) in antibody-containing solution formulations. Preferably, the method for inhibiting antibody aggregation (aggregate formation) in antibody-containing solution formulations includes the above-described method of adding histidine-aspartate buffer, Poloxamer 188, and arginine to the solution.

[0059] In addition, the above-mentioned method for inhibiting antibody aggregation (aggregation) in antibody-containing preparations includes adding histidine-aspartate buffer, Poloxamer 188, and arginine to the solution. The preferred antibody concentration is 20-180 mg / mL, the histidine-aspartate buffer concentration is 10 mM-40 mM, the Poloxamer 188 concentration is 0.2-1 mg / mL, the arginine concentration is 100 mM-300 mM, and the pH value is 4.5-6.5. More preferably, the antibody concentration is 20-180 mg / mL, the histidine-aspartate buffer concentration is 20 mM, the Poloxamer 188 concentration is 0.5 mg / mL, the arginine concentration is 150 mM, and the pH value is 6.

[0060] Another embodiment of the present invention is a method for reducing the charge-opposite component in an antibody-containing preparation. Preferably, the method for reducing the charge-opposite component in an antibody-containing preparation includes the above-described method of adding histidine-aspartate buffer to a solution. More preferably, the method for reducing the charge-opposite component in an antibody-containing preparation includes adding histidine-aspartate buffer to a solution, and includes setting the concentration of the histidine-aspartate buffer to 10 mM~40 mM, or 20 mM, as described above. Furthermore, another embodiment of the present invention is a method for reducing the charge-opposite components in antibody-containing preparations, comprising adding histidine-aspartate buffer, Poloxamer 188 and arginine to a solution. More preferably, a method for reducing the charge-biased components in antibody preparations includes adding histidine-aspartate buffer, Poloxamer 188, and arginine to a solution, wherein the antibody concentration is 20-180 mg / mL, the histidine-aspartate buffer concentration is 10 mM-40 mM, the Poloxamer 188 concentration is 0.2-1 mg / mL, the arginine concentration is 100 mM-300 mM, and the pH value is 4.5-6.5; more preferably, the antibody concentration is 20-180 mg / mL, the histidine-aspartate buffer concentration is 20 mM, the Poloxamer 188 concentration is 0.5 mg / mL, the arginine concentration is 150 mM, and the pH value is 6.

[0061] Among the above-mentioned methods for stabilizing antibodies, inhibiting antibody aggregation (forming aggregates), and reducing the charge-isotropic components, the preferred antibody is a bispecific antibody, and more preferably Emicizumab (ACE910).

[0062] In the description of this invention, the state expressed by "comprising" includes the state expressed by "essentially consisting of" and the state expressed by "consisting of".

[0063] The numerical values ​​described in this invention, such as the machine and measurement conditions, can be adjusted within a certain range according to the operator's technology. To achieve the purpose of this invention, the values ​​can be adjusted within a range, such as to a degree of 10%.

[0064] All patents and references cited in this invention description can be obtained by referring to this invention description. The present invention is illustrated by the following embodiments, but is not limited to the following embodiments. [ [Example]

[0065] Example 1: The inhibitory effect of histidine on aggregates in the heat-accelerated preservation of humanized IgG4 antibody ACE910 (1) Materials ACE910 is a dual-specific antibody that can recognize both coagulation factor IX and coagulation factor X. It is a humanized IgG4 antibody that is expected to prevent bleeding in hemophilia A by replacing the function of coagulation factor VIII.

[0066] (2) Test samples Prepare solutions containing 100 mg / mL ACE910, 150 mmol / L NaCl, pH 6, and any one of 20 mmol / L phosphate buffer, or 20 mmol / L citrate buffer, or 20 mmol / L acetate buffer, or 20 mmol / L histidine buffer as a buffer solution, and fill each solution into a glass bottle with 5-15 μL. The humanized antibody solution prepared in this way was placed in a constant temperature bath at 25°C for 8 weeks and then used as a test sample.

[0067] (3) Methods for determining and calculating the aggregate size of ACE910 The amount of aggregates was determined by size sieving chromatography (SEC) using a column (Tosoh-G3000SWXL), with 50 mmol / L phosphate buffer (pH 7.0) and 300 mmol / L sodium chloride as the mobile phase, at a flow rate of 0.5 mL / min. Among the detected peaks, the one with the largest area and height is considered a single entity, and the collective peaks detected before the single entity are called an aggregate (HMWS). For all the peaks, calculate the area and calculate the peak area ratio of the target object peaks according to the following formula.

[0068] (4) Results The results are shown in Table 1.

[0069] Table 1. Increase in aggregate size (%) after storage at 25℃ formula ΔHMW(%) 25℃-2W 25℃-4W 25℃-8W phosphate 0.20 0.27 0.41 citrate 0.14 0.21 0.32 acetate 0.24 0.38 0.63 histidine 0.08 0.13 0.22

[0070] As shown in Table 1, the samples with added 20 mmol / L histidine achieved a high aggregate inhibition effect after 8 weeks of thermal acceleration at 25℃.

[0071] Example 2: The effect of salt concentration and arginine on the inhibition of aggregates in the accelerated heat preservation and freeze-thaw process of humanized IgG4 antibody ACE910. (1) Materials The antibody described in Example 1 was used.

[0072] (2) Test samples Prepare solutions containing 100 mg / mL ACE910, 20 mmol / L histidine, pH 6, and any one of 50 mmol / L NaCl, 75 mmol / L NaCl, 150 mmol / L NaCl, or 150 mmol / L arginine as additives, and fill 5-15 μL of each solution into a glass bottle. The humanized antibody solution prepared in this way was placed in a constant temperature bath at 25°C for 8 weeks, or subjected to repeated freeze-thaw cycles (F / T) (5°C↔-20°C) 10 times, and then used as a test sample.

[0073] (3) Methods for determining and calculating the aggregate size of ACE910 Perform according to the method described in Example 1.

[0074] (4) Results The results obtained are shown in Table 2.

[0075] Table 2. Increase in aggregate size (%) after storage at 25℃ and after freeze-thaw. formula ΔHMW(%) 5F / T 10F / T 25℃-2W 25℃-4W 25℃-8W 50 mM NaCl 1.00 2.07 0.09 0.16 0.26 75 mM NaCl 0.62 1.43 0.07 0.13 0.23 150 mM NaCl 0.11 0.28 0.08 0.13 0.22 150 mM Arg 0.03 0.05 0.03 0.06 0.11

[0076] As shown in Table 2, the samples with added 150 mmol / L arginine showed high aggregate inhibition in both the samples after 8 weeks of thermal acceleration at 25°C and the samples after freeze-thaw.

[0077] Example 3: The inhibitory effect of aspartic acid on aggregates during the freeze-thaw reaction of humanized IgG4 antibody ACE910 (1) Materials Using the antibody described in Example 1

[0078] (2) Test samples Prepare solutions containing 100 mg / mL ACE910, 20 mmol / L histidine, pH 6, and either 150 mmol / L NaCl or 150 mmol / L sodium L-aspartic acid as a relative ion, and fill each solution into a glass bottle with 5–15 μL of each solution. The humanized antibody solution prepared in this way was repeatedly frozen and thawed (5℃↔-20℃) 10 times and used as the test sample.

[0079] (3) Methods for determining and calculating the aggregate size of ACE910 Perform according to the method described in Example 1.

[0080] (4) Results The results are shown in Table 3.

[0081] Table 3. Increase in aggregate size (%) after freeze-thaw cycle formula ΔHMW(%) 5F / T 10F / T NaCl 0.11 0.28 Na aspartic acid 0.05 0.13

[0082] As shown in Table 3, the samples with added aspartic acid achieved a high aggregate inhibition effect in the samples after freeze-thaw.

[0083] Example 4: The effect of pH value on inhibiting aggregates and charge-isomer components during heat-accelerated preservation of humanized IgG4 antibody ACE910 (1) Materials Using the antibody described in Example 1

[0084] (2) Test samples Prepare solutions containing 100 mg / mL ACE910, 20 mmol / L histidine-aspartic acid, and 150 mmol / L arginine-aspartic acid, with pH values ​​of 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, and 7.5, and fill each solution with 5–15 μL into a glass bottle. The humanized antibody solution prepared in this way was placed in a constant temperature bath at 25°C for 8 weeks and then used as a test sample.

[0085] (3) Methods for determining and calculating the aggregate size of ACE910 Perform according to the method described in Example 1.

[0086] (4) Methods for determining and calculating the charge-displacement component of ACE910 The amount of the oppositely charged component was determined by ion exchange chromatography (IEC) using a YMC BioPro QA-F column, with 20 mmol / L Tris-HCl buffer (pH 7.8) as mobile phase A and 20 mmol / L Tris-HCl buffer (pH 7.8) and 500 mmol / L sodium chloride as mobile phase B, at a flow rate of 0.5 mL / min. The peak with the largest area and height among the detected peaks is called the main peak. The peaks detected after the main peak are collectively called acidic peaks. For all wave crests, calculate their areas, and then calculate the wave crest area ratio of the target object using the following formula.

[0087] (5) Results The results are shown in Table 4.

[0088] Table 4. Increase in aggregate size (%) and increase in acid peak-1 size (%) after storage at 25℃ formula ΔHMW(%) ΔAcidity -1 (%) 25℃-2W 25℃-4W 25℃-8W 25℃-2W 25℃-4W 25℃-8W pH 4.5 0.05 0.10 0.24 0.21 2.43 3.41 pH 5.0 0.09 0.11 0.20 0.46 0.86 2.57 pH 5.5 0.07 0.08 0.15 0.36 0.68 2.10 pH 6.0 0.07 0.11 0.17 0.38 0.47 3.23 pH 6.5 0.09 0.14 0.25 0.24 1.17 3.90 pH 7.0 0.13 0.17 0.30 1.25 2.44 5.39 pH 7.5 0.18 0.32 0.79 3.09 5.01 9.49

[0089] As shown in Table 4, samples with pH 4.5 to pH 6.5, especially pH 5.5 and pH 6.0, showed high inhibition of aggregates and charged components after storage at 25°C.

[0090] Example 5: The effect of histidine concentration on the inhibition of aggregates and charge-isomer components during heat-accelerated preservation of humanized IgG4 antibody ACE910 (1) Materials The antibody described in Example 1 was used.

[0091] (2) Test samples Prepare solutions containing 100 mg / mL ACE910, 150 mmol / L arginine, and histidine-aspartic acid at pH 6.0, 5 mmol / L, 10 mmol / L, 20 mmol / L, or 40 mmol / L, and fill 5–15 μL of each solution into glass vials. The humanized antibody solution prepared in this way was placed in a constant temperature bath at 25°C for 8 weeks and then used as a test sample.

[0092] (3) Methods for determining ACE910 aggregate size and methods for calculating aggregate size Perform according to the method described in Example 1.

[0093] (4) Methods for determining and calculating the charge-distribution component of ACE910 Perform according to the method described in Example 4.

[0094] (5) Results The results obtained are shown in Table 5.

[0095] Table 5. Increase in aggregate size (%) and increase in acid peak-1 size (%) after storage at 25℃ histidine concentration ΔHMW(%) ΔAcidity -1 (%) 25℃-2W 25℃-4W 25℃-8W 25℃-2W 25℃-4W 25℃-8W 5 mmol / L 0.04 0.09 0.17 0.87 3.01 9.19 10 mmol / L 0.02 0.05 0.14 0.39 1.50 7.09 20 mmol / L 0.01 0.04 0.12 0.06 1.04 7.11 40 mmol / L 0.01 0.02 0.07 0.52 1.13 6.74

[0096] As shown in Table 5, samples containing more than 10 mmol / L of histidine-aspartic acid exhibited high inhibition of aggregates and charge-isotropic components after storage at 25°C.

[0097] Example 6: The effect of arginine concentration on aggregate inhibition during freeze-thaw, accelerated heat preservation, and cryopreservation of humanized IgG4 antibody ACE910 (1) Materials The antibody described in Example 1 was used.

[0098] (2) Test samples Prepare various solutions containing 100 mg / mL ACE910, 20 mmol / L histidine-aspartic acid, pH 6.0, 75 mg / mL, or 100 mmol / L, or 150 mmol / L, or 200 mmol / L, or 300 mmol / L arginine, and fill 5-15 μL into glass bottles respectively. The humanized antibody solution prepared in this way was repeatedly frozen and thawed (5℃↔-20℃) 10 times, or placed in a constant temperature bath at 25℃ for 8 weeks, or placed in a constant temperature bath at -20℃ for 6 months, and used as test samples.

[0099] (3) Methods for determining ACE910 aggregate size and methods for calculating aggregate size Perform according to the method described in Example 1.

[0100] (4) Results The results are shown in Table 6.

[0101] Table 6. Increase in aggregate size (%) after freeze-thaw and storage at 25°C and -20°C Arginine concentration ΔHMW(%) 5F / T 10F / T 25℃-2W 25℃-4W 25℃-8W -20℃-3W -20℃-6W 75 mmol / L 0.10 0.10 0.02 0.06 0.17 0.19 0.53 100 mmol / L 0.04 0.04 0.01 0.04 0.12 0.08 0.02 150 mmol / L 0.01 0.01 -0.01 0.01 0.04 0.00 0.00 200 mmol / L 0.01 0.01 -0.01 0.00 0.02 0.00 -0.01 300 mmol / L 0.00 0.00 -0.02 -0.02 0.00 0.00 -0.01

[0102] As shown in Table 6, samples containing arginine at concentrations of 100 mmol / L or higher exhibited high aggregation inhibition effects after freezing and thawing, storage at 25°C, and storage at -20°C.

[0103] Example 7: Inhibition of insoluble impurities and insoluble microparticle formation caused by Poloxamer 188 during 5°C storage of humanized IgG4 antibody ACE910. (1) Materials Use the antibody described in Example 1.

[0104] (2) Test samples Prepare solutions containing 80 mg / mL ACE910, 20 mmol / L histidine-aspartic acid, 150 mmol / L arginine, pH 6.0, and 0 mg / mL Poloxamer 188, or 0.2 mg / mL Poloxamer 188, or 0.5 mg / mL Poloxamer 188, or 1.0 mg / mL Poloxamer 188, or 0.05 mg / mL Polysorbate 20, or 1.0 mg / mL Polysorbate 20 as additives, and fill each solution to 1.0 mL into a glass bottle. The humanized antibody solution prepared in this way was left to stand in a 5°C refrigerator for 5 months and then used as a test sample.

[0105] (3) Insoluble impurity observation method Place the sample on the sample stage of the visual inspection station, rotate the sample stage, and observe to confirm the presence of insoluble impurities.

[0106] (4) Determination of insoluble microparticles A solution microparticle counter (Hach Ultra Analytics, Model 9703) was used to count the number of insoluble microparticles in the solution.

[0107] (5) Results The results are shown in Table 7.

[0108] Table 7. Number of insoluble microparticles (microparticles / mL) and detection rate of insoluble impurities (%) after storage at 5℃ PX188 0.2 mg / mL 0.5 mg / mL 1.0 mg / mL initial 1M 3M 5M initial 1M 3M 5M initial 1M 3M 5M Number of insoluble microparticles in solution (Number of microparticles / mL) ≥5 μm 31 - 61 188 9 - 18 27 7 - 7 43 ≥10 μm 5 - 4 15 5 - 3 2 5 - 3 5 ≥25 μm 1 - 1 1 1 - 0 0 0 - 1 1 Insoluble impurity detection rate (number of impurity samples / number of samples tested) 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 PS20 0.05 mg / mL 1.0 mg / mL initial 1M 3M 5M initial 1M 3M 5M Number of insoluble microparticles in solution (Number of microparticles / mL) ≥5 μm 13 - 36 89 twenty four - 238 411 ≥10 μm 5 - 3 10 9 - 49 66 ≥25 μm 0 - 0 0 1 - 3 2 Insoluble impurity detection rate (number of impurity samples / number of samples tested) 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 0 / 5 5 / 5 5 / 5

[0109] As shown in Table 7, the samples containing 0.05 mg / mL PS20 and the samples containing 0.2 mg / mL Poloxamer 188 exhibited high inhibition effects on insoluble impurities and insoluble particles after storage at 5℃.

[0110] Example 8: Inhibition effect of Poloxamer 188 on the formation of insoluble impurities and insoluble microparticles during the shaking and freeze-thaw storage of humanized IgG4 antibody ACE910. (1) Materials The antibody described in Example 1 was used.

[0111] (2) Test samples Preparations were made containing 150 mg / mL ACE910, 20 mmol / L histidine-aspartic acid, 150 mg / mL arginine-aspartic acid, pH 6.0, and 0 mg / mL or 0.2 mg / mL or 0.5 mg / mL or 0.8 mg / mL or 0.8 mg / mL or 0.9 mL of each formulation as an additive, and each formulation was filled into a glass vial. The humanized antibody solution prepared in this way was shaken at 200 strokes / min for 24 hours at room temperature, or subjected to repeated freeze-thaw cycles (5℃↔-20℃) 10 times, and used as test samples.

[0112] (3) Observation methods for insoluble impurities Perform according to the method described in Example 7.

[0113] (4) Detection method for insoluble impurities Perform according to the method described in Example 7.

[0114] (5) Results The results obtained are shown in Table 8 and Figures 1 and 2.

[0115] Table 8. Detection rate (%) of insoluble impurities after shaking and freeze-thaw. Poloxamer 188 concentration Insoluble impurity detection rate (%) (number of impurity samples / number of samples tested) initial 30 minutes of fluctuation 24-hour fluctuations Freeze and thaw 0 mg / mL 0% (0 / 10) 50% (5 / 10) 100% (10 / 10) 0% (0 / 10) 0.2 mg / mL 0% (0 / 10) 0% (0 / 10) 0% (0 / 10) 0% (0 / 10) 0.5 mg / mL 0% (0 / 10) 0% (0 / 10) 0% (0 / 10) 0% (0 / 10) 0.8 mg / mL 0% (0 / 10) 0% (0 / 10) 0% (0 / 10) 0% (0 / 10)

[0116] As shown in Table 8 and Figures 1 and 2, samples containing 0.2 mg / mL or higher of Poloxamer 188 exhibited high inhibition of insoluble impurities and insoluble particles after oscillation stress and freeze-thaw preservation.

[0117] Example 9: Stability of humanized IgG4 antibody ACE910 concentration during accelerated heat preservation and freeze-thaw preservation (1) Materials Use the antibody described in Example 1. (2) Test samples Prepare ACE910 solutions containing 20 mmol / L histidine-aspartic acid, 150 mmol / L arginine-aspartic acid, pH 6.0, and 0.5 mmol / L Poloxamer 188 as ACE910 solutions of 20 mg / mL, 30 mg / mL, 40 mg / mL, 120 mg / mL, 150 mg / mL, or 180 mg / mL, and fill each solution into a glass bottle with 0.65 mL of each solution. The humanized antibody solution prepared in this way was placed in a constant temperature bath at 40°C for 8 weeks, or subjected to repeated freeze-thaw cycles (25°C↔-20°C) 5 and 10 times, and used as test samples.

[0118] (3) Methods for measuring and calculating ACE910 aggregate size Perform according to the method described in Example 1.

[0119] (4) Methods for determining and calculating the charge-distribution component of ACE910 The amount of the oppositely charged components was determined by anion exchange chromatography (AIEC) using a Waters TSKgeL Q-STAT column, with 50 mmol / L Tris-HCl buffer (pH 8.0) as mobile phase A and 50 mmol / L Tris-HCl buffer (pH 8.0) and 200 mmol / L sodium chloride as mobile phase B, at a flow rate of 0.5 mL / min. Among the detected peaks, the one with the largest area and height is designated as the main peak. The peaks detected before the main peak are collectively referred to as basic peaks, and the peaks detected after the main peak are collectively referred to as acidic peaks. In addition, the amount of the oppositely charged component was determined by cation exchange chromatography (CIEC) using a Thermo Scientific ProPac WCX-10G column, with 9.6 mmol / L Tris, 6.0 mmol / L piperazine, and 11.0 mmol / L imidazole buffer (pH 6.0) as mobile phase A and 9.6 mmol / L Tris, 6.0 mmol / L piperazine, 11.0 mmol / L imidazole, and 100 mmol / L sodium chloride buffer (pH 10.1) as mobile phase B, at a flow rate of 0.5 mL / min. Among the detected peaks, the one with the largest area and height is called the BiAb peak. Peaks detected before the BiAb peak are collectively called pre-peaks, and peaks detected after the BiAb peak are collectively called post-peaks. For all wave peaks, calculate the area, and calculate the target object according to the following formula. The ratio of the area of ​​one peak to the area of ​​another peak.

[0120] (5) Results The results obtained are shown in Table 9. Furthermore, SE represents the results of size sieve chromatography, AE represents the results of anion exchange chromatography, and CE represents the results of cation exchange chromatography.

[0121] Table 9. Aggregate content (%) and charge-opposite component (%) after storage at 40℃ and freeze-thaw. ACE910 concentration (mg / mL) 20 30 40 120 150 180 SE-HPLC %HMWS initial 0.10 0.11 0.11 0.13 0.14 0.14 40℃ in 2 weeks 0.14 0.15 0.16 0.26 0.28 0.31 4 weeks 40℃ 0.16 0.17 0.20 0.34 0.38 0.44 5-cycle F / T 0.11 0.11 0.12 0.13 0.14 0.14 10-cycle F / T 0.11 0.11 0.11 0.13 0.13 0.14 SE-HPLC %monomer initial 99.90 99.89 99.89 99.87 99.86 99.86 40℃ in 2 weeks 99.86 99.85 99.84 99.74 99.72 99.69 4 weeks 40℃ 99.84 99.83 99.80 99.66 99.62 99.56 5-cycle F / T 99.89 99.89 99.88 99.87 99.86 99.86 10-cycle F / T 99.89 99.89 99.89 99.87 99.87 99.86 AE-HPLC % alkaline region initial 16.1 16.2 16.1 16.3 16.0 16.3 40℃ in 2 weeks 14.3 14.6 14.4 14.5 14.5 14.5 4 weeks 40℃ 12.7 12.8 12.8 12.8 12.7 12.7 5-cycle F / T 16.1 16.2 16.4 16.5 16.5 16.5 10-cycle F / T 15.9 16.0 16.2 16.4 16.5 16.4 AE-HPLC %Main Area initial 71.7 71.6 71.8 71.5 72.1 71.6 40℃ in 2 weeks 65.0 64.6 64.7 64.5 63.9 64.4 4 weeks 40℃ 58.3 57.8 57.8 57.2 57.3 57.5 5-cycle F / T 72.3 72.2 72.0 71.6 71.6 71.6 10-cycle F / T 72.7 72.4 72.1 71.5 71.6 71.7 AE-HPLC % acidic area initial 12.1 12.2 12.1 12.2 11.9 12.1 40℃ in 2 weeks 20.7 20.8 20.9 20.9 21.6 21.1 4 weeks 40℃ 29.0 29.4 29.4 30.0 30.0 29.8 5-cycle F / T 11.6 11.6 11.6 11.9 11.9 12.0 10-cycle F / T 11.5 11.6 11.7 12.1 12.0 11.9 CE-HPLC %Pre-peaks initial 3.2 3.2 3.3 3.3 3.3 3.3 40℃ in 2 weeks 3.6 3.8 3.8 3.7 3.7 3.7 4 weeks 40℃ 4.3 4.3 4.3 4.3 4.2 4.3 5-cycle F / T 3.4 3.0 3.2 3.1 3.0 3.1 10-cycle F / T 3.1 3.2 2.8 3.1 3.1 3.1 CE-HPLC %BiAb initial 96.8 96.8 96.7 96.6 96.6 96.6 40℃ in 2 weeks 96.4 96.2 96.3 96.3 96.3 96.2 4 weeks 40℃ 95.6 95.7 95.6 95.7 95.8 95.8 5-cycle F / T 96.6 97.0 96.8 96.9 97.0 96.9 10-cycle F / T 96.8 96.7 97.2 96.8 96.9 96.9 CE-HPLC %Post-peaks initial 0.0 0.0 0.0 0.1 0.1 0.1 40℃ in 2 weeks 0.0 0.0 0.0 0.0 0.0 0.0 4 weeks 40℃ 0.0 0.0 0.0 0.0 0.0 0.0 5-cycle F / T 0.0 0.0 0.0 0.0 0.0 0.0 10-cycle F / T 0.0 0.0 0.0 0.0 0.0 0.0

[0122] As shown in Table 9, the samples of ACE910 with concentrations ranging from 20 mg / mL to 180 mg / mL exhibited the same and adequate stability after storage at 40°C and after freeze-thaw cycles. [Industrial applicability]

[0123] Compared to previous formulations, the antibody-containing solution formulation of the present invention is a formulation with good stability in the solution state, and is characterized by inhibiting the aggregation of antibody molecules and other proteins after storage at low temperature, room temperature, or high temperature, as well as after freeze-thaw cycles. This antibody solution formulation of the present invention, which is not prone to degradation, can be used, for example, for the treatment of hemophilia A administered subcutaneously.

[0124] none

[0125] <![CDATA[ <110> Chugai Seiyaku Kabushiki Kaisha Co., Ltd. <![CDATA[ <120> Antibody preparations <![CDATA[ <130> C1-A1602-TW]]> <![CDATA[ <150> JP 2016-090590 <![CDATA[ <151> 2016-04-28 <![CDATA[<16]]> 0> 15 ]]> <![CDATA[ <170> PatentIn version 3.5]]> <![CDATA[ <210> 1]]> <![CDATA[ <211> 5]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Heavy chain variable region CDR1]]> <![CDATA[ <400> 1]]> <![CDATA[Tyr Tyr Asp Ile Gln 1 5 <![CDATA[ <210> 2]]> <![CDATA[ <211> 17]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Heavy chain variable region CDR2]]> <![CDATA[ <400> 2]]> Ser Ile Ser Pro Ser Gly Gln Ser Thr Tyr Tyr Tyr Arg Arg Glu Val Lys 1 5 10 15 Gly <![CDATA[ <210> 3]]> <![CDATA[ <211> 14]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Heavy chain variable region CDR3]]> <![CDATA[ <400> 3]]> Arg Thr Gly Arg Glu Tyr Gly Gly Gly Trp Tyr Phe Asp Tyr 1 5 10 <![CDATA[ <210> 4]]> <![CDATA[ <211> 5]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Heavy chain variable region CDR1]]> <![CDATA[ <400> 4]]> Asp Asn Asn Met Asp 1 5 <![CDATA[ <210> 5]]> <![CDATA[ <211> 17]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Heavy chain variable region CDR2]]> <![CDATA[ <400> 5]]> Asp Ile Asn Thr Arg Ser Gly Gly Ser Ile Tyr Asn Glu Glu Phe Gln 1 5 10 15 Asp <![CDATA[ <210> 6]]> <![CDATA[ <211> 10]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Heavy chain variable region CDR3]]> <![CDATA[ <400> 6]]> Arg Lys Ser Tyr Gly Tyr Tyr Leu Asp Glu 1 5 10 <![CDATA[ <210> 7]]> <![CDATA[ <211> 11]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Light chain variable region CDR1]]> <![CDATA[ <400> 7]]> Lys Ala Ser Arg Asn Ile Glu Arg Gln Leu Ala 1 5 10 <![CDATA[ <210> 8]]> <![CDATA[ <211> 7]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Light chain variable region CDR2]]> <![CDATA[ <400> 8]]> Gln Ala Ser Arg Lys Glu Ser 1 5 <![CDATA[ <210> 9]]> <![CDATA[ <211> 9]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Light chain variable region CDR3]]> <![CDATA[ <400> 9]]> Gln Gln Tyr Ser Asp Pro Pro Leu Thr 1 5 <![CDATA[ <210> 10]]> <![CDATA[ <211> 448]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Heavy chain]]> <![CDATA[<400> 10]]> Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Tyr 20 25 30 Asp Ile Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Pro Ser Gly Gln Ser Thr Tyr Tyr Arg Arg Glu Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Thr Gly Arg Glu Tyr Gly Gly Gly Trp Tyr Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Thr Cys Asn Val 195 200 205 Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys 210 215 220 Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 260 265 270 Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Gln Lys Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn Arg Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <![CDATA[ <210> 11]]> <![CDATA[ <211> 444]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Heavy chain <![CDATA[ <400> 11]]> Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Asn 20 25 30 Asn Met Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asp Ile Asn Thr Arg Ser Gly Gly Ser Ile Tyr Asn Glu Glu Phe 50 55 60 Gln Asp Arg Val Ile Met Thr Val Asp Lys Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr His Cys 85 90 95 Ala Arg Arg Lys Ser Tyr Gly Tyr Tyr Leu Asp Glu Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260 265 270 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 <![CDATA[ <210> 12]]> <![CDATA[ <211> 214]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Light chain <![CDATA[ <400> 12]]> Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Arg Asn Ile Glu Arg Gln 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Glu Leu Leu Ile 35 40 45 Tyr Gln Ala Ser Arg Lys Glu Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Arg Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Asp Pro Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <![CDATA[ <210> 13]]> <![CDATA[ <211> 123]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Heavy chain variable region <![CDATA[ <400> 13]]> Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Tyr 20 25 30 Asp Ile Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Pro Ser Gly Gln Ser Thr Tyr Tyr Arg Arg Glu Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Thr Gly Arg Glu Tyr Gly Gly Gly Trp Tyr Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <![CDATA[ <210> 14]]> <![CDATA[ <211> 119]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Heavy chain variable region <![CDATA[ <400> 14]]> Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Asn 20 25 30 Asn Met Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asp Ile Asn Thr Arg Ser Gly Gly Ser Ile Tyr Asn Glu Glu Phe 50 55 60 Gln Asp Arg Val Ile Met Thr Val Asp Lys Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr His Cys 85 90 95 Ala Arg Arg Lys Ser Tyr Gly Tyr Tyr Leu Asp Glu Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <![CDATA[<210> 15]]> <![CDATA[<211> 107]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Light chain variable region]]> <![CDATA[ <400> 15]]> Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Arg Asn Ile Glu Arg Gln 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Glu Leu Leu Ile 35 40 45 Tyr Gln Ala Ser Arg Lys Glu Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Arg Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Asp Pro Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105

[0126]

Claims

1. An antibody solution formulation, comprising: A dual-specific antibody consisting of a first and third polypeptide pairing at a concentration of 20–180 mg / mL, and a second and fourth polypeptide pairing. The first polypeptide is composed of an H-chain containing the amino acid sequences 1, 2, and 3 of the H-chain CDR (Q499); the second polypeptide contains the amino acid sequences 1, 2, and 3 of the H-chain CDR (J327); and the third and fourth polypeptides are composed of a common L-chain containing the amino acid sequences 1, 2, and 3 of the L-chain CDR (L404). Histidine-aspartate buffer, wherein the histidine concentration in the buffer is 10 mM–40 mM, and 0.2–1 mg / mL Poloxamer is used. 188, and 100 mM to 300 mM L-arginine, with a pH of 4.5 to 6.

5.

2. The antibody solution formulation as described in claim 1, wherein the dual-specific antibody is a dual-specific antibody formed by pairing a first polypeptide with a third polypeptide, and pairing a second polypeptide with a fourth polypeptide, and is formed by the first polypeptide being an H chain formed by the amino acid sequence of sequence identification number 10, the second polypeptide being an H chain formed by the amino acid sequence of sequence identification number 11, and the third and fourth polypeptides being a common L chain of sequence identification number 12.

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

4. The antibody solution preparation as described in claim 1, wherein the pH value is 6.

0.

5. The antibody solution formulation as described in claim 1, wherein the concentration of the histidine-aspartate buffer solution is 20 mM.

6. The antibody solution formulation as described in claim 1, wherein the concentration of L-arginine is 150 mM.

7. The antibody solution preparation described in claim 1 does not actually contain chloride ions and acetate ions.

8. An antibody solution formulation, comprising: A dual-specific antibody consisting of a first and third polypeptide forming a pair, and a second and fourth polypeptide forming a pair, at a concentration of 20–180 mg / mL. The first polypeptide is an H-chain formed by the amino acid sequence of sequence identification number 10, the second polypeptide is an H-chain formed by the amino acid sequence of sequence identification number 11, and the third and fourth polypeptides are a common L-chain formed by the amino acid sequence of sequence identification number 12. The antibody also includes an L-histidine-aspartate buffer, wherein the histidine concentration in the buffer is 20 mM, 0.5 mg / mL Poloxamer 188, and 150 mM L-arginine, with a pH of 6.

9. The antibody solution preparation as described in claim 1 or 8 is intended for subcutaneous administration.

10. The antibody solution preparation described in claim 1 or 8 is for the treatment of hemophilia A.

11. A method for stabilizing an antibody in an antibody-containing solution formulation, comprising adding histidine-aspartate buffer, Poloxamer 188, and L-arginine to the solution, wherein the concentration of histidine in 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 L-arginine is 100 mM to 300 mM, wherein the antibody is a dual-specific antibody formed by pairing a first polypeptide with a third polypeptide and pairing a second polypeptide with a fourth polypeptide, and wherein the first polypeptide is an H chain containing the H chain CDR1, 2, 3 amino acid sequence of sequence identification numbers: 1, 2, 3 (Q499 H chain CDR), and the second polypeptide contains sequence identification numbers: 4, 5, 6 The H chain of (J327's H chain CDR) contains amino acid sequences 1, 2, and 3. The third and fourth peptides are bispecific antibodies formed from the common L chain containing sequence identification numbers 7, 8, and 9 of (L404's L chain CDR) containing amino acid sequences 1, 2, and 3.

12. A method for inhibiting antibody aggregation (aggregation) in an antibody-containing solution formulation, comprising adding histidine-aspartate buffer, Poloxamer 188, and L-arginine to the solution, wherein the concentration of histidine in the histidine-aspartate buffer is 10 mM to 40 mM, the concentration of Poloxamer 188 is 0.2 to 1 mg / mL, the concentration of L-arginine is 100 mM to 300 mM, and the antibody is a dual-specific antibody formed by pairing a first polypeptide with a third polypeptide and pairing a second polypeptide with a fourth polypeptide, wherein the first polypeptide is an H chain containing the H chain CDR1, 2, 3 amino acid sequence of sequence identification numbers: 1, 2, 3 (Q499 H chain CDR), and the second polypeptide contains sequence identification numbers: 4, 5, 6 The H chain of (J327's H chain CDR) contains amino acid sequences 1, 2, and 3. The third and fourth peptides are bispecific antibodies formed from the common L chain containing sequence identification numbers 7, 8, and 9 of (L404's L chain CDR) containing amino acid sequences 1, 2, and 3.

13. A method for reducing the charge-isomer component in an antibody-containing solution formulation, comprising adding a histidine-aspartate buffer to the solution, wherein the concentration of histidine in the histidine-aspartate buffer is 10 mM to 40 mM, wherein the antibody is a dual-specific antibody formed by pairing a first polypeptide with a third polypeptide and pairing a second polypeptide with a fourth polypeptide, and wherein the first polypeptide is an H chain containing the H chain CDR1, 2, 3 amino acid sequence of sequence identification number: 1, 2, 3 (H chain CDR of Q499), the second polypeptide is an H chain containing the H chain CDR1, 2, 3 amino acid sequence of sequence identification number: 4, 5, 6 (H chain CDR of J327), and the third and fourth polypeptides contain the H chain CDR1, 2, 3 amino acid sequence of sequence identification number: 7, 8, 9. A bispecific antibody formed from the common L chain of amino acid sequences 1, 2, and 3 of the L chain CDR of (L404).

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