Antibody-containing formulations

Abstract

The present invention relates to a stable antibody-containing solution formulation in which aggregate formation of Emicizumab (ACE910), a bispecific antibody that substitutes for functional FVIII, is inhibited. Specifically, the present invention relates to the above antibody-containing solution formulation at pH 4.5 to pH 6.5, which contains 20 to 180 mg / mL of the above bispecific antibody, 10 mM to 40 mM of a histidine-aspartate salt buffer, 0.2 to 1 mg / mL of poloxamer 188, and 100 mM to 300 mM of arginine.

Description

[0001] This application is a divisional application of Chinese application number 201780020233.X, filed on April 27, 2017, entitled "Antibody-containing Preparation". [Technical Field]

[0002] This invention relates to formulations comprising bispecific antibodies that bind coagulation factor IX (FIX) and / or activated coagulation factor IX (FIXa) and a functional substitute coagulation factor VIII (FVIII) that bind coagulation factor X (FX). [Background Technology]

[0003] Bispecific antibodies that functionally replace FVIII have been discovered, binding to coagulation factor IX (FIX) and / or activated coagulation factor IX (FIXa) and binding to coagulation factor X (FX) (Non-Patent Literature 1 and 2; Patent Literature 1 to 3). The bispecific antibody Emicizumab (ACE910) improves the reduction in coagulation reactions caused by FVIII deficiency and dysfunction by functionally replacing FVIII; therefore, clinical trials are underway in patients with hemophilia A.

[0004] Numerous antibody solution formulations have been developed. To date, high-concentration antibody solution formulations reported include those using histidine and arginine (Patent Document 4) and those using histidine / aspartate salt buffer (Patent Document 5). Meanwhile, stable liquid drug antibody formulations containing amyloid (A) using histidine / histidine-HCl as a buffer have been reported (Patent Document 6).

[0005] However, for solution formulations containing the aforementioned bispecific antibodies, there are no reports of stable solution formulations in which aggregate formation and / or components with charge heterogeneity are inhibited.

[0006] [List of Citations]

[0007] [Patent Literature]

[0008] [Patent Document 1] WO2005 / 035756

[0009] [Patent Document 2] WO2006 / 109592

[0010] [Patent Document 3] WO2012 / 067176

[0011] [Patent Document 4] WO2002 / 030463

[0012] [Patent Document 5] WO2011 / 090088

[0013] [Patent Document 6] WO2013 / 131866

[0014] [Non-patent literature]

[0015] [Non-Patent Literature 1] Nat Med. 2012; 18(10):1570-74

[0016] [Non-Patent Literature 2] PLoS One. 2013; 8(2):e57479 [Invention Overview]

[0017] [Problems to be solved]

[0018] The object of this invention is to provide a stable solution formulation comprising Emicizumab (ACE910), wherein Emicizumab is a functionally substituted FVIII binding FIX and / or FIXa binding FX bispecific antibody.

[0019] [Problem-solving methods]

[0020] As a result of specific research aimed at achieving the above objectives, the inventors discovered that a solution formulation with pH 4.5 to 6.5 (containing 20 to 180 mg / mL of the above-mentioned bispecific antibody, 10 mM to 40 mM histidine / aspartate buffer, 0.2 to 1 mg / mL poloxamer 188, and 100 mM to 300 mM arginine) can be a stable antibody-containing solution formulation in which aggregate formation and / or components with charge heterogeneity are inhibited, thereby completing the present invention.

[0021] Specifically, the present invention provides the following:

[0022] [1] An antibody solution formulation with pH 4.5 to 6.5, comprising:

[0023] A bispecific antibody at concentrations of 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 contains an H chain containing the amino acid sequences of H chain CDR 1, 2, and 3 of SEQ ID NO:1, 2, and 3 (H chain CDR of Q499); the second polypeptide contains an H chain containing the amino acid sequences of H chain CDR 1, 2, and 3 of SEQ ID NO:4, 5, and 6 (H chain CDR of J327); and the third and fourth polypeptides contain a common L chain containing the amino acid sequences of L chain CDR 1, 2, and 3 of SEQ ID NO:7, 8, and 9 (L chain CDR of L404);

[0024] 10mM to 40mM histidine / aspartate buffer;

[0025] 0.2 to 1 mg / mL poloxamer 188; and

[0026] 100mM to 300mM arginine.

[0027] [2][1] Antibody solution formulation, wherein in the bispecific antibody, 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 contains an H chain containing the amino acid sequence of SEQ ID NO:10; the second polypeptide contains an H chain containing the amino acid sequence of SEQ ID NO:11, and the third polypeptide and the fourth polypeptide contain a common L chain of SEQ ID NO:12.

[0028] [3][1] or [2] antibody solution preparations, wherein the concentration of poloxamer 188 is 0.5 mg / mL.

[0029] An antibody solution preparation of any one of [4][1]-[3], wherein the pH is 6.0.

[0030] The antibody solution preparation of any one of [5][1]-[4], wherein the concentration of histidine / aspartate buffer is 20 mM.

[0031] An antibody solution preparation of any one of [6][1]-[5], wherein the concentration of arginine is 150 mM.

[0032] The antibody solution preparation of any one of [7][1] to [6] is substantially free of chloride or acetate ions.

[0033] [8] An antibody solution preparation with pH 6, comprising:

[0034] A bispecific antibody at concentrations of 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 contains an H chain containing the amino acid sequence of SEQ ID NO:10; the second polypeptide contains an H chain containing the amino acid sequence of SEQ ID NO:11; and the third polypeptide and the fourth polypeptide contain a common L chain of SEQ ID NO:12.

[0035] 20 mM L-histidine / aspartate buffer;

[0036] 0.5 mg / mL poloxamer 188; and

[0037] 150mM L-arginine.

[0038] An antibody solution preparation of any one of [9][1]-[8] for subcutaneous application.

[0039] An antibody solution preparation of any one of

[10] [1]-[9], used to treat hemophilia A.

[0040]

[11] A method for stabilizing an antibody in a solution containing an antibody, comprising adding histidine / aspartate buffer, poloxamer 188 and arginine to the solution, wherein 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.

[0041]

[12] A method for inhibiting antibody association (aggregate formation) in a solution containing antibodies, comprising adding histidine / aspartate buffer, poloxamer 188 and arginine to a 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.

[0042]

[13] A method for inhibiting a component with charge heterogeneity in an antibody-containing preparation, comprising adding a histidine / aspartate buffer to a solution, wherein the concentration of the histidine / aspartate buffer is from 10 mM to 40 mM.

[0043] [Effects of the Invention]

[0044] This invention provides antibody-containing formulations exhibiting excellent stability. Furthermore, this invention also provides antibody-containing formulations in which aggregate formation and / or components exhibiting charge heterogeneity are inhibited in their solution state. [Brief description of the attached diagram]

[0045] Figure 1 Photographs of insoluble foreign matter present after the shaking test in Example 8 are shown (a: 0 mg / mL poloxamer 188; b: 0.5 mg / mL poloxamer 188).

[0046] Figure 2 The graph shows the number of insoluble particles (particles / mL) present after the shaking test and freeze-thaw cycle of Example 8. [Means for carrying out the present invention]

[0047] The present invention will now be described in detail.

[0048] This invention provides a solution formulation with pH 4.5-6.5 comprising: 20-180 mg / mL of Emicizumab (ACE910) (which is a functionally substituted FVIII-binding FIX and / or FIXa-binding FX-bispecific antibody); 10 mM to 40 mM of histidine / aspartate salt buffer; 0.2 to 1 mg / mL of poloxamer 188; and 100 mM to 300 mM of arginine.

[0049] The bispecific antibody Emicizumab (ACE910) is described below.

[0050] A bispecific antibody (Q499-z121 / J327-z119 / L404-k) 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 contains an H chain, which contains the amino acid sequences of H chain CDR 1, 2 and 3 of SEQ ID NO:1, 2 and 3 (H chain CDR of Q499); the second polypeptide contains an H chain, which contains the amino acid sequences of H chain CDR 1, 2 and 3 of SEQ ID NO:4, 5 and 6 (H chain CDR of J327); and the third polypeptide and the fourth polypeptide contain a common L chain, which contains the amino acid sequences of L chain CDR 1, 2 and 3 of SEQ ID NO:7, 8 and 9 (L chain CDR of L404).

[0051] More specifically, the above-mentioned bispecific antibody is a bispecific antibody in which 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 contains an H chain containing the amino acid sequence of the H chain variable region of SEQ ID NO:13; the second polypeptide contains an H chain containing the amino acid sequence of the H chain variable region of SEQ ID NO:14; and the third polypeptide and the fourth polypeptide share an L chain containing the amino acid sequence of the L chain variable region of SEQ ID NO:15.

[0052] More specifically, the aforementioned bispecific antibody is the following bispecific antibody (Q499-z121 / J327-z119 / L404-k), wherein 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 contains an H chain containing the amino acid sequence of SEQ ID NO:10; the second polypeptide contains an H chain containing the amino acid sequence of SEQ ID NO:11; and the third polypeptide and the fourth polypeptide contain a common L chain of SEQ ID NO:12. Such antibodies can be obtained by the methods described in WO2005 / 035756, WO2006 / 109592, WO2012 / 067176, etc.

[0053] The antibody concentration in the formulations of the present invention is not particularly limited, but is preferably between 20 mg / mL and 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 antibody concentration in the formulations of the present invention is not particularly limited, but is typically 250 mg / mL.

[0054] The antibodies used in this invention are not particularly limited, as long as they bind to the desired antigen, and they can be polyclonal or monoclonal antibodies. Monoclonal antibodies are preferred because they can stably produce homogeneous antibodies.

[0055] The amino acids contained in the amino acid sequence of this invention may be post-translational modified (e.g., modifying N-terminal glutamine to pyroglutamic acid by pyroglutamylation is well known to those skilled in the art). Of course, such post-translational modified amino acids are included in the antibodies used in this invention.

[0056] In this invention, the phrase "functionally substituted FVIII" means recognizing FIX or FIXa and recognizing FX, and promoting FX activation via FIXa (promoting FXa production via FIXa). The activity promoting FXa production can be assessed using, for example, a measurement system comprising FXIa, FX, the synthetic substrate S-2222 (a synthetic substrate of FXa), and phospholipids. Such a measurement system has shown correlation with disease severity and clinical symptoms in hemophilia A cases (Rosen S, Andersson M, Blombach M, et al. Clinical applications of a chromogenic substrate method for determination of FVIII activity. Thromb Haemost 1985; 54:811-23).

[0057] In this invention, the term "common L-chain" refers to an L-chain capable of pairing with each of two or more different H-chains and exhibiting binding ability against each antigen. Hereinafter, 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 with amino acid sequences that differ from each other. For example, a common L-chain can be obtained according to the method described in WO 2006 / 109592.

[0058] In this invention, the term "stable antibody-containing formulation" refers to a formulation in which it is difficult to generate aggregates and / or charge-heterogeneous components from proteins such as antibodies, i.e., a formulation in which it is difficult to undergo deterioration reactions (including insoluble aggregates, soluble aggregates, and charge-heterogeneous components) in solution.

[0059] "Components with charge heterogeneity" refers to protein surface charges that differ from those of the main component due to processes such as deamidation, oxidation, and hydrolysis.

[0060] In this invention, "polypeptide" generally refers to peptides and proteins with a length of about 10 amino acids or longer. Typically, they are biologically derived polypeptides, but are not particularly limited thereto, and can be, for example, polypeptides containing artificially designed sequences. Furthermore, they can be any naturally occurring polypeptide, synthetic polypeptide, recombinant polypeptide, etc. Additionally, fragments of the aforementioned polypeptides are also included in the polypeptides of this invention.

[0061] The term “antibody” is used in the broadest sense and includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), antibody derivatives, and modified antibodies (Miller K et al. J Immunol. 2003, 170(9), 4854-61), provided they exhibit the desired biological activity. Antibodies can be mouse antibodies, human antibodies, humanized antibodies, chimeric antibodies, or antibodies derived from other species, or synthetically produced antibodies. The antibodies disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules. Immunoglobulins can be derived from any species (e.g., human, mouse, or rabbit). The terms “antibody,” “immunoglobulin,” and “immunoglobulin” are used interchangeably in a broad sense.

[0062] A "bispecific antibody" is an antibody with two variable regions, each recognizing a different epitope, wherein the variable regions are present in the same antibody molecule. Bispecific antibodies can be antibodies that recognize two or more different antigens, or antibodies that recognize two or more different epitopes on the same antigen. Bispecific antibodies can include not only intact antibodies but also antibody derivatives.

[0063] Recombinant antibodies, produced using genetic engineering techniques, can be used as antibodies. Recombinant antibodies can be obtained by cloning the DNA encoding the antibody from a hybridoma or antibody-producing cell (e.g., antibody-sensitized lymphocytes), inserting it into a vector, and then introducing it into a host (host cell) to produce antibodies.

[0064] Bispecific antibodies are not limited to the IgG type; for example, IgG bispecific antibodies can be secreted from hybridomas (tetraploids) 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 co-expressed genes into the cell from the L-chain and H-chain genes that make up the two target IgGs, i.e., a total of four genes.

[0065] The antibodies of the present invention can be produced using methods known to those skilled in the art. Specifically, DNA encoding the target antibody is inserted into an expression vector. The insertion of the expression vector is performed such that expression is controlled by expression regulatory regions, such as enhancers and promoters. Next, host cells are transformed using the expression vector to express the antibody. In this case, a suitable combination of host and expression vector can be used.

[0066] The antibodies obtained in this way can be isolated from or outside host cells (culture medium, etc.) and purified into substantially pure homogeneous antibodies. Antibodies can be isolated and purified using methods commonly used for antibody isolation and purification, and the methods are not limited in any way. For example, antibodies can be isolated and purified by appropriate selection and combination of column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc.

[0067] In a preferred aspect, the histidine / aspartate salt buffer in the formulation of the present invention is a buffer prepared by titrating a solution, for example, an aqueous solution supplemented with histidine as a free amino acid, with a liquid, such as an aqueous solution containing an aqueous solution of aspartic acid as a free amino acid. Alternatively, the buffer can be prepared by adding amino acids in reverse order, or by direct titration with powder.

[0068] The inventors conducted freeze-thaw tests, accelerated heat treatment tests, long-term storage tests, and frozen storage tests to evaluate the effects of various additives on the stability of samples containing the aforementioned bispecific antibodies during storage. As a result, the inventors found that, compared to phosphate buffer, citrate buffer, and acetate buffer, the use of histidine buffer inhibited aggregate formation and / or components with charge heterogeneity.

[0069] Furthermore, the inventors discovered that aggregate formation and / or components with charge heterogeneity are inhibited by using aspartic acid (which is an acidic amino acid and serves as a counterion species for buffering), i.e., by using a histidine / aspartate salt buffer as a buffer.

[0070] The concentration (amount) of histidine / aspartate buffer in the formulation of the present invention is preferably 10 to 100 mM, more preferably 10 to 40 mM. Examples of histidine / aspartate buffer concentrations (amounts) are 10 mM, 20 mM, and 40 mM.

[0071] Furthermore, compared with sodium chloride, which has been reported as a stabilizer for antibody-containing preparations, the addition of arginine was found to exhibit a higher stabilizing effect (i.e., the effect of inhibiting aggregate formation and the effect of inhibiting components with charge heterogeneity).

[0072] The concentration (amount) of arginine in the formulation of the present invention is preferably from 100 mM to 300 mM. Examples of arginine concentrations (amounts) include 100 mM, 150 mM, 200 mM and 300 mM.

[0073] The solution pH 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.

[0074] The surfactants included in the formulations of the present invention are, for example, polysorbate 20 (PS20) and Pronick F-68 (poloxamer 188: polyethylene (160) polyoxypropylene (30) glycol), with poloxamer 188 being particularly preferred. The amount of poloxamer 188 (or PX188) added to the formulations of the present invention is preferably from 0.2 mg / mL to 1 mg / mL. Examples of amounts of poloxamer 188 added to the formulations include 0.2 mg / mL, 0.5 mg / mL, 0.8 mg / mL, and 1 mg / mL.

[0075] The histidine used in this invention can be histidine itself or a derivative thereof, with L-histidine being particularly preferred. The arginine used in this invention can be arginine itself, its derivatives, or its salts, with L-arginine or its salts being particularly preferred. Preferred arginine salts include aspartate and glutamate.

[0076] The formulations of the present invention may also contain amino acids. Preferred amino acids used in the present invention are natural amino acids or amino acid derivatives, with particularly preferred amino acids being L-methionine and L-proline.

[0077] The formulations of the present invention may further contain sugars. Preferred sugars for use in the present invention are sucrose, trehalose, meglumine, and sorbitol.

[0078] The amount of amino acids or sugars added to the formulations of the present invention is typically from 1 mM to 1000 mM, preferably from 5 mM to 500 mM, and more preferably from 10 mM to 300 mM.

[0079] The formulations of the present invention may also contain inorganic salts. Preferred inorganic salts for use in the present invention are magnesium salts and calcium salts.

[0080] Furthermore, the formulations of the present invention preferably do not contain counterions other than aspartic acid, which serve as buffers or stabilizers. In one aspect, examples of such formulations include those substantially free of chloride or acetate ions. "Substantially free of chloride or acetate ions" means that the concentrations of chloride and acetate ions are, for example, 5 mM or lower, preferably 2 mM or lower, more preferably 1 mM or lower. By using aspartic acid, which has a large stabilizing effect, as a counterion and substantially free of chloride or acetate ions, which have a small stabilizing effect, highly stable antibody-containing formulations can be produced without increasing osmotic pressure.

[0081] If desired, the formulations of the present invention may additionally contain suitable cryoprotectants, suspending agents, solubilizers, isotonic agents, preservatives, adsorption inhibitors, diluents, excipients, pH adjusters, analgesics, sulfur-containing reducing agents, antioxidants, etc.

[0082] Cryoprotectants include, for example, sugars such as trehalose, sucrose, and sorbitol.

[0083] Solubilizers include, for example, polyoxyethylene cured castor oil, polysorbate 80, nicotinamide, polyoxyethylene dehydrated sorbitol monolaurate, polyethylene glycol, and castor oil fatty acid ethyl ester.

[0084] Isotonic agents include, for example, sodium chloride, potassium chloride, and calcium chloride.

[0085] Preservatives include, for example, methylparaben, ethylparaben, sorbic acid, phenol, cresol, and chlorocresol.

[0086] Adsorption inhibitors include, for example, human serum albumin, lecithin, dextran, ethylene oxide / propylene oxide copolymer, hydroxypropyl cellulose, methylcellulose, polyoxyethylene-cured castor oil, and polyethylene glycol.

[0087] Sulfur-containing reducing agents include, for example, those containing a thiol group, such as N-acetylcysteine, N-acetylhomocysteine, lipoic acid, thiodiethylene glycol, thioethanolamine, thioglycerol, thiosorbose, thioglycolic acid and its salts, sodium thiosulfate, glutathione, and thioalkyl acids having one to seven carbon atoms.

[0088] 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, trimethyl gallate, propyl gallate, and chelating agents such as disodium ethylenediaminetetraacetate (EDTA), sodium pyrophosphate, and sodium metaphosphate.

[0089] In the implementation scheme, the formulation of the present invention is as follows:

[0090] An antibody solution formulation with pH 6, comprising:

[0091] A bispecific antibody at concentrations of 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 contains an H chain containing the amino acid sequence of SEQ ID NO:10; the second polypeptide contains an H chain containing the amino acid sequence of SEQ ID NO:11; and the third polypeptide and the fourth polypeptide contain a common L chain of SEQ ID NO:12.

[0092] 20 mM L-histidine / aspartate buffer;

[0093] 0.5 mg / mL poloxamer 188; and

[0094] 150mM L-arginine;

[0095] or

[0096] An antibody solution formulation with pH 6, comprising:

[0097] Emicizumab (ACE910), a bispecific antibody, at concentrations ranging from 20 to 180 mg / mL.

[0098] 20 mM L-histidine / aspartate buffer;

[0099] 0.5 mg / mL poloxamer 188; and

[0100] 150mM L-arginine.

[0101] In another embodiment, the formulation of the present invention is as follows:

[0102] An antibody solution formulation with pH 6, comprising:

[0103] A bispecific antibody at concentrations of 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 contains an H chain containing the amino acid sequence of SEQ ID NO:10; the second polypeptide contains an H chain containing the amino acid sequence of SEQ ID NO:11; and the third polypeptide and the fourth polypeptide contain a common L chain of SEQ ID NO:12.

[0104] 20 mM L-histidine / aspartate buffer;

[0105] 0.05 mg / mL PS20; and

[0106] 150mM L-arginine;

[0107] or

[0108] An antibody solution formulation with pH 6, comprising:

[0109] Emicizumab (ACE910), a bispecific antibody, at concentrations ranging from 20 to 180 mg / mL.

[0110] 20 mM L-histidine / aspartate buffer;

[0111] 0.05 mg / mL PS20; and

[0112] 150mM L-arginine.

[0113] The antibody-containing formulation of the present invention can be administered to a patient via any suitable route, such as by bolus injection or continuous infusion over a period of time, intravenously, intramuscularly, or subcutaneously. Intravenous or subcutaneous administration is preferred.

[0114] The dosage of Emicizumab (ACE910) is, for example, 0.001 to 1000 mg / kg, administered at intervals of at least one day or longer.

[0115] More specifically, for example, after administering Emicizumab (ACE910) at an initial dose of 1 mg / kg, Emicizumab (ACE910) may 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) may 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) may be administered at a continuous dose of 3 mg / kg once weekly.

[0116] The antibody-containing formulations of the present invention can be used for diseases that occur and / or progress due to reduced or absent FVIII activity and / or activated coagulation factor VIII (FVIIIa). For example, they can be used for hemophilia A, including hemophilia A for which inhibitors against FVIII / FVIIIa have been developed, acquired hemophilia A, von Willebrand's disease, but are not particularly limited thereto.

[0117] 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 adding histidine / aspartate buffer, poloxamer 188, and arginine to the solution.

[0118] Another embodiment of the present invention is a method for reducing antibody association (aggregate formation) in antibody-containing solution formulations. Preferably, the method for reducing antibody association (aggregate formation) in antibody-containing solution formulations includes adding histidine / aspartate salt buffer, poloxamer 188, and arginine to the solution.

[0119] Furthermore, the methods for stabilizing antibodies and reducing antibody association (aggregate formation) described above include adding histidine / aspartate buffer, poloxamer 188, and arginine to the solution. Preferably, the antibody concentration is 20-180 mg / mL, the histidine / aspartate buffer concentration is 10 mM to 40 mM, the poloxamer 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 more preferably, the antibody concentration is 20 to 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 is 6.

[0120] Another embodiment of the present invention is a method for reducing components with charge heterogeneity in an antibody-containing formulation. Preferably, the method for reducing components with charge heterogeneity in an antibody-containing formulation includes adding a histidine / aspartate buffer solution to the solution. More preferably, the method for reducing components with charge heterogeneity in an antibody-containing formulation includes adding a histidine / aspartate buffer solution to the solution, wherein the concentration of the histidine / aspartate buffer solution is 10 mM-40 mM, or 20 mM.

[0121] In another embodiment of the invention, a method for reducing the charge heterogeneity of components in an antibody-containing formulation includes adding histidine / aspartate buffer, poloxamer 188, and arginine to a solution. More preferably, the method for reducing the charge heterogeneity of components in an antibody-containing formulation includes adding histidine / aspartate buffer, poloxamer 188, and arginine to a solution, wherein the antibody concentration is preferably 20-180 mg / mL, the histidine / aspartate buffer concentration is 10 mM to 40 mM, the poloxamer 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 more preferably, the antibody concentration is 20 to 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 is 6.

[0122] In the above-described methods for stabilizing antibodies, reducing antibody association (aggregate formation), and reducing components with charge heterogeneity, the antibody is preferably a bispecific antibody, more preferably Emicizumab (ACE910).

[0123] As used herein, aspects referred to by the expression “contains” include those referenced by the expression “consistently of…” as well as those referenced by the expression “consisting of…”.

[0124] The numerical values ​​described herein may vary within a certain range, for example, depending on the instrument or equipment, measurement conditions, and procedures used by those skilled in the art, and may contain deviations of approximately 10%, as long as they are within the range that allows the objectives of the invention to be achieved.

[0125] All patents and references explicitly cited herein are incorporated herein by reference in their entirety.

[0126] The invention is further illustrated by the following embodiments, but should not be construed as being limited thereto. [Example]

[0127] [Example 1]

[0128] Histidine's effect on aggregate inhibition during the thermally accelerated storage of humanized IgG4 antibody ACE910

[0129] (1) Materials

[0130] ACE910 is a bispecific humanized IgG4 antibody that recognizes blood coagulation factor IX and blood coagulation factor X. It is expected to prevent bleeding in hemophilia A by functionally replacing activated coagulation factor VIII.

[0131] (2) Test Sample

[0132] Prepare a liquid composition at pH 6.0 containing 100 mg / mL ACE910, 150 mmol / L NaCl, and any one of the following buffers: 20 mmol / L phosphate buffer; 20 mmol / L citrate buffer; 20 mmol / L acetate buffer; or 20 mmol / L histidine buffer. Dispense 5 to 15 μL of the composition into separate glass vials.

[0133] The solution containing humanized antibodies prepared therefrom was left to stand in a 25°C heat-conditioning bath for 8 weeks before being used as a test sample.

[0134] (3) Methods for measuring and calculating ACE910 aggregate size

[0135] The amount of aggregates in the sample was determined by size exclusion chromatography (SEC) using a G3000SW. XLThe measurement was performed using a Tosoh column, with phosphate buffer (50 mmol / L, pH 7.0) containing 300 mmol / L sodium chloride as the mobile phase at a flow rate of 0.5 mL / min.

[0136] Among the detected peaks, the peak with the largest area and height is identified as the monomer, and the peaks detected earlier than the monomers are collectively referred to as aggregate peaks (high molecular weight substances, HMWS).

[0137] Calculate the peak area of ​​all peaks, and use the following equation to calculate the peak area ratio of the target peak:

[0138] The peak area ratio of the target peak (%) = 100 × (peak area of ​​the target peak) / (peak area of ​​the target peak + total peak area of ​​other peaks)

[0139] (4) Results

[0140] The results are shown in Table 1.

[0141] [Table 1]

[0142] The percentage increase in aggregate size after storage at 25°C.

[0143]

[0144] As can be clearly seen from Table 1, when histidine was supplemented at 20 mmol / L, the samples showed a high aggregation inhibition effect after thermal acceleration at 25°C for 8 weeks.

[0145] [Example 2]

[0146] The effect of salt concentration and arginine on aggregate inhibition during heat-accelerated storage and freeze-thaw cycles of humanized IgG4 antibody ACE910

[0147] (1) Materials

[0148] Use the antibody described in Example 1.

[0149] (2) Test Sample

[0150] Prepare a liquid composition with pH 6.0 containing 100 mg / mL ACE910, 20 mmol / L histidine, and any one of the following additives: 50 mmol / L NaCl; 75 mmol / L NaCl; 150 mmol / L NaCl; and 150 mmol / L arginine. Dispense 5 to 15 μL of the composition into glass vials.

[0151] The solution formulation containing humanized antibodies prepared therefrom is left to stand in a 25°C heat-conditioning bath for 8 weeks, or subjected to 10 freeze-thaw cycles (F / T) (5°C / -20°C), and then used as a test sample.

[0152] (3) Methods for measuring and calculating ACE910 aggregate size

[0153] The method is performed as described in Example 1.

[0154] (4) Results

[0155] The results are shown in Table 2.

[0156] [Table 2]

[0157] The percentage of aggregates increased after storage at 25°C and after freeze-thaw cycles.

[0158]

[0159] As can be clearly seen from Table 2, when arginine was supplemented at 150 mmol / L, the samples showed a high aggregate inhibition effect after 8 weeks of accelerated thermal testing at 25°C and freeze-thaw cycles.

[0160] [Example 3]

[0161] The aggregation inhibition effect of aspartic acid during the freeze-thaw process of humanized IgG4 antibody ACE910

[0162] (1) Materials

[0163] Use the antibody described in Example 1.

[0164] (2) Test Sample

[0165] Prepare a liquid composition with pH 6.0 containing 100 mg / mL ACE910, 20 mmol / L histidine, and 150 mmol / L NaCl or 150 mmol / L sodium L-aspartate as an anti-counterion. Dispense 5 to 15 μL of the composition into separate glass vials.

[0166] The solution formulation containing humanized antibodies prepared therefrom was subjected to 10 freeze-thaw cycles (5℃ / -20℃) and then used as a test sample.

[0167] (3) Methods for measuring and calculating ACE910 aggregate size

[0168] The method is performed as described in Example 1.

[0169] (4) Results

[0170] The results are shown in Table 3.

[0171] [Table 3]

[0172] The percentage increase in aggregates after freeze-thaw cycles.

[0173]

[0174] As can be clearly seen from Table 3, when aspartic acid was supplemented, the samples showed a high aggregate inhibition effect after freeze-thaw.

[0175] [Example 4]

[0176] The effect of pH on inhibiting aggregates and components with charge heterogeneity during the heat-accelerated storage of humanized IgG4 antibody ACE910.

[0177] (1) Materials

[0178] Use the antibody described in Example 1.

[0179] (2) Test Sample

[0180] Prepare a liquid composition containing 100 mg / mL ACE910, 20 mmol / L histidine-aspartic acid, and 150 mmol / L arginine-aspartic acid, with a pH of 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, or 7.5. Dispense 5 to 15 μL of the composition into glass vials.

[0181] The solution containing humanized antibodies prepared therefrom was left to stand in a 25°C heat-conditioning bath for 8 weeks before being used as a test sample.

[0182] (3) Methods for measuring and calculating ACE910 aggregate size

[0183] The method is performed as described in Example 1.

[0184] (4) Methods for measuring and calculating ACE910 components with charge heterogeneity

[0185] The amount of charged heterogeneous components in a sample was measured by ion exchange chromatography (IEC) using a BioPro QA-F column (YMC), with 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.

[0186] Among the detected peaks, the peak with the largest area and height is identified as the main peak, and the peaks detected after the main peak are collectively referred to as acid peaks.

[0187] Calculate the peak area of ​​all peaks, and use the following equation to calculate the peak area ratio of the target peak:

[0188] The peak area ratio of the target peak (%) = 100 × (peak area of ​​the target peak) / (peak area of ​​the target peak + total peak area of ​​other peaks)

[0189] (5) Results

[0190] The results are shown in Table 4.

[0191] [Table 4]

[0192] The percentage increase in aggregate size and the percentage increase in acid peak-1 were observed after storage at 25°C.

[0193]

[0194] As can be clearly seen from Table 4, the samples at pH 4.5 to 6.5, especially at pH 5.5 and pH 6.0, showed a high effect in inhibiting aggregates and components with charge heterogeneity after storage at 25°C.

[0195] [Example 5]

[0196] The effect of histidine concentration on inhibiting aggregates and components with charge heterogeneity during the heat-accelerated storage of humanized IgG4 antibody ACE910.

[0197] (1) Materials

[0198] Use the antibody described in Example 1.

[0199] (2) Test Sample

[0200] Prepare a liquid composition at pH 6.0 containing 100 mg / mL ACE910, 150 mmol / L arginine, and 5 mmol / L, 10 mmol / L, 20 mmol / L, or 40 mmol / L histidine-aspartic acid. Dispense 5 to 15 μL of the composition into glass vials.

[0201] The solution containing humanized antibodies prepared therefrom was left to stand in a 25°C heat-conditioning bath for 8 weeks before being used as a test sample.

[0202] (3) Methods for determining and calculating ACE910 aggregate size

[0203] The method is performed as described in Example 1.

[0204] (4) Methods for measuring and calculating ACE910 components with charge heterogeneity

[0205] The method is performed as described in Example 4.

[0206] (5) Results

[0207] The results are shown in Table 5.

[0208] [Table 5]

[0209] The percentage increase in aggregate size and the percentage increase in acid peak-1 were observed after storage at 25°C.

[0210]

[0211] As can be clearly seen from Table 5, samples containing 10 mmol / L or more of histidine-aspartic acid showed a high effect in inhibiting aggregates and components with charge heterogeneity after storage at 25°C.

[0212] [Example 6]

[0213] The effect of arginine concentration on aggregate inhibition during freeze-thaw, heat-accelerated storage, and cryogenic storage of humanized IgG4 antibody ACE910

[0214] (1) Materials

[0215] Use the antibody described in Example 1.

[0216] (2) Test Sample

[0217] Prepare a liquid composition with pH 6.0 containing 100 mg / mL ACE910, 20 mmol / L histidine-aspartic acid, and 75 mmol / L, 100 mmol / L, 150 mmol / L, 200 mmol / L, or 300 mmol / L arginine. Dispense 5 to 15 μL of the composition into glass vials.

[0218] The solution formulation containing humanized antibodies prepared therefrom is subjected to 10 freeze-thaw cycles (5°C / -20°C), or left to stand in a 25°C heat-controlled bath for 8 weeks or at -20°C for 6 months, and then used as a test sample.

[0219] (3) Methods for measuring and calculating ACE910 aggregate size

[0220] The method is performed as described in Example 1.

[0221] (4) Results

[0222] The results are shown in Table 6.

[0223] [Table 6]

[0224] The percentage increase in aggregates after freeze-thaw cycles, storage at 25°C, and storage at 20°C.

[0225]

[0226] As shown in Table 6, samples containing 100 mmol / L or more of arginine exhibited high aggregate inhibition effects after freeze-thaw cycles, storage at 25°C, and storage at -20°C.

[0227] [Example 7]

[0228] The effect of poloxamer 188 on inhibiting insoluble foreign matter and insoluble microparticles during storage of humanized IgG4 antibody ACE91 at 5℃.

[0229] (1) Materials

[0230] Use the antibody described in Example 1.

[0231] (2) Test Sample

[0232] Prepare a liquid composition with pH 6.0 containing 80 mg / mL ACE910, 20 mmol / L histidine-aspartic acid, 150 mmol / L arginine, and any one of the following additives: 0 mg / mL poloxamer 188; 0.2 mg / mL poloxamer 188; 0.5 mg / mL poloxamer 188; 1.0 mg / mL poloxamer 188; 0.05 mg / mL polysorbate 20; and 1.0 mg / mL polysorbate 20. Dispense 1.0 mL of the composition into separate glass vials.

[0233] The solution preparation containing humanized antibodies was thus stored in a refrigerator at 5°C for 5 months and then used as a test sample.

[0234] (3) Methods for observing insoluble foreign matter

[0235] The presence of insoluble foreign matter is assessed by placing the sample on the sample platform of a visual inspection table for vials, rotating the sample platform, and observing the vials.

[0236] (4) Methods for measuring insoluble particles

[0237] The number of insoluble particles in the solution was determined using a liquid particle counter (Hach Ultra Analytics, Model 9703).

[0238] (5) Results

[0239] The results are shown in Table 7.

[0240] [Table 7]

[0241]

[0242] As can be clearly seen from Table 7, samples containing 0.05 mg / mL PS20 and samples containing 0.2 mg / mL or higher poloxamer 188 showed a high effect in inhibiting the formation of insoluble particles and insoluble foreign matter after storage at 5°C.

[0243] [Example 8]

[0244] The effect of poloxamer 188 on inhibiting insoluble foreign matter and insoluble microparticles during oscillation stress and freeze-thaw storage of humanized IgG4 antibody ACE910

[0245] (1) Materials

[0246] Use the antibody described in Example 1.

[0247] (2) Test Sample

[0248] Prepare a liquid composition with pH 6.0 containing 150 mg / mL ACE910, 20 mmol / L histidine-aspartic acid, 150 mmol / L arginine-aspartic acid, and any one of the following additives: 0 mg / mL poloxamer 188; 0.2 mg / mL poloxamer 188; 0.5 mg / mL poloxamer 188; and 0.8 mg / mL poloxamer 188. Dispense 0.9 mL of the composition into separate glass vials.

[0249] The solution containing the humanized antibody prepared in this way was shaken at 200 times / minute for 24 hours at room temperature, or 10 freeze-thaw cycles (5℃ / -20℃), and then used as a test sample.

[0250] (3) Methods for observing insoluble matter

[0251] The method is performed as described in Example 7.

[0252] (4) Methods for measuring insoluble particles

[0253] The method is performed as described in Example 7.

[0254] (5) Results

[0255] The results are shown in Table 8 and Figure 1 and 2 middle.

[0256] [Table 8]

[0257] Detection rate (%) of insoluble foreign matter after shaking and freeze-thaw storage

[0258]

[0259] From Table 8 and Figure 1and 2 It can be clearly seen that samples containing 0.2 mg / mL or higher of poloxamer 188 showed a high effect in inhibiting the formation of insoluble particles and insoluble foreign matter after being subjected to oscillating stress and freeze-thaw storage.

[0260] [Example 9]

[0261] Effect of humanized IgG4 antibody ACE910 concentration on stability during heat-accelerated storage and freeze-thaw storage

[0262] (1) Materials

[0263] Use the antibody described in Example 1.

[0264] (2) Test Sample

[0265] A liquid composition with pH 6.0 was prepared, containing 20 mmol / L histidine-aspartic acid, 150 mmol / L arginine-aspartic acid, 0.5 mg / mL poloxamer 188, and 20 mg / mL, 30 mg / mL, 40 mg / mL, 120 mg / mL, 150 mg / mL, or 180 mg / mL ACE910. 0.65 mL of the composition was dispensed into separate glass vials.

[0266] The solution formulation containing humanized antibodies prepared therefrom is left to stand in a 40°C thermoregulatory bath for 8 weeks, or subjected to 5 or 10 freeze-thaw cycles (25°C / -20°C), and then used as a test sample.

[0267] (3) Methods for measuring and calculating ACE910 aggregate size

[0268] The method is performed as described in Example 1.

[0269] (4) Methods for measuring and calculating ACE910 components with charge heterogeneity

[0270] The amount of charged heterogeneous components in a sample was measured by anion exchange chromatography (AIEC) using a TSKgel Q-STAT column (Waters). The mobile phase A was Tris-HCl buffer (50 mmol / L, pH 8.0) and the mobile phase B was Tris-HCl buffer (50 mmol / L, pH 8.0) containing sodium chloride (200 mmol / L). The flow rate was 0.5 mL / min.

[0271] Among the detected peaks, the peak with the largest area and height is identified as the main peak, and the peaks detected before the main peak are collectively referred to as base peaks, while the peaks detected after the main peak are collectively referred to as acid peaks.

[0272] In addition, the amount of components with charge heterogeneity was measured by cation exchange chromatography (CIEC) using a ProPac WCX-10G column (Thermo Scientific). Mobile phase A was prepared with a buffer solution (pH 6.0) containing 9.6 mmol / L Tris, 6.0 mmol / L piperazine, and 11.0 mmol / L imidazole, and mobile phase B (pH 10.1) containing 9.6 mmol / L Tris, 6.0 mmol / L piperazine, 11.0 mmol / L imidazole, and 100 mmol / L NaCl. The flow rate was 0.5 mL / min.

[0273] Among the detected peaks, the peak with the largest area and height is identified as the BiAb peak, and the peaks detected before the BiAb peak are collectively referred to as the preceding peaks, and the peaks detected after the BiAb peak are collectively referred to as the following peaks.

[0274] Calculate the peak area of ​​all peaks, and use the following equation to calculate the peak area ratio of the target peak:

[0275] The peak area ratio of the target peak (%) = 100 × (peak area of ​​the target peak) / (peak area of ​​the target peak + total peak area of ​​other peaks)

[0276] (5) Results

[0277] The results are shown in Table 9. “SE”, “AE” and “CE” represent the results of size exclusion chromatography, anion exchange chromatography and cation exchange chromatography, respectively.

[0278] [Table 9]

[0279] The amount (%) of aggregates after storage at 40°C and after freeze-thaw cycles, and the amount (%) of components with charge heterogeneity.

[0280]

[0281] As can be clearly seen from Table 9, comparing samples containing ACE910 at concentrations ranging from 20 mg / mL to 180 mg / mL, the results show that the samples exhibit considerable and sufficient stability after storage at 40°C and after freeze-thaw cycles.

[0282] [Industrial Applicability]

[0283] Compared to conventional formulations, the antibody solution formulation of the present invention exhibits excellent stability in the solution state and shows inhibition of protein, such as antibody molecule, aggregation formation after storage at low temperatures, ambient temperatures, and high temperatures, as well as after freeze-thaw cycles. The antibody solution formulation of the present invention, which is resistant to deterioration, can be used, for example, for the treatment of hemophilia A by subcutaneous administration.

Claims

1. Use of an antibody solution formulation with pH 4.5 to 6.5 in the preparation of a medicament for treating hemophilia A, wherein said antibody solution formulation comprises: A bispecific antibody at a concentration of 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 is an H-chain with an amino acid sequence as shown in SEQ ID NO: 10; the second polypeptide is an H-chain with an amino acid sequence as shown in SEQ ID NO: 11, and the third polypeptide and the fourth polypeptide are a common L-chain with an amino acid sequence as shown in SEQ ID NO: 12; 20 mM histidine-aspartic acid buffer, where aspartic acid is the counterion used in the buffer. 0.5 to 1 mg / mL poloxamer 188; and 100 mM to 300 mM arginine.

2. The use of claim 1, wherein the concentration of poloxamer 188 is 0.5 mg / mL.

3. The use of claim 1, wherein the pH is 6.

4. The use of claim 1, wherein the concentration of arginine is 150 mM.

5. The use of claim 1, wherein the antibody solution formulation is substantially free of chloride or acetate ions, wherein being substantially free of chloride or acetate ions means that the concentrations of chloride and acetate ions are 5 mM or lower.

6. Use of a pH 6 antibody solution formulation in the preparation of a medicament for treating hemophilia A, wherein the antibody solution formulation comprises: A bispecific antibody at concentrations of 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 is an H-chain with an amino acid sequence as shown in SEQ ID NO: 10; the second polypeptide is an H-chain with an amino acid sequence as shown in SEQ ID NO: 11; and the third polypeptide and the fourth polypeptide are each a common L-chain with an amino acid sequence as shown in SEQ ID NO:

12. 20 mM histidine-aspartic acid buffer, where aspartic acid is the counterion used in the buffer. 0.5 mg / mL poloxamer 188; and 150 mM L-arginine.

7. The use of claim 1 or 6, wherein the drug is for subcutaneous administration.

8. A method for stabilizing an antibody in a solution containing an antibody, comprising adding histidine-aspartate buffer, poloxamer 188 and arginine to the solution, wherein the concentration of the histidine-aspartate buffer is 20 mM, wherein aspartic acid is an anti-ion species used in the buffer, the concentration of poloxamer 188 is 0.5 to 1 mg / mL, and the concentration of arginine is 100 mM to 300 mM, wherein the antibody is a bispecific antibody, 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 is an H chain with an amino acid sequence as shown in SEQ ID NO: 10; the second polypeptide is an H chain with an amino acid sequence as shown in SEQ ID NO: 11, and the third polypeptide and the fourth polypeptide are a common L chain with an amino acid sequence as shown in SEQ ID NO:

12.

9. A method for inhibiting antibody aggregate formation in an antibody-containing solution formulation, comprising adding histidine-aspartate buffer, poloxamer 188, and arginine to the solution, wherein the concentration of the histidine-aspartate buffer is 20 mM, wherein aspartic acid is an anti-ion species used in the buffer, the concentration of poloxamer 188 is 0.5 to 1 mg / mL, and the concentration of arginine is 100 mM to 300 mM, wherein the antibody is a bispecific antibody, 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 is an H chain with an amino acid sequence as shown in SEQ ID NO: 10; the second polypeptide is an H chain with an amino acid sequence as shown in SEQ ID NO: 11, and the third polypeptide and the fourth polypeptide are a common L chain with an amino acid sequence as shown in SEQ ID NO:

12.

10. A method for inhibiting a component with charge heterogeneity in an antibody-containing preparation, comprising adding a histidine-aspartic acid buffer to the solution, wherein the concentration of the histidine-aspartic acid buffer is 20 mM, wherein aspartic acid is an anti-ion species used in the buffer, wherein the antibody is a bispecific antibody, 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 is an H chain with an amino acid sequence as shown in SEQ ID NO: 10; the second polypeptide is an H chain with an amino acid sequence as shown in SEQ ID NO: 11, and the third polypeptide and the fourth polypeptide are a common L chain with an amino acid sequence as shown in SEQ ID NO: 12.

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