Method for preparing antibody-containing formulation

Abstract

To provide a pharmaceutical formulation that reduces the formation of particles including an anti-blood coagulation factor IXa / X antibody (bispecific monoclonal antibody) used as a substitution for coagulation factor VIII or an anti-IL-6 receptor antibody that inhibits binding with a receptor for interleukin 6.SOLUTION: Provided is a pharmaceutical formulation including an aqueous solution containing a polyoxyethylene polyoxypropylene glycol (poloxamer), the poloxamer being represented by formula I: HO(C2H4O)a(C3H6O)b(C2H4O)cH(I) [where, a and c each independently represent an integer selected from 75 to 85, b represents an integer selected from 22 to 33, and a, b, and c are each an average with respect to the whole of the poloxamer]. The poloxamer contains a poloxamer molecule including 34 or more of (C3H6O) within the molecule, at a proportion of 3%(w / w) or more with respect to the whole poloxamer.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to stable pharmaceutical formulations containing antibodies. [Background technology]

[0002] In recent years, various antibody preparations have been developed and put to practical use, but the formation of particles in aqueous solutions is a problem in antibody-containing preparations. The particles that are formed include sub-visible particles (SVPs), which are aggregates derived from antibodies and are generally difficult to see with the naked eye, and visible particles (VPs, larger than 100 μm), which can be detected visually at standard illuminance (approximately 2,000-3,000 lx). Although the visual detection rate of visible particles in pharmaceutical preparations varies greatly between practitioners, it has been reported that at the standard illuminance (approximately 2,000-3,000 lx) specified in the Pharmacopoeia, the detection sensitivity of particles with a diameter of 100 μm is approximately 40%, the detection sensitivity of particles with a diameter of 150 μm is approximately 70%, and the detection sensitivity of particles with a diameter of 200 μm is almost 100% (Non-Patent Document 1). Furthermore, by increasing the illuminance for observing the pharmaceutical preparation or lengthening the observation time, it is actually possible to visually detect particles with even smaller diameters, down to approximately 40 μm.

[0003] It is known that surfactants are used to reduce particle formation. Examples of such surfactants include nonionic surfactants, including poloxamers such as poloxamer 188 (PX188) and polysorbates such as polysorbate 20 and polysorbate 80. However, the ability to reduce particle formation varies depending on the type and grade of the surfactant (Non-Patent Documents 2-4, Patent Document 1). It is also known that even surfactants of the same type have heterogeneous polymer structures. Poloxamer 188 is known to vary between lots, and methods for providing more homogeneous poloxamer 188 are being studied (Non-Patent Document 2, Patent Documents 2-5). Furthermore, it has been reported that differences in hydrophobicity resulting from the heterogeneity of surfactants may affect particle formation (Non-Patent Document 2). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2020-534260 [Patent Document 2] Special Publication No. 2017-523828 [Patent Document 3] Special Publication No. 2019-511606 [Patent Document 4] Special Publication No. 2009-508132 [Patent Document 5] Special Publication No. 2014-502656 [Non-patent literature]

[0005] [Non-Patent Document 1] James A. Melchore, AAPS PharmSciTech; 2011; 12(1): 215-221. [Non-Patent Document 2] Chen et al., J. Chromatogr. A 1652 (2021) 462353. [Non-Patent Document 3] Grapentin et al., J. Pharm. Sci. 109 (2020) 2393-2404. [Non-Patent Document 4] Vaclaw et al., J. Pharm. Sci. 110 (2021) 746-759. Summary of the Invention [Problem to be solved by the invention]

[0006] It is not known what level of hydrophobicity of the surfactant is required to reduce particle formation in anti-blood coagulation factor IXa / X antibodies (bispecific monoclonal antibodies) that replace coagulation factor VIII or anti-IL-6 receptor antibodies that inhibit the binding of interleukin 6 to the receptor. In addition, the effects of the surface tension of a solution containing a surfactant and impurities contained in the surfactant (e.g., unreacted intermediate compounds having unsaturated bonds) on particle formation were unknown. There is a need for better surfactants to suppress particle generation. [Means for solving the problem]

[0007] The present inventors have found that the addition of a surfactant such as poloxamer, which contains a long polypropylene oxide block and a highly hydrophobic moiety, is effective in reducing particle formation in pharmaceutical formulations containing certain antibodies.

[0008] [1-1] A pharmaceutical preparation comprising an aqueous solution containing a monoclonal antibody selected from an antibody having an H chain of SEQ ID NO: 1 and 2 and an L chain of SEQ ID NO: 3, or an antibody having an H chain of SEQ ID NO: 4 and an L chain of SEQ ID NO: 5, and polyoxyethylene polyoxypropylene glycol (poloxamer), Poloxamers have the formula I: HO(C2H4O) a (C3H6O) b (C2H4O) c H (I) [In the formula, a and c are independently numbers selected from 75 to 85, b is a number selected from 22 to 40; a, b and c are average values ​​for the whole poloxamer] It is expressed as A pharmaceutical preparation in which the peak area at elution times after 17 minutes is 3% or more of the total peak area in high performance liquid chromatography under the conditions defined below: [High performance liquid chromatography conditions] (1) Column: HPLC column packed with macroporous styrene-divinylbenzene (1000 Å, 5 μm, 50 × 2.1 mm) (2) Mobile phase: Mobile phase A: Ultrapure water Mobile phase B: Acetonitrile (3) Elution gradient program From 0 min to 16.0 min: Mobile phase B 58% to 64% From 16.0 to 18.5 min: Mobile phase B 64% to 90% From 18.5 to 21.5 min: Fixed at 90% mobile phase B From 21.5 to 23.5 min: Mobile phase B 90% to 100% From 23.5 to 30.0 min: Fixed at 100% mobile phase B From 30.0 to 30.1 min: Mobile phase B 100% to 58% From 30.1 to 40.0 min: Mobile phase B fixed at 58% (4)Flow rate: 0.2mL / min (5) Detection method: Evaporative light scattering detection (drift tube temperature: 50±25°C, nebulizer heating power level: 75%, gain value: 250, gas pressure: 20 psi) (6) Column temperature: 65±5℃ (7) Poloxamer concentration (in ultrapure water): 0.5 mg / mL.

[0009] [1-2] The pharmaceutical formulation according to [1-1], wherein b is a number selected from 22 to 33. [1-3] The pharmaceutical formulation according to [1-1], wherein b is a number selected from 25 to 30. [1-4] The pharmaceutical formulation according to [1-1], wherein b is a number selected from 35 to 40.

[0010] [1-5] The pharmaceutical formulation according to any one of [1-1] to [1-4], wherein the peak area after 17 minutes is 6% or more, 19% or more, 33% or more, or 35% or more. [1-6] An aqueous solution comprising a monoclonal antibody selected from an antibody having an H chain of SEQ ID NO: 1 and 2 and an L chain of SEQ ID NO: 3, or an antibody having an H chain of SEQ ID NO: 4 and an L chain of SEQ ID NO: 5, and polyoxyethylene polyoxypropylene glycol (poloxamer). A pharmaceutical formulation comprising: Poloxamers have the formula I: HO(C2H4O) a (C3H6O) b (C2H4O) c H (I) [In the formula, a and c are independently numbers selected from 75 to 85, b is a number selected from 22 to 40; a, b and c are average values ​​for the whole poloxamer] It is expressed as A pharmaceutical preparation in which the peak area at elution times 17 minutes or later is 3% or more of the total peak area at elution times 1.5 minutes or later in high performance liquid chromatography under the conditions defined below: [High performance liquid chromatography conditions] (1) Column: HPLC column packed with macroporous styrene-divinylbenzene (1000 Å, 5 μm, 50 × 2.1 mm) (2) Mobile phase: Mobile phase A: Ultrapure water Mobile phase B: Acetonitrile (3) Elution gradient program From 0 min to 16.0 min: Mobile phase B 58% to 64% From 16.0 to 18.5 min: Mobile phase B 64% to 90% From 18.5 to 21.5 min: Fixed at 90% mobile phase B From 21.5 to 23.5 min: Mobile phase B 90% to 100% From 23.5 to 30.0 min: Fixed at 100% mobile phase B From 30.0 to 30.1 min: Mobile phase B 100% to 58% From 30.1 to 40.0 min: Mobile phase B fixed at 58% (4)Flow rate: 0.2mL / min (5) Detection method: Evaporative light scattering detection (drift tube temperature: 50±25°C, nebulizer heating power level: 75%, gain value: 250, gas pressure: 20 psi) (6) Column temperature: 65±5℃ (7) Poloxamer concentration (in ultrapure water): 0.5 mg / mL.

[0011] [1-7] The pharmaceutical formulation according to [1-6], wherein b is a number selected from 22 to 33. [1-8] The pharmaceutical formulation according to [1-6], wherein b is a number selected from 25 to 30. [1-9] The pharmaceutical formulation according to [1-6], wherein b is a number selected from 35 to 40.

[0012] [1-10] The pharmaceutical formulation according to any one of [1-6] to [1-9], wherein the peak area after 17 minutes is 6% or more, 20% or more, 36% or more, or 46% or more. [1-11] The pharmaceutical preparation according to any one of [1-1] to [1-10], wherein the HPLC column packed with macroporous styrenedivinylbenzene is a PLRP-S column.

[0013] [1-12] A pharmaceutical preparation comprising an aqueous solution containing a monoclonal antibody selected from an antibody having an H chain of SEQ ID NO: 1 and 2 and an L chain of SEQ ID NO: 3, or an antibody having an H chain of SEQ ID NO: 4 and an L chain of SEQ ID NO: 5, and polyoxyethylene polyoxypropylene glycol (poloxamer), Poloxamers have the formula I: HO(C2H4O) a (C3H6O) b (C2H4O) c H (I) [In the formula, a and c are independently numbers selected from 75 to 85, b is a number selected from 22 to 40; a, b and c are average values ​​for the whole poloxamer] It is expressed as A pharmaceutical preparation comprising poloxamer molecules containing 34 or more (CHO) units in the molecule at a ratio of 3% (w / w) or more relative to the total poloxamer.

[0014] [1-13] The pharmaceutical formulation according to [1-12], wherein b is a number selected from 22 to 33. [1-14] The pharmaceutical formulation according to [1-12], wherein b is a number selected from 25 to 30.

[0015] [1-15] The pharmaceutical formulation according to [1-12], wherein b is a number selected from 35 to 40. [1-16] A pharmaceutical preparation according to any one of [1-12] to [1-15], containing poloxamer molecules containing 34 or more (C3H6O) units in the molecule at a ratio of 6%, 20%, 29%, or 36% (w / w) or more relative to the total poloxamer.

[0016] [1-17] The pharmaceutical formulation according to any one of [1-1] to [1-16], wherein the number average molecular weight of the poloxamer is within the range of 7,680 to 9,510. [1-18] The pharmaceutical preparation according to any one of [1-1] to [1-17], wherein the concentration of poloxamer in the aqueous solution is 0.001 to 100 mg / mL, 0.01 to 10 mg / mL, 0.05 to 5 mg / mL, or 0.1 to 1 mg / mL.

[0017] [1-19] The pharmaceutical preparation according to any one of [1-1] to [1-18], wherein the concentration of the antibody in the aqueous solution is 10 to 300 mg / mL, 20 to 200 mg / mL, or 30 to 150 mg / mL.

[0018] [1-20] The pharmaceutical formulation according to any one of [1-1] to [1-19], wherein the aqueous solution contains one or more pharma- ceutically acceptable excipients selected from sugars, sugar alcohols, buffers, preservatives, carriers, antioxidants, chelating agents, natural polymers, synthetic polymers, cryoprotectants, bulking agents, and stabilizers.

[0019] [1-21] The pharmaceutical formulation according to any one of [1-1] to [1-20], wherein the poloxamer is poloxamer 188 or poloxamer 237. [2-1] A pharmaceutical preparation comprising a monoclonal antibody selected from an antibody having an H chain of SEQ ID NO: 1 and 2 and an L chain of SEQ ID NO: 3, or an antibody having an H chain of SEQ ID NO: 4 and an L chain of SEQ ID NO: 5, and an aqueous solution containing a surfactant, The pharmaceutical preparation, wherein the surfactant is a surfactant such that an aqueous solution containing the surfactant at a concentration of 0.5 mg / mL has a surface tension of 52.3 mN / m or less.

[0020] [2-2] The pharmaceutical formulation according to [2-1], wherein the surfactant is a surfactant having a surface tension of 52 mN / m or less, 51 mN / m or less, 50.7 mN / m or less, 50.5 mN / m or less, or 39 mN / m or less in an aqueous solution containing the surfactant at a concentration of 0.5 mg / mL.

[0021] [2-3] The pharmaceutical formulation according to [2-1] or [2-2], wherein the surfactant is selected from poloxamer or fatty acid polyoxyethylene sorbitan (polysorbate). [2-4] The pharmaceutical formulation according to any one of [2-1] to [2-3], wherein the surfactant is a poloxamer represented by formula I according to any one of [1-1] to [1-4] or [1-21].

[0022] [2-5] The pharmaceutical formulation according to [2-3], wherein the poloxamer is poloxamer 188 or poloxamer 237. [2-6] The pharmaceutical formulation according to [2-4] or [2-5], wherein the number average molecular weight of the poloxamer is within the range of 7680 to 9510.

[0023] [2-7] The pharmaceutical preparation according to any one of [2-1] to [2-6], wherein the surfactant is a polysorbate selected from polysorbate 20, polysorbate 60, polysorbate 65, or polysorbate 80.

[0024] [2-8] The pharmaceutical preparation according to any one of [2-1] to [2-7], wherein the surfactant is a sorbate selected from polysorbate 20 and polysorbate 80. [2-9] The pharmaceutical formulation according to any one of [2-1] to [2-8], wherein the surfactant is polysorbate 80.

[0025] [2-10] The pharmaceutical preparation according to any one of [2-1] to [2-9], wherein the concentration of the surfactant in the aqueous solution is 0.001 to 100 mg / mL, 0.01 to 10 mg / mL, 0.05 to 5 mg / mL, or 0.1 to 1 mg / mL.

[0026] [2-11] The pharmaceutical preparation according to any one of [2-1] to [2-10], wherein the concentration of the antibody in the aqueous solution is 10 to 300 mg / mL, 20 to 200 mg / mL, or 30 to 150 mg / mL.

[0027] [2-12] The pharmaceutical formulation according to any one of [2-1] to [2-11], wherein the aqueous solution contains one or more pharma- ceutically acceptable excipients selected from sugars, sugar alcohols, buffers, preservatives, carriers, antioxidants, chelating agents, natural polymers, synthetic polymers, cryoprotectants, bulking agents, and stabilizers.

[0028] [3-1] A pharmaceutical preparation comprising an aqueous solution containing a monoclonal antibody selected from an antibody having an H chain of SEQ ID NO: 1 and 2 and an L chain of SEQ ID NO: 3, or an antibody having an H chain of SEQ ID NO: 4 and an L chain of SEQ ID NO: 5, and polyoxyethylene polyoxypropylene glycol (poloxamer), Poloxamers have the formula I: HO(C2H4O) a (C3H6O) b (C2H4O) c H (I) [In the formula, a and c are independently numbers selected from 75 to 85, b is a number selected from 22 to 40; a, b and c are average values ​​for the whole poloxamer] It is expressed as A pharmaceutical formulation, wherein the poloxamer has a degree of unsaturation of less than 0.018 mEq / g.

[0029] [3-2] The pharmaceutical formulation according to [3-1], wherein b is a number selected from 22 to 33. [3-3] The pharmaceutical formulation according to [3-1], wherein b is a number selected from 25 to 30. [3-4] The pharmaceutical formulation according to [3-1], wherein b is a number selected from 35 to 40.

[0030] [3-5] A pharmaceutical formulation according to any one of [3-1] to [3-4], wherein the number average molecular weight of the poloxamer is within the range of 7,680 to 9,510. [3-6] The pharmaceutical preparation according to any one of [3-1] to [3-5], wherein the concentration of poloxamer in the aqueous solution is 0.001 to 100 mg / mL, 0.01 to 10 mg / mL, 0.05 to 5 mg / mL, or 0.1 to 1 mg / mL.

[0031] [3-7] The pharmaceutical preparation according to any one of [3-1] to [3-6], wherein the concentration of the antibody in the aqueous solution is 10 to 300 mg / mL, 20 to 200 mg / mL, or 30 to 150 mg / mL.

[0032] [3-8] Aqueous solutions of sugars, sugar alcohols, buffers, preservatives, carriers, antioxidants, chelates, etc. The pharmaceutical formulation according to any one of [3-1] to [3-7], further comprising one or more pharma- ceutical acceptable excipients selected from a lysing agent, a natural polymer, a synthetic polymer, a cryoprotectant, a bulking agent, and a stabilizer.

[0033] [3-9] The pharmaceutical formulation according to any one of [3-1] to [3-8], wherein the poloxamer is poloxamer 188 or poloxamer 237. [4-1] A method for reducing particle generation in an aqueous solution in a pharmaceutical preparation containing a monoclonal antibody selected from an antibody having an H chain of SEQ ID NO: 1 and 2 and an L chain of SEQ ID NO: 3, or an antibody having an H chain of SEQ ID NO: 4 and an L chain of SEQ ID NO: 5, comprising: adding polyoxyethylene polyoxypropylene glycol (poloxamer) to the aqueous solution; The poloxamer has formula I: HO(C2H4O) a (C3H6O) b (C2H4O) c H (I) [In the formula, a and c are independently numbers selected from 75 to 85, b is a number selected from 22 to 40; a, b and c are average values ​​for the whole poloxamer] and wherein the peak area at elution time after 17 minutes is 3% or more of the total peak area in high performance liquid chromatography under the conditions defined below, [High performance liquid chromatography conditions] (1) Column: HPLC column packed with macroporous styrene-divinylbenzene (1000 Å, 5 μm, 50 × 2.1 mm) (2) Mobile phase: Mobile phase A: Ultrapure water Mobile phase B: Acetonitrile (3) Elution gradient program From 0 min to 16.0 min: Mobile phase B 58% to 64% From 16.0 to 18.5 min: Mobile phase B 64% to 90% From 18.5 to 21.5 min: Fixed at 90% mobile phase B From 21.5 to 23.5 min: Mobile phase B 90% to 100% From 23.5 to 30.0 min: Fixed at 100% mobile phase B From 30.0 to 30.1 min: Mobile phase B 100% to 58% From 30.1 to 40.0 min: Mobile phase B fixed at 58% (4)Flow rate: 0.2mL / min (5) Detection method: Evaporative light scattering detection (drift tube temperature: 50±25°C, nebulizer heating power level: 75%, gain value: 250, gas pressure: 20 psi) (6) Column temperature: 65±5℃ (7) Poloxamer concentration (in ultrapure water): 0.5 mg / mL.

[0034] [4-2] A method for reducing particle generation in an aqueous solution in a pharmaceutical preparation containing a monoclonal antibody selected from an antibody having an H chain of SEQ ID NO: 1 and 2 and an L chain of SEQ ID NO: 3, or an antibody having an H chain of SEQ ID NO: 4 and an L chain of SEQ ID NO: 5, comprising: adding polyoxyethylene polyoxypropylene glycol (poloxamer) to the aqueous solution; The poloxamer has formula I: HO(C2H4O) a (C3H6O) b (C2H4O) c H (I) [In the formula, a and c are independently numbers selected from 75 to 85, b is a number selected from 22 to 40; a, b and c are average values ​​for the whole poloxamer] The elution time in high performance liquid chromatography under the conditions defined below is 1 adding to an aqueous solution a poloxamer having a peak area after 7 minutes that is 3% or more of the total peak area after 1.5 minutes; [High performance liquid chromatography conditions] (1) Column: HPLC column packed with macroporous styrene-divinylbenzene (1000 Å, 5 μm, 50 × 2.1 mm) (2) Mobile phase: Mobile phase A: Ultrapure water Mobile phase B: Acetonitrile (3) Elution gradient program From 0 min to 16.0 min: Mobile phase B 58% to 64% From 16.0 to 18.5 min: Mobile phase B 64% to 90% From 18.5 to 21.5 min: Fixed at 90% mobile phase B From 21.5 to 23.5 min: Mobile phase B 90% to 100% From 23.5 to 30.0 min: Fixed at 100% mobile phase B From 30.0 to 30.1 min: Mobile phase B 100% to 58% From 30.1 to 40.0 min: Mobile phase B fixed at 58% (4)Flow rate: 0.2mL / min (5) Detection method: Evaporative light scattering detection (drift tube temperature: 50±25°C, nebulizer heating power level: 75%, gain value: 250, gas pressure: 20 psi) (6) Column temperature: 65±5℃ (7) Poloxamer concentration (in ultrapure water): 0.5 mg / mL.

[0035] [4-3] The method according to [4-1] or [4-2], wherein the HPLC column packed with macroporous styrene-divinylbenzene is a PLRP-S column. [4-4] A method for reducing particle generation in an aqueous solution in a pharmaceutical preparation containing a monoclonal antibody selected from an antibody having an H chain of SEQ ID NO: 1 and 2 and an L chain of SEQ ID NO: 3, or an antibody having an H chain of SEQ ID NO: 4 and an L chain of SEQ ID NO: 5, comprising: adding polyoxyethylene polyoxypropylene glycol (poloxamer) to the aqueous solution; The poloxamer has formula I: HO(C2H4O) a (C3H6O) b (C2H4O) c H (I) [In the formula, a and c are independently numbers selected from 75 to 85, b is a number selected from 22 to 40; a, b and c are average values ​​for the whole poloxamer] and the poloxamer contains poloxamer molecules containing 34 or more (C3H6O) groups in the molecule at a ratio of 3% (w / w) or more relative to the total poloxamer.

[0036] [4-5] The method according to any one of [4-1] to [4-4], wherein b is a number selected from 22 to 33. [4-6] The method according to any one of [4-1] to [4-4], wherein b is a number selected from 25 to 30.

[0037] [4-7] The method according to any one of [4-1] to [4-4], wherein b is a number selected from 35 to 40. [4-8] The method according to any one of [4-1] to [4-7], wherein the poloxamer is poloxamer 188 or poloxamer 237.

[0038] [4-9] A method for reducing particle generation in an aqueous solution in a pharmaceutical preparation containing a monoclonal antibody selected from an antibody having an H chain of SEQ ID NO: 1 and 2 and an L chain of SEQ ID NO: 3, or an antibody having an H chain of SEQ ID NO: 4 and an L chain of SEQ ID NO: 5, comprising: A method comprising adding, to an aqueous solution, as a surfactant, a surfactant having a surface tension of 52.3 mN / m or less in an aqueous surfactant solution at a concentration of 0.5 mg / mL.

[0039] [4-10] The method according to [4-9], wherein the surfactant is selected from poloxamer or fatty acid polyoxyethylene sorbitan (polysorbate). [4-11] The method according to [4-9] or [4-10], wherein the surfactant is a poloxamer represented by formula I described in any one of [1-1] to [1-4] or [1-21].

[0040] [4-12] The method according to [4-11], wherein the number average molecular weight of poloxamer is in the range of 7680 to 9510. [4-13] The method according to any one of [4-9] to [4-12], wherein the surfactant is a polysorbate selected from polysorbate 20, polysorbate 60, polysorbate 65, or polysorbate 80.

[0041] [4-14] The method according to any one of [4-9] to [4-13], wherein the surfactant is a polysorbate selected from polysorbate 20 and polysorbate 80. [4-15] The method according to any one of [4-9] to [4-14], wherein the surfactant is polysorbate 80.

[0042] [4-16] The method according to any one of [4-1] to [4-15], wherein the surfactant is added to the aqueous solution to a concentration of 0.001 to 100 mg / mL, 0.01 to 10 mg / mL, 0.05 to 5 mg / mL, or 0.1 to 1 mg / mL.

[0043] [4-17] The method according to any one of [4-1] to [4-16], wherein the concentration of the antibody in the aqueous solution is 10 to 300 mg / mL, 20 to 200 mg / mL, or 30 to 150 mg / mL.

[0044] [4-18] The method according to any one of [4-1] to [4-17], wherein the aqueous solution contains one or more pharma- ceutically acceptable excipients selected from sugars, sugar alcohols, buffers, preservatives, carriers, antioxidants, chelating agents, natural polymers, synthetic polymers, cryoprotectants, bulking agents, and stabilizers.

[0045] [4-19] The method according to any one of [4-1] to [4-18], wherein the particles are derived from a protein. [4-20] The method according to any one of [4-1] to [4-19], wherein the particles have a particle size of 40 μm or more. [5-1] A pharmaceutical preparation comprising an aqueous solution containing a monoclonal antibody selected from an antibody having an H chain of SEQ ID NO: 1 and 2 and an L chain of SEQ ID NO: 3, or an antibody having an H chain of SEQ ID NO: 4 and an L chain of SEQ ID NO: 5, and polyoxyethylene polyoxypropylene glycol (poloxamer), A pharmaceutical formulation, wherein the number average molecular weight of the poloxamer is in the range of 7680 to 9510. [5-2] [5-1] The pharmaceutical formulation according to Poloxamers have the formula I: HO(C2H4O) a (C3H6O) b (C2H4O) c H (I) [In the formula, a and c are independently numbers selected from 60 to 85, b is a number selected from 22 to 40; a, b and c are average values ​​for the whole poloxamer] A pharmaceutical preparation represented by the formula: [5-3] The pharmaceutical formulation described in [5-2], wherein a and c are independently numbers selected from 60 to 68. [5-4] The pharmaceutical formulation according to [5-3], wherein b is a number selected from 22 to 33. [5-5] The pharmaceutical formulation according to [5-3], wherein b is a number selected from 25 to 30. [5-6] The pharmaceutical formulation according to [5-3], wherein b is a number selected from 35 to 40. [5-7] The pharmaceutical formulation described in [5-2], wherein a and c are independently numbers selected from 75 to 85. [5-8] The pharmaceutical formulation according to [5-7], wherein b is a number selected from 22 to 33. [5-9] The pharmaceutical formulation according to [5-7], wherein b is a number selected from 25 to 30. [5-10] The pharmaceutical formulation according to [5-7], wherein b is a number selected from 35 to 40. [5-11] The pharmaceutical formulation according to [5-2], wherein a and c are independently numbers selected from 75 to 85, and b is a number selected from 25 to 30. [5-12] The pharmaceutical formulation according to [5-2], wherein a and c are independently numbers selected from 60 to 68, and b is a number selected from 35 to 40. [5-13] The pharmaceutical preparation according to any one of [5-1] to [5-12], wherein the peak area at an elution time of 17 minutes or later is 3% or more of the total peak area at an elution time of 1.5 minutes or later in high performance liquid chromatography under the conditions defined below: [High performance liquid chromatography conditions] (1) Column: HPLC column packed with macroporous styrene-divinylbenzene (1000 Å, 5 μm, 50 × 2.1 mm) (2) Mobile phase: Mobile phase A: Ultrapure water Mobile phase B: Acetonitrile (3) Elution gradient program From 0 min to 16.0 min: Mobile phase B 58% to 64% From 16.0 to 18.5 min: Mobile phase B 64% to 90% From 18.5 to 21.5 min: Fixed at 90% mobile phase B From 21.5 to 23.5 min: Mobile phase B 90% to 100% From 23.5 to 30.0 min: Fixed at 100% mobile phase B From 30.0 to 30.1 min: Mobile phase B 100% to 58% From 30.1 to 40.0 min: Mobile phase B fixed at 58% (4)Flow rate: 0.2mL / min (5) Detection pharmaceutical formulation: Evaporative light scattering detection (drift tube temperature: 50±25°C, nebulizer heating power level: 75%, gain value: 250, gas pressure: 20 psi) (6) Column temperature: 65±5℃ (7) Poloxamer concentration (in ultrapure water): 0.5 mg / mL. [5-14] The pharmaceutical formulation according to [5-13], wherein the peak area at an elution time of 17 minutes or later is 3% or more of the total peak area in high performance liquid chromatography. [5-15] The pharmaceutical formulation according to [5-13] or [5-14], wherein the peak area after 17 minutes is 6% or more, 19% or more, 20% or more, 33% or more, 35% or more, 36% or more, or 46% or more. [5-16] The pharmaceutical preparation according to any one of [5-13] to [5-15], wherein the HPLC column packed with macroporous styrene-divinylbenzene is a PLRP-S column. [5-17] A pharmaceutical preparation according to any one of [5-1] to [5-16], wherein the poloxamer contains poloxamer molecules containing 34 or more (C3H6O) in the molecule at a ratio of 3% (w / w) or more relative to the total poloxamer. [5-18] A pharmaceutical preparation according to any one of [5-1] to [5-17], wherein the poloxamer contains poloxamer molecules containing 34 or more (C3H6O) in the molecule at a ratio of 6%, 20%, 29%, or 36% (w / w) or more relative to the total poloxamer. [5-19] The pharmaceutical preparation according to any one of [5-1] to [5-18], wherein the degree of unsaturation of poloxamer is less than 0.018 mEq / g. [5-20] The pharmaceutical formulation according to any one of [5-1] to [5-19], wherein the number average molecular weight of the poloxamer is within the range of 6840 to 8830. [5-21] The pharmaceutical preparation according to any one of [5-1] to [5-20], wherein the concentration of poloxamer in the aqueous solution is 0.001 to 100 mg / mL, 0.01 to 10 mg / mL, 0.05 to 5 mg / mL, or 0.1 to 1 mg / mL. [5-22] The pharmaceutical preparation according to any one of [5-1] to [5-21], wherein the concentration of the antibody in the aqueous solution is 10 to 300 mg / mL, 20 to 200 mg / mL, or 30 to 150 mg / mL. [5-23] The pharmaceutical formulation according to any one of [5-1] to [5-22], wherein the aqueous solution contains one or more pharma- ceutically acceptable excipients selected from sugars, sugar alcohols, buffers, preservatives, carriers, antioxidants, chelating agents, natural polymers, synthetic polymers, cryoprotectants, bulking agents, and stabilizers. [5-24] The pharmaceutical preparation according to any one of [5-1] to [5-23], wherein the poloxamer is poloxamer 188 or poloxamer 237. [6-1] A method for reducing particle generation in an aqueous solution in a pharmaceutical preparation containing a monoclonal antibody selected from an antibody having an H chain of SEQ ID NO: 1 and 2 and an L chain of SEQ ID NO: 3, or an antibody having an H chain of SEQ ID NO: 4 and an L chain of SEQ ID NO: 5, comprising: adding polyoxyethylene polyoxypropylene glycol (poloxamer) to the aqueous solution; A method in which the number average molecular weight of the poloxamer is in the range of 7680 to 9510. [6-2] [6-1], Poloxamers have the formula I: HO(C2H4O) a (C3H6O) b (C2H4O) c H (I) [In the formula, a and c are independently numbers selected from 60 to 85, b is a number selected from 22 to 40; a, b and c are average values ​​for the whole poloxamer] The method is represented by [6-3] The method according to [6-2], wherein a and c are independently numbers selected from 60 to 68. [6-4] The method according to [6-3], wherein b is a number selected from 22 to 33. [6-5] The method according to [6-3], wherein b is a number selected from 25 to 30. [6-6] The method according to [6-3], wherein b is a number selected from 35 to 40. [6-7] The method according to [6-2], wherein a and c are independently numbers selected from 75 to 85. [6-8] The method according to [6-7], wherein b is a number selected from 22 to 33. [6-9] The method according to [6-7], wherein b is a number selected from 25 to 30. [6-10] The method according to [6-7], wherein b is a number selected from 35 to 40. [6-11] The method according to [6-2], wherein a and c are independently numbers selected from 75 to 85, and b is a number selected from 25 to 30. [6-12] The method according to [6-2], wherein a and c are independently numbers selected from 60 to 68, and b is a number selected from 35 to 40. [6-13] A method according to any one of [6-1] to [6-12], comprising adding to an aqueous solution a poloxamer having a peak area at an elution time of 17 minutes or later of 3% or more of the total peak area at an elution time of 1.5 minutes or later in high performance liquid chromatography under the conditions defined below: [High performance liquid chromatography conditions] (1) Column: HPLC column packed with macroporous styrene-divinylbenzene (1000 Å, 5 μm, 50 × 2.1 mm) (2) Mobile phase: Mobile phase A: Ultrapure water Mobile phase B: Acetonitrile (3) Elution gradient program From 0 min to 16.0 min: Mobile phase B 58% to 64% From 16.0 to 18.5 min: Mobile phase B 64% to 90% From 18.5 to 21.5 min: Fixed at 90% mobile phase B From 21.5 to 23.5 min: Mobile phase B 90% to 100% From 23.5 to 30.0 min: Fixed at 100% mobile phase B From 30.0 to 30.1 min: Mobile phase B 100% to 58% From 30.1 to 40.0 min: Mobile phase B fixed at 58% (4)Flow rate: 0.2mL / min (5) Detection method: Evaporative light scattering detection (drift tube temperature: 50±25°C, nebulizer heating power level: 75%, gain value: 250, gas pressure: 20 psi) (6) Column temperature: 65±5℃ (7) Poloxamer concentration (in ultrapure water): 0.5 mg / mL. [6-14] The method according to [6-13], in which the peak area after an elution time of 17 minutes in high performance liquid chromatography is 3% or more of the total peak area. [6-15] The method according to [6-13] or [6-14], wherein the peak area after 17 minutes is 6% or more, 19% or more, 20% or more, 33% or more, 35% or more, 36% or more, or 46% or more. [6-16] The method according to any one of [6-13] to [6-15], wherein the HPLC column packed with macroporous styrene-divinylbenzene is a PLRP-S column. [6-17] A method according to any of [6-1] to [6-16], wherein the poloxamer contains poloxamer molecules containing 34 or more (C3H6O) groups in the molecule at a ratio of 3% (w / w) or more relative to the total poloxamer. [6-18] A method according to any of [6-1] to [6-17], wherein the poloxamer contains poloxamer molecules containing 34 or more (C3H6O) groups in the molecule at a ratio of 6%, 20%, 29%, or 36% (w / w) or more relative to the total poloxamer. [6-19] The method according to any of [6-1] to [6-18], wherein the degree of unsaturation of poloxamer is less than 0.018 mEq / g. [6-20] The method according to any of [6-1] to [6-19], wherein the number average molecular weight of the poloxamer is within the range of 6840 to 8830. [6-21] The method according to any one of [6-1] to [6-20], wherein the poloxamer is poloxamer 188 or poloxamer 237. [6-22] A method for reducing particle generation in an aqueous solution in a pharmaceutical preparation containing a monoclonal antibody selected from an antibody having an H chain of SEQ ID NO: 1 and 2 and an L chain of SEQ ID NO: 3, or an antibody having an H chain of SEQ ID NO: 4 and an L chain of SEQ ID NO: 5, comprising: A method comprising adding, to an aqueous solution, as a surfactant, a surfactant having a surface tension of 52.3 mN / m or less in an aqueous surfactant solution at a concentration of 0.5 mg / mL. [6-23] The method according to [6-22], wherein the surfactant is selected from poloxamer or fatty acid polyoxyethylene sorbitan (polysorbate). [6-24] The method according to [6-22] or [6-23], wherein the surfactant is a poloxamer represented by formula I described in any one of [6-2] to [6-12] or [6-21]. [6-25] The method according to any one of [6-22] to [6-24], wherein the surfactant is a polysorbate selected from polysorbate 20, polysorbate 60, polysorbate 65, or polysorbate 80. [6-26] The method according to any one of [6-22] to [6-25], wherein the surfactant is a polysorbate selected from polysorbate 20 and polysorbate 80. [6-27] The method according to any one of [6-22] to [6-25], wherein the surfactant is polysorbate 80. [6-28] The method according to any one of [6-1] to [6-27], wherein the surfactant is added to the aqueous solution to a concentration of 0.001 to 100 mg / mL, 0.01 to 10 mg / mL, 0.05 to 5 mg / mL, or 0.1 to 1 mg / mL. [6-29] The method according to any one of [6-1] to [6-28], wherein the concentration of the antibody in the aqueous solution is 10 to 300 mg / mL, 20 to 200 mg / mL, or 30 to 150 mg / mL. [6-30] The method according to any one of [6-1] to [6-29], wherein the aqueous solution contains one or more pharma- ceutically acceptable excipients selected from sugars, sugar alcohols, buffers, preservatives, carriers, antioxidants, chelating agents, natural polymers, synthetic polymers, cryoprotectants, bulking agents, and stabilizers. [6-31] The method according to any one of [6-1] to [6-30], wherein the particles are derived from a protein. [6-32] The method according to any one of [6-1] to [6-31], wherein the particles have a particle size of 40 μm or more. Effect of the Invention

[0046] According to one aspect of the present invention, there is provided a pharmaceutical formulation which comprises an aqueous solution containing an anti-blood coagulation factor IXa / X antibody (bispecific monoclonal antibody) which replaces coagulation factor VIII or an anti-IL-6 receptor antibody which inhibits the binding of interleukin-6 to its receptor, and which has reduced particle formation, and a surfactant. [Brief description of the drawings]

[0047] [Figure 1] FIG. 1 is a chromatogram showing the results of analysis of seven components of PX188 by reversed-phase chromatography. [Diagram 2] FIG. 2 is a graph showing the measurement results of the surface tension values ​​of seven types of PX188 and one type of PS80. [Diagram 3] FIG. 3 is a graph showing the correlation between the percentage of slow extractables in the components contained in PX188 and the surface tension value. [Figure 4] FIG. 4 shows the application of mechanical stress (including one or four sets of vibration stress). [Diagram 5] FIG. 5 shows the operation of applying mechanical stress. [Figure 6]FIG. 6 shows Raman spectra of representative particles consisting of only protein (Protein-only) and particles consisting of a complex of protein and polydimethylsiloxane (PDMS) (Protein-PDMS). [Figure 7] FIG. 7 is a graph showing the correlation between the percentage of slow elution of PX188 and the particle generation rate of mAb1 under heat stress conditions. [Figure 8] FIG. 8 is a graph showing the correlation between the percentage of slow eluents for PX188 and the incidence of visually detectable particles under heat stress conditions for mAb2. [Figure 9] FIG. 9 is a graph showing the correlation of PX188 % Slow Elutant, Unsaturation, and Synthesis variables, respectively, with the incidence of visually detectable particles under mechanical stress conditions for mAb1. [Figure 10] FIG. 10 is a graph showing the correlation of the percent late eluent, unsaturation, and synthesis variables of PX188, respectively, with the incidence of visually detectable particles under mechanical stress conditions for mAb2. [Figure 11] FIG. 11 is an image of antibody protection by interfacial adsorption of PX188. [Figure 12] FIG. 12 is a graph of the surface tension values ​​of PX188. [Figure 13] FIG. 13 shows the results of component analysis of PX237 by reverse phase chromatography. [Figure 14] FIG. 14 is a graph showing the measurement results of the surface tension value of PX237. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] 1. Antibodies The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0049] The "class" of an antibody refers to the type of constant domain or constant region present in the antibody's heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0050] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies that make up the population are identical and / or bind to the same epitope, except for possible variant antibodies (e.g., variant antibodies including naturally occurring variants or variant antibodies that arise during the manufacture of a monoclonal antibody preparation, which are usually present in small amounts). In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be made by a variety of techniques, including, but not limited to, hybridoma techniques, recombinant DNA techniques, phage display techniques, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, and such methods and other exemplary methods for making monoclonal antibodies are described herein.

[0051] The term "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions.

[0052] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies usually have a similar structure, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, e.g., Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007).) One VH or VL domain may be sufficient to confer antigen-binding specificity. In addition, antibodies that bind to a particular antigen may be isolated by screening a complementary library of VL or VH domains, respectively, with a VH or VL domain from an antibody that binds that antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0053] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences typically appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0054] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence (the "complementarity determining region" or "CDR") and / or forms structurally defined loops (the "hypervariable loops") and / or contains antigen-contacting residues (the "antigen contacts"). Typically, antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3).

[0055] In one aspect of the invention, the antibody is a bispecific antibody that specifically binds to (a) coagulation factor IX (FIX) and / or activated coagulation factor IX (FIXa) and (b) coagulation factor X (FX) and / or activated coagulation factor FX (FXa) and mimics the cofactor function of coagulation factor VIII (FVIII) (e.g., emicizumab), or an anti-IL-6 receptor antibody that inhibits the binding of interleukin-6 to its receptor (e.g., satralizumab (SA237)).

[0056] Emicizumab is a bispecific antibody having heavy chains of SEQ ID NOs: 1 and 2 and a light chain of SEQ ID NO:3. The anti-IL-6 receptor antibody used in the present invention binds to the IL-6 receptor, thereby inhibiting the binding of IL-6 to the IL-6 receptor and blocking the transmission of the biological activity of IL-6 into cells. An example of such an anti-IL-6 receptor antibody is a monoclonal antibody (satralizumab (SA237)) having an H chain of SEQ ID NO: 4 and an L chain of SEQ ID NO: 5.

[0057] In one aspect of the invention, the antibody is a monoclonal antibody, including a chimeric, humanized, or human antibody. In one embodiment, the antibody is an antibody fragment, such as, for example, an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a full-length antibody, such as, for example, a complete IgG1, IgG2, IgG3, and IgG4 antibody, or other antibody class or isotype as defined herein.

[0058] In one aspect of the present invention, the monoclonal antibody used includes not only monoclonal antibodies derived from animals such as humans, mice, rats, hamsters, rabbits, sheep, camels, and monkeys, but also artificially modified recombinant antibodies such as chimeric antibodies, humanized antibodies, and bispecific antibodies.Furthermore, recombinant antibodies in which the constant region of an antibody has been artificially modified to modify the physical properties of the antibody molecule for the purpose of improving blood retention or pharmacokinetics (specifically, modification of the isoelectric point (p1), modification of the affinity of Fc receptors, etc.) are also included.

[0059] In one aspect of the present invention, the antibody used can be prepared by a known method. Hybridomas producing monoclonal antibodies can basically be prepared using known techniques as follows. That is, a desired antigen or cells expressing the desired antigen are used as a sensitizing antigen, which is immunized according to a conventional immunization method, the resulting immune cells are fused with a known parent cell by a conventional cell fusion method, and monoclonal antibody-producing cells (hybridomas) are screened by a conventional screening method. Hybridomas can be prepared, for example, according to the method of Milstein et al. (Kohler, G. and Milstein, C., Methods Enzymol. (1981) 73: 3-46) or the like. The antigen When the immunogenicity is low, the antibody may be bound to a macromolecule having immunogenicity such as albumin and then immunized.

[0060] Alternatively, a recombinant antibody can be used which is produced by cloning an antibody gene from a hybridoma, incorporating it into an appropriate vector, and introducing the vector into a host cell using recombinant gene technology (see, for example, Carl, A. K. Borrebaeck, James, W. Larrick, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LT). D, 1990). Specifically, cDNA of the variable region (V region) of an antibody is synthesized from the mRNA of a hybridoma using reverse transcriptase. Once DNA encoding the V region of the target antibody is obtained, it is linked to DNA encoding the constant region (C region) of the desired antibody, and this is incorporated into an expression vector. Alternatively, DNA encoding the V region of an antibody may be incorporated into an expression vector containing DNA of the antibody C region. It is incorporated into the expression vector so that it is expressed under the control of an expression control region, for example, an enhancer or promoter. Next, host cells can be transformed with this expression vector to express the antibody.

[0061] In one aspect of the present invention, a genetically engineered antibody that has been artificially modified for the purpose of reducing heterologous antigenicity against humans, for example, a chimeric antibody or a humanized antibody, can be used. These modified antibodies can be produced using known methods. A chimeric antibody is an antibody that consists of the heavy and light chain variable regions of an antibody from a mammal other than human, for example, a mouse, and the heavy and light chain constant regions of a human antibody, and can be obtained by linking DNA encoding the variable region of a mouse antibody to DNA encoding the constant region of a human antibody, incorporating this into an expression vector, and introducing the vector into a host for production.

[0062] A humanized antibody is also called a reshaped human antibody, and is a human antibody that is made by replacing the complementarity determining region (CDR) of a non-human mammal, such as a mouse antibody, with that of a human antibody. The human antibody is a mouse antibody that has been transplanted into a CDR region of a human antibody, and a general genetic recombination method for the same is also known. Specifically, a DNA sequence designed to link the CDR of a mouse antibody and the framework region (FR) of a human antibody is synthesized by PCR from several oligonucleotides that have been prepared so as to have overlapping portions at the ends. The resulting DNA is linked to a DNA encoding a human antibody constant region, which is then incorporated into an expression vector, which is then introduced into a host for production (see European Patent Application Publication No. 239400 and WO 96 / 02576). The FR of the human antibody that is linked via the CDR is selected from those that form a good antigen-binding site in the CDR region. If necessary, amino acids in the framework region of the variable region of the antibody may be replaced so that the CDR of the reshaped human antibody forms an appropriate antigen-binding site (Sato, K. et al., Cancer Res. (1993) 53, 851-856).

[0063] Techniques for substituting amino acids in an antibody to improve the activity, physical properties, pharmacokinetics, safety, etc. of the antibody are known, for example, the techniques described below. In one aspect of the present invention, the antibodies used also include antibodies with such amino acid substitutions (including deletions and additions).

[0064] Technologies for making amino acid substitutions in the variable regions of IgG antibodies include humanization (Tsurushita N, Hinton PR, Kumar S. , Design of humanized antibodies: from anti-Tac to Zenapax., Methods.2005 May;36(1):69-83.), affinity maturation by amino acid substitution in the complementarity determining region (CDR) to enhance binding activity (Rajpal A, Beyaz N, Haber L, Cappuccilli G, Yee H, Bhatt RR, Takeuchi T, Lerner RA, Crea R. , A general method for greatly improving the affinity of antibodies by using combinatorial libraries., Proc Natl Acad Sci US A. 2005 Jun 14;102(24):8466-71.), and improvement of physicochemical stability by amino acid substitution in the framework (FR) (Ewert S, Honegger A, Pluckthun A. , Stability improvement of antibodies for extracellular and intracellular applications: CDR grafting to stable frameworks and structure-based framework engineering. , Methods. 2004 Oct;34(2):184-99. Review) has been reported. Techniques for enhancing antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) are known to involve amino acid substitution in the target region (Kim SJ, Park Y, Hong HJ. Antibody engineering for the development of therapeutic antibodies., Mol Cells. 2005 Aug 31;20(1):17-29. Review.). Furthermore, A technique for amino acid substitution in Fc that not only enhances the antibody's activity but also improves its half-life in blood has been reported (Hinton PR, Xiong JM, Johlfs MG, Tang MT, Keller S, Tsurushita N., An engineered human IgG1 antibody with longer serum half-life., J Immunol. 2006 Jan 1;176(1):346-56., Ghetie V, Popov S, Borvak J, Radu C, Matesoi D, Medesan C, Ober RJ, Ward ES., Increasing the serum persistence of an IgG fragment by random mutagenesis., Nat Biotechnol. 1997 Jul;15(7):637-40.). Furthermore, various amino acid substitution techniques in the constant region for the purpose of improving the physical properties of antibodies are also known (WO 09 / 41613).

[0065] Methods for obtaining human antibodies are also known. For example, human lymphocytes can be sensitized in vitro with a desired antigen or cells expressing the desired antigen, and the sensitized lymphocytes can be fused with human myeloma cells, such as U266, to obtain a desired human antibody having binding activity to the antigen (see Japanese Patent Publication No. 1-59878). Also, a desired human antibody can be obtained by immunizing a transgenic animal having a full repertoire of human antibody genes with an antigen (see WO 93 / 12227, WO 92 / 03918, WO 94 / 02602, WO 94 / 25585, WO 96 / 34096, and WO 96 / 33735). Furthermore, a technique for obtaining a human antibody by panning using a human antibody library is also known. For example, the variable region of a human antibody can be expressed on the surface of a phage as a single chain antibody (scFv) by phage display, and a phage that binds to the antigen can be selected. By analyzing the genes of the selected phage, the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined. Once the DNA sequence of the scFv that binds to the antigen is identified, a suitable expression vector containing the sequence can be prepared to obtain a human antibody. These methods are already well known, and reference can be made to WO 92 / 01047, WO 92 / 20791, WO 93 / 06213, WO 93 / 11236, WO 93 / 19172, WO 95 / 01438, and WO 95 / 15388. In one aspect of the invention, the antibodies used also include such human antibodies.

[0066] When the antibody gene is isolated and then introduced into a suitable host to produce the antibody, a suitable combination of host and expression vector can be used. When eukaryotic cells are used as hosts, animal cells, plant cells, and fungal cells can be used. Known animal cells include (1) mammalian cells, such as CHO, COS, myeloma, BHK (baby hamster kidney), HeLa, and Vero, (2) amphibian cells, such as Xenopus oocytes, and (3) insect cells, such as sf9, sf21, and Tn5. Known plant cells include cells derived from the genus Nicotiana, such as Nicotiana tabacum, which can be cultured as callus. Known fungal cells include yeasts, such as the genus Saccharomyces, e.g., Saccharomyces cerevisiae, and filamentous fungi, such as the genus Aspergillus, e.g., Aspergillus niger. When prokaryotic cells are used, bacteria are used. There are production systems that use cells. As bacterial cells, E. coli and Bacillus subtilis are known. The desired antibody gene is introduced into these cells by transformation, and the transformed cells are cultured in vitro to obtain the antibody.

[0067] Furthermore, the antibodies used in pharmaceutical preparations include modified antibodies. For example, antibodies conjugated with various molecules such as polyethylene glycol (PEG) or cytotoxic drugs can also be used (Farmaco. 1999 Aug 30; 54(8): 497-516., Cancer J. 2008 May-Jun; 14(3): 154-69). Such modified antibodies can be obtained by chemically modifying the antibodies. These methods are already established in the field.

[0068] In one aspect of the present disclosure, an antibody may be a chimeric antibody. Chimeric antibodies are described, for example, in US Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). Chimeric antibodies contain non-human variable regions (e.g., monkey or from mice, rats, hamsters, or rabbits. The antibody may comprise a human variable region and a human constant region.

[0069] In one aspect of the present invention, the antibody of the present disclosure may be a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity in humans while maintaining the specificity and affinity of the parent non-human antibody. Typically, a humanized antibody comprises one or more variable regions, in which there are HVRs, e.g., CDRs (or portions thereof) derived from a non-human antibody, and FRs (or portions thereof) derived from a human antibody sequence. The humanized antibody may optionally comprise at least a portion of a human constant region. In one embodiment, amino acid residues of FRs in a humanized antibody may be substituted with corresponding amino acid residues of a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to maintain or improve the specificity or affinity of the antibody.

[0070] Humanized antibodies and methods for making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008) and further described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); US Patent Nos. 5, 821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991). (describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the "guided selection" approach to FR shuffling).

[0071] In one aspect of the invention, human frameworks to be used for humanization are selected using, for example, the "best-fit" method (Sims et al. J. Immunol. 151:2296 (1993)). frameworks derived from consensus sequences of human antibodies of a certain subgroup of heavy or light chain variable regions (Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992) and Presta et al. J. Immunol., 151:2623 (1993)); It may also contain framework regions derived from screening (Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618(1996)).

[0072] In one aspect of the invention, the antibody of the present disclosure may be a human antibody. Human antibodies can be produced by a variety of techniques. Human antibodies are reviewed, for example, in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-374 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008). Human antibodies are antibodies that respond to antigens and contain fully human antibodies or human variable regions. Antibodies that are human may be prepared by administering an immunogen to a transgenic animal that has been engineered to produce a complete antibody that is capable of producing a specific antibody. Such animals typically contain all or a portion of a human immunoglobulin locus, which either replaces an endogenous immunoglobulin locus or is present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci are usually inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See, for example, US Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE™ technology; US Patent No. 5,770,429, which describes HUMAB® technology; US Patent No. 7,041,870, which describes KM MOUSE® technology; and US Patent No. 7,041,870, which describes VELOCIMOUSE® technology. See US 2007 / 0061900 describing the use of ELISA (Promega.TM.) technology. The human variable regions from whole antibodies produced by such animals may be further modified, for example, by combining with different human constant regions.

[0073] In another aspect of the invention, human antibodies can also be produced using hybridoma-based methods. Human myeloma cells and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies are described below (e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991)). Human antibodies generated via human B cell hybridoma technology are described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Other methods include, for example, those described in U.S. Patent No. 7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (human- Human hybridoma techniques (trioma techniques) are described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0074] In another aspect of the invention, human antibodies can be generated by isolating Fv clone variable domain sequences selected from a human-derived phage display library. Such variable region sequences can then be combined with the desired human constant regions. See below for techniques for selecting human antibodies from antibody libraries.

[0075] In one aspect of the invention, antibodies of this disclosure may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. Methods for generating phage display libraries and screening such libraries for antibodies with the desired binding characteristics are known in the art. Such methods are reviewed by Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and further described in, e.g., McCafferty et al., Nature 348:552-554;Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992);Marks and Bradbury, Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003);Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004);Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004);Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004);Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

[0076] In one aspect of the present invention, in a particular phage display method, a repertoire of VH and VL sequences is generated by polymerase chain reaction (PCR). The isolated antibodies can be cloned separately and randomly recombined into a phage library, which can be constructed using the methods described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). In this manner, the antigen-binding phages may be screened for. The phages display antibody fragments, such as scFv or Fab. Libraries from immunized sources can provide high affinity antibodies to immunogens without the need to construct hybridomas. In another embodiment, naive repertoires can be cloned (e.g., from humans) without immunization to provide a wide range of single-origin antibodies to non-self or self antigens, as described in Griffiths et al., EMBO J, 12: 725-734 (1993). In yet another embodiment, naive libraries can be synthetically generated by cloning unrearranged V-gene segments from stem cells and using PCR primers that encode the hypervariable region CDR3 and contain random sequences to achieve rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent documents describing human antibody phage libraries include, for example, US Patent Nos. 5,750,373, US2005 / 0079574, US2005 / 0119455, US2005 / 0266000, US2007 / 0117126, US2007 / 0160598, US2007 / 0237764, US2007 / 0292936, and US2009 / 0002360.

[0077] Antibodies or antibody fragments isolated from a human antibody library are referred to herein as human antibodies or is considered a human antibody fragment. In one aspect of the present invention, the antibody of this disclosure is a multispecific antibody (e.g., a bispecific antibody). A multispecific antibody is an antibody (e.g., a monoclonal antibody) that has binding specificities at at least two different sites. In one embodiment, one of the binding specificities is for an antigen and the other is for another antigen. In another embodiment, a bispecific antibody may bind to two different epitopes of an antigen. Bispecific antibodies may be used to localize cytotoxic agents to cells expressing the antigen. Bispecific antibodies may be prepared as full length antibodies or as antibody fragments.

[0078] Techniques for producing multispecific antibodies include, but are not limited to, recombinant coexpression of two immunoglobulin heavy chain-light chain pairs with different specificities (e.g., Milstein and Cuello, Nature 305: 537 (1983), WO93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and and knob-in-hole technology (e.g., U.S. Patent No. 5,731,168). These include engineering electrostatic steering effects to create Fc heterodimeric molecules (e.g., WO2009 / 089004A1); crosslinking two or more antibodies or antibody fragments; using bridging (e.g., U.S. Patent No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to create antibodies with two specificities (e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology to create bispecific antibody fragments (e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); using scFv dimers (e.g., Gruber et al., J. Immunol., 152:5368 (1994)); preparing trispecific antibodies (e.g., Tutt et al. J. Immunol. 147: 60 (1991)). Additionally, antibodies may be engineered to have three or more functional antigen binding sites, including "octopus antibodies" (e.g., US2006 / 0025576).

[0079] In one aspect of the invention, the antibody or antibody fragment thereof of the present disclosure is a "dual-acting Fab" or "antibody fragment" that contains one antigen-binding site that binds to an antigen and another distinct antigen. DAF" (e.g., US2008 / 0069820).

[0080] In one aspect of the present invention, modified (variant) amino acid sequences of antibodies of the present disclosure may be prepared by introducing appropriate modifications into a nucleic acid encoding the antibody molecule or by synthesizing a peptide. Such modifications may be made by any one or more appropriate combinations of deletion, insertion, and substitution of any amino acid (residue) into the amino acid sequence. Any combination of deletion, insertion, and substitution may be used as long as the final construct has the desired characteristics (e.g., antigen binding).

[0081] In one aspect of the present invention, when an antibody variant (mutant) with one or more amino acid substitutions is provided, target sites for substitutional mutagenesis may include HVRs and FRs. In one aspect of the present invention, the concentration of the antibody in the aqueous solution may be in the range of 10 to 300 mg / mL, 20 to 200 mg / mL, or 30 to 150 mg / mL.

[0082] 2. Surfactants In one aspect of the invention, the pharmaceutical formulation may comprise as surfactant a poloxamer such as Poloxamer 188, a polysorbate such as Polysorbate 20 and Polysorbate 80, Triton X such as Triton® X-100 and Triton X-114, Brij such as Brij®-35 and Brij-58, Nonidet® P-40, octyl glucoside, octyl thioglucose, octyl thioglycerol ... Further include, but are not limited to, non-ionic surfactants such as glucosides.

[0083] In one aspect of the present invention, the amount of surfactant added to the pharmaceutical preparation is generally 0.001 to 100 mg / mL, preferably 0.01 to 10 mg / mL, and more preferably 0. The concentration is preferably 0.05 to 5 mg / mL, and more preferably 0.1 to 1 mg / mL.

[0084] (1) Poloxamer As used herein, poloxamers are block copolymers of ethylene oxide and propylene oxide. As used herein, "polyethylene oxide / polypropylene oxide copolymer," "PPC," "PX," or "poloxamer" refers to a block copolymer having the following formula Ia: HO(CH2CH2O) a ─[CH(CH3)CH2O] b ─(CH2CH2O) c H (Ia) and means a block copolymer having a polypropylene oxide (PPO) central block flanked on either side by blocks of polyethylene oxide (PEO). a and c are equal to or less than the number a and c, which may be the same or different. Each of a and c is a number such that the hydrophilic portion represented by (C2H4O) (i.e., the polyethylene oxide portion of the copolymer) constitutes about 60% to 90% by weight of the copolymer, for example, 70% to 90% by weight of the copolymer; b is a number such that the hydrophilic portion represented by (C3H6O) (i.e., the polyethylene oxide portion of the copolymer) constitutes about 60% to 90% by weight of the copolymer, for example, 70% to 90% by weight of the copolymer; bThe hydrophobic portion (i.e., the polypropylene oxide portion of the copolymer) represented by has a molecular weight of approximately 950-4,000 Daltons (Da), e.g., about 1,200-3,500 Da, e.g., 1,200-2,300 Da, 1,500-2,100 Da, 1,400-2,000 Da, or 1,700-1,900 Da. For example, the molecular weight of the hydrophilic portion can be 5,000-15,000 Da. Exemplary poloxamers having the above general formula include poloxamers in which a or c is a number between 5 and 150 and b is a number between 15 and 75, e.g., a and c are numbers between about 60 and 85, preferably between 75 and 85, e.g., 79, and b is a number between 22 and 40, preferably between 22 and 33, 25 and 30, or 35 and 40, and most preferably between 22 and 33. The average total molecular weight of the compound is approximately 6,840 to 9,510 Da, preferably 6,840 to 8,830 Da or 7,680 to 9,510 Da; For example, generally 8,400 to 8,800 Da, for example about 8,400 Da or 8,400 Da. Poloxamers include poloxamer 188 (e.g., Pluronic® F-68, Synperonic™ PE / F 68, Flocor®, Kolliphor® and Lutrol®), Poloxamer 188 EMPROVE® EXPERT, Pronon® )). The quality of commercially available grades of poloxamer 188 varies depending on the supplier. Poloxamer 188 may also vary in quality from lot to lot, even within the same grade from the same supplier. Further poloxamers include poloxamer 237 (sold, for example, under the names Pluronic® F-87 and Synperonic™ PE / F 87). Examples of such cases include those that have been

[0085] The nomenclature of polyethylene oxide / polypropylene oxide copolymers is related to the monomer composition. The first two digits of the poloxamer number are multiplied by 100 to give the approximate molecular weight of the hydrophobic polypropylene oxide block. The last digit is multiplied by 10 to give the approximate weight percent of the hydrophilic polyethylene oxide content. For example, poloxamer 188 represents a polymer containing about 1,800 Da of polypropylene oxide hydrophobe with a hydrophilic polyethylene oxide block content of about 80% of the total molecular weight. Poloxamer 237 represents a polymer containing about 2,300 Da of polypropylene oxide hydrophobe with a hydrophilic polyethylene oxide block content of about 70% of the total molecular weight. Here, polyethylene oxide is also known as polyoxyethylene, and polypropylene oxide is also known as polyoxypropylene.

[0086] Poloxamers can be synthesized in two steps by first constructing a polypropylene oxide core and then adding polyethylene oxide to the ends of the polypropylene oxide core. Due to variations in the polymerization rates during both steps, poloxamers contain heterogeneous polymer species of various molecular weights. The distribution of polymer species can include, but is not limited to: Characterization can be performed using standard techniques, including gel permeation chromatography (GPC).

[0087] In one embodiment of the present invention, the average number of polyethylene oxide and polypropylene oxide blocks contained in various poloxamers and the number average molecular weight are shown in Table 1.

[0088] [Table 1]

[0089] Poloxamers are represented herein by Formula I: HO(C2H4O) a (C3H6O) b (C2H4O) c H (I) In the above, a, c, and b are the average number of (C2H4O) units and (C3H6O) units, respectively, in the poloxamer. Here, (C2H4O) means ((CH2)2O) and is also called ethylene oxide (EO) units. (C3H6O) means (CH(CH3)CHO) and is also called propylene oxide (PO) units. The average of the ethylene oxide units and propylene oxide units can be calculated by obtaining the EO / PO ratio of the target poloxamer fraction by NMR, and then performing MALDI-FTICR-MS on that fraction to obtain the molecular weight as an input value. The EO / PO ratio is calculated by multiplying the area integral I of the methyl group peak by the propylene oxide peak in NMR. PO The combined area integral of ethylene oxide (hydrogen, 4 atoms) and propylene oxide (hydrogen, 3 atoms) is I EO+PO It is calculated based on the following formula (Non-Patent Document 2).

[0090] EO / PO=(I EO+PO / I PO -1) × (3 / 4) In one embodiment of the present invention, poloxamer 188 (also referred to as P-188, PX188 or P188) has the following formula Ia: HO(CH2CH2O) a -[CH(CH3)CH2O] b -(CH2CH2O) c H (Ia) where a, b and c are average numbers, a and c can be the same or different, each such that the hydrophilic portion represented by (C2H4O) (i.e., the polyethylene oxide portion of the copolymer) constitutes about 60% to 90%, for example about 80%, and b is a number such that the hydrophobic portion represented by (C3H6O) has a molecular weight of about 1,300 to 2,300 Da, for example 1,400 to 2,000 Da, for example about 1,750 Da. For example, a and c are numbers between about 75 and 85, for example 79, and b is a number between 25 and 30, or preferably a number between 22 and 33, for example 28. The average total molecular weight of the compound is about 7,680 to 1,750 Da. 9,510 Da, e.g., generally 8,400-8,800 Da, e.g., about 8,400 Da or 8,400 Da. Poloxamer 188 may include a heterogeneous distribution of polymer species that differ primarily in the overall chain length of the polymer, but also includes truncated polymer chains with unsaturation, and some low molecular weight glycols. Poloxamer 188 also includes those that exhibit a species profile (e.g., as determined by GPC) that includes a main peak and a "shoulder" peak on either side, representing low molecular weight (LMW) and high molecular weight (HMW) polymer species. In one aspect of the invention, the poloxamer may be a poloxamer obtained by purifying poloxamer 188 to remove or reduce species other than the main component.

[0091] Poloxamer 188 is sometimes referred to as "polyoxyethylene (160) polyoxypropylene (30) glycol" in the standards of the Japanese Pharmacopoeia. As used herein, "major component" or "main peak" in reference to poloxamer 188 refers to a species of copolymer molecule having a molecular weight less than about 13,000 Da and greater than about 4,500 Da, with a number average molecular weight of about 7,680-9,510 Da, e.g., generally 8,400-8,800 Da, e.g., about 8,400 Da or 8,400 Da. Main peak species include those eluting by gel permeation chromatography (GPC) at 14-15 minutes depending on the chromatographic conditions (see U.S. Pat. No. 5,696,298). In one embodiment of the present invention, poloxamer 237 (also referred to as P-237, PX237 or P237) has the following formula Ia: HO(CH2CH2O) a -[CH(CH3)CH2O] b -(CH2CH2O) c H (Ia) where a, b and c are average numbers, a and c may be the same or different, each being such that the hydrophilic portion represented by (C2H4O) (i.e., the polyethylene oxide portion of the copolymer) constitutes about 60% to 80%, for example about 70%, and b is a number such that the hydrophobic portion represented by (C3H6O) has a molecular weight of about 1,800 to 2,800 Da, for example 2,000 to 2,600 Da, for example about 2,300 Da. For example, a and c are numbers between about 60 and 68, for example 64, and b is a number between 35 and 40, or preferably a number between 32 and 43, for example 37. The average total molecular weight of the compound is about 6,840 to 8,830 Da, for example generally 7,500 to 8,000 Da, for example about 7,800 Da or 7,800 Da. Poloxamer 237 may contain a heterogeneous distribution of polymer species that differ primarily in overall polymer chain length, but also contain truncated polymer chains with unsaturation, and some low molecular weight glycols. Poloxamer 237 also includes species profiles (e.g., as determined by GPC) that include a main peak and two "shoulder" peaks representing low molecular weight (LMW) and high molecular weight (HMW) polymer species. In one aspect of the invention, the poloxamer may be a poloxamer obtained by purifying poloxamer 237 to remove or reduce species other than the main component.

[0092] (2) Polysorbate Polysorbates are useful as non-ionic surfactants and protein stabilizers and have the following chemical formula II or III:

[0093] [ka]

[0094] [In the formula, w+x+y+z is an integer selected from 15 to 25, R is independently an alkyl or alkenyl having 11 or more carbon atoms. Polysorbate 20, Polysorbate 40, Polysorbate 60, and Polysorbate 80 are widely used as stabilizers and emulsifiers in the pharmaceutical, cosmetic, and food industries. Polysorbate 20 mainly contains the monolaurate ester of polyoxyethylene (20) sorbitan. Polysorbate 40 mainly contains the monopalmitate ester of polyoxyethylene (20) sorbitan. Polysorbate 60 mainly contains the monostearate ester of polyoxyethylene (20) sorbitan. Polysorbate 80 mainly contains the monooleate ester of polyoxyethylene (20) sorbitan.

[0095] Polysorbates are often mixtures of various chemicals, consisting mostly of polyoxyethylene (20) sorbitan monoesters, with occasional isosorbide ester contaminants. They may also contain, for example, polyethylene glycols (PEGs), intermediate structures, and fatty acid reactants. The head group (in this case polyoxyethylene (20) sorbitan) comprises sorbitan (1,4-anhydrosorbitol, 1,5-anhydrosorbitol, and 1,4,3,6-dianhydrosorbitol) substituted with three of its alcohol groups forming ether bonds with three polyethylene oxide groups.

[0096] 3. Other additives In one aspect of the present invention, the pharmaceutical preparation can be prepared as a solution preparation by mixing with an appropriate pharma- ceutically acceptable carrier, vehicle, etc., as necessary. The solvent of the solution preparation is water or a pharma- ceutically acceptable organic solvent. Examples of such organic solvents include propylene glycol (1,2-propanediol), polyethylene glycol 300, polyethylene glycol 400, ethanol, glycerol, and acetic acid. Examples of suitable pharma- ceutically acceptable carriers and vehicles include sterilized water, physiological saline, stabilizers, antioxidants (ascorbic acid, etc.), buffers (phosphate, citric acid, histidine, other organic acids, etc.), preservatives, chelating agents (EDTA, etc.), binders, etc. It may also contain other low molecular weight polypeptides, proteins such as serum albumin, gelatin, and immunoglobulin, amino acids such as glycine, glutamine, asparagine, glutamic acid, aspartic acid, methionine, arginine, and lysine, sugars and carbohydrates such as polysaccharides and monosaccharides, and sugar alcohols such as mannitol and sorbitol.When used as a solution for injection, examples include physiological saline, isotonic solutions containing glucose and other auxiliary drugs, for example, D-sorbitol, D-mannose, D-mannitol, and sodium chloride, and may be used in combination with an appropriate solubilizing agent, for example, alcohol (ethanol, etc.), polyalcohol (propylene glycol, PEG, etc.), etc.

[0097] In one aspect of the present invention, the buffer used in the solution formulation is prepared using a substance for maintaining the pH of the solution. In one aspect of the present invention, in a high-concentration antibody-containing solution formulation, the pH of the solution is preferably 4.5 to 7.5, more preferably 5.0 to 7.0, and even more preferably 5.5 to 6.5. In one aspect of the present invention, a buffer that can be used is one that can adjust the pH to this range and is medicamentally acceptable. Such buffers are known to those skilled in the art in the field of solution formulation, and examples of such buffers include inorganic salts such as phosphate (sodium or potassium) and sodium bicarbonate; organic acid salts such as citrate (sodium or potassium), sodium acetate, and sodium succinate; and acids such as phosphoric acid, carbonic acid, citric acid, succinic acid, malic acid, and gluconic acid. Furthermore, Good's buffers such as Tris, MES, MOPS, and HEPES, histidine (e.g., histidine hydrochloride), and glycine may be used.

[0098] The concentration of the buffer is generally 1 to 500 mmol / L, preferably 5 to 100 mmol / L, and more preferably 10 to 20 mmol / L. When a histidine buffer is used, the buffer preferably contains 5 to 25 mmol / L of histidine, and more preferably 10 to 20 mmol / L of histidine.

[0099] In one aspect of the present invention, the high-concentration antibody-containing solution formulation is preferably stabilized by adding a stabilizer appropriate for the antibody, which is the active ingredient. In one aspect of the present invention, a "stable" high-concentration antibody-containing solution formulation shows no significant change at least 12 months, preferably 2 years, more preferably 3 years, at refrigerated temperature (2-8°C); or at least 3 months, preferably 6 months, more preferably 1 year, at room temperature (22-28°C). For example, the total amount of dimer and decomposition product after storage at 5°C for 2 years is 5.0% or less, preferably 2% or less, more preferably 1.5% or less, or the total amount of dimer and decomposition product after storage at 25°C for 6 months is 5.0% or less, preferably 2% or less, more preferably 1.5% or less.

[0100] Furthermore, cryoprotectants, suspending agents, solubilizing agents, isotonicity agents, preservatives, adsorption inhibitors, diluents, excipients, pH adjusters, soothing agents, sulfur-containing reducing agents, antioxidants and the like can be appropriately added to the formulations of the present invention, as necessary.

[0101] Examples of cryoprotectants include sugars such as trehalose, sucrose, and sorbitol. Examples of solution aids include polyethylene oxide hydrogenated castor oil, polysorbate 80, nicotinamide, polyethylene oxide sorbitan monolaurate, macrogol, castor oil fatty acid ethyl ester, and the like.

[0102] Examples of the isotonic agent include sodium chloride, potassium chloride, calcium chloride, and the like. Examples of preservatives include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, sorbic acid, phenol, cresol, and chlorocresol.

[0103] Examples of anti-adsorption agents include human serum albumin, lecithin, dextran, ethylene oxide-propylene oxide copolymer, hydroxypropyl cellulose, methyl cellulose, polyethylene oxide hydrogenated castor oil, and polyethylene glycol.

[0104] Examples of sulfur-containing reducing agents include N-acetylcysteine, N-acetylhomocysteine, thioctic acid, thiodiglycol, thioethanolamine, thioglycerol, and thiosorbate. Examples of the sulfhydryl group-containing antioxidant include thioglycolic acid and its salts, sodium thiosulfate, glutathione, and thioalkanoic acids having 1 to 7 carbon atoms.

[0105] Examples of antioxidants include erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, α-tocopherol, tocopherol acetate, L-ascorbic acid and its salts, L-ascorbyl palmitate, L-ascorbyl stearate, sodium hydrogen sulfite, sodium sulfite, triamyl gallate, propyl gallate, and chelating agents such as disodium ethylenediaminetetraacetate (EDTA), sodium pyrophosphate, and sodium metaphosphate.

[0106] 4. Surfactants that reduce particle generation (1) Hydrophobicity In one aspect of the present invention, the generation of particles in pharmaceutical formulations containing antibodies can be reduced by using highly hydrophobic surfactants.

[0107] Poloxamers are three-block amphiphilic copolymers consisting of one hydrophobic polypropylene oxide (PPO) chain and two hydrophilic polyethylene oxide (PEO) chains on either side, the PPO chains having an average of 22-40 propylene oxide units, preferably 22-33, 25-30, or 35-40, most preferably 22-33, and the PEO chains each having an average of 75-85 ethylene oxide units. Propylene oxide is the hydrophobic moiety, and ethylene oxide is the hydrophilic moiety. Poloxamers containing more hydrophobic moieties are more hydrophobic.

[0108] In one aspect of the invention, the poloxamer comprises a poloxamer containing a PPO chain consisting of 34 or more propylene oxide units in a ratio of 3%, 6%, 20%, or 29% (w / w) or more of the total poloxamer.

[0109] In one aspect of the present invention, the difference in hydrophobicity of poloxamers can be confirmed by reversed-phase chromatography. In reversed-phase chromatography, the length distribution of PPO blocks can be specified by setting the PEO block not to interact with the stationary phase. This method can also be used to analyze the difference in hydrophobicity of poloxamers (Non-Patent Document 2). In one aspect of the present invention, the conditions for reversed-phase chromatography of poloxamers are set as follows, with reference to Non-Patent Document 2. In reversed-phase chromatography, an HPLC column packed with macroporous styrene-divinylbenzene, for example, a PLRP-S column from Agilent Technologies, is used.

[0110] [High performance liquid chromatography conditions] (1) Column: PLRP-S column (1000 Å, 5 μm, 50 × 2.1 mm; Agilent Technologies) (2) Mobile phase: Mobile phase A: Ultrapure water Mobile phase B: Acetonitrile (3) Elution gradient program From 0 min to 16.0 min: Mobile phase B 58% to 64% From 16.0 to 18.5 min: Mobile phase B 64% to 90% From 18.5 to 21.5 min: Fixed at 90% mobile phase B From 21.5 to 23.5 min: Mobile phase B 90% to 100% From 23.5 to 30.0 min: Fixed at 100% mobile phase B From 30.0 to 30.1 min: Mobile phase B 100% to 58% From 30.1 to 40.0 min: Mobile phase B fixed at 58% (4)Flow rate: 0.2mL / min (5) Detection method: Evaporative light scattering detection (drift tube temperature: 50±25°C, nebulizer heating power level: 75%, gain value: 250, gas pressure: 20 psi) (6) Column temperature: 65±5℃ (7) Poloxamer concentration (in ultrapure water): 0.5 mg / mL.

[0111] In one aspect of the invention, in the chromatography of poloxamer under the above conditions, the elution time after 17 minutes corresponds to a highly hydrophobic poloxamer, and the peak area after elution time after 17 minutes is 3% or more, 6% or more, 19% or more, 33% or more, or 35% or more of the total peak area.

[0112] In one aspect of the present invention, in the chromatography of poloxamer under the above conditions, the peak area after elution time 17 minutes is 3% or more, 6% or more, 20% or more, 36% or more, or 46% or more of the total peak area after elution time 1.5 minutes.

[0113] (2)Surface tension In one aspect of the present invention, the generation of particles in an aqueous solution of a pharmaceutical preparation containing an antibody can be reduced by using a surfactant that reduces the surface tension of the aqueous solution containing the surfactant. In one aspect of the present invention, surfactants that can reduce the generation of particles include poloxamer 188, which was confirmed in the examples of the present invention, and further include poloxamer 237, polysorbate 20, polysorbate 60, polysorbate 65, and polysorbate 80. The higher the surfactant's surface activity, the lower the surface tension of the aqueous solution containing the surfactant. There is a correlation between the hydrophobicity index of poloxamer and the surface tension value.

[0114] Surface tension is measured using the Wilhelmy method (plate method, vertical plate method) and the du Nouy method (ring method, ring method). The viscosity can be measured by commonly known methods such as, but not limited to, the pendant drop method or the maximum bubble pressure method.

[0115] In one aspect of the invention, an aqueous solution containing a surfactant at a concentration of 0.5 mg / mL has a surface tension of 52.3 mN / m or less, 52 mN / m or less, 51 mN / m or less, 50.7 mN / m or less, 50.5 mN / m or less, or 39 mN / m or less.

[0116] (3) Degree of unsaturation In one aspect of the invention, the use of less unsaturated poloxamers as surfactants can reduce particulate generation in pharmaceutical formulations containing certain antibodies.

[0117] The degree of unsaturation can be determined using mercuric acetate solution according to the method specified in the United States Pharmacopoeia. How to prepare mercury(II) acetate solution: Place 50 g of mercury(II) acetate in a 1000 mL volumetric flask and dissolve in approximately 900 mL of methanol with 0.5 mL of glacial acetic acid. Dilute to volume with methanol and mix. If the solution is yellow, discard. If it is cloudy, filter. If it is still cloudy, discard. If the solution needs to be prepared repeatedly, use fresh reagent. Store the solution in a brown bottle in the dark and protect it from light.

[0118] Steps for calculating the degree of unsaturation of poloxamer: Transfer approximately 15.0 g of poloxamer to a 250 mL Erlenmeyer flask. Pipette 50 mL of the solution into the flask and mix with a magnetic stirrer until completely dissolved. Leave for 30 minutes, swirling occasionally. Add 10 g of sodium bromide crystals and stir with a magnetic stirrer for about 2 minutes. Without delay, add about 1 mL of phenophthalein test solution and titrate the liberated acetic acid with 0.1N methanolic potassium hydroxide titrant. A control test is performed in the same manner. Measure the initial acidity. Dissolve 15.0 g of poloxamer in 75 mL of methanol neutralized to the phenolphthalein end point with methanolic potassium hydroxide. Then add about 1 mL of phenolphthalein test solution and titrate with 0.1N methanolic potassium hydroxide titrant under nitrogen flow.

[0119] Calculate the degree of unsaturation (mEq / g) according to the following formula: (V U -V B -V A )N / 15 Here, VU , V B and V A are the volumes (mL) of 0.1N methanolic potassium hydroxide used in the titration of the test sample, blank, and initial acidity, respectively, and N is the normality of the titrant.

[0120] In one aspect of the invention, the poloxamer used as a surfactant has a degree of unsaturation of less than 0.018 mEq / g. 5. Reduced particle generation (1) Reduction of particle generation In one aspect of the present invention, "reducing particle generation" refers to preventing the generation of visually detectable particles or reducing the number of particles generated in an aqueous solution of a pharmaceutical preparation that generates visually detectable particles under certain conditions. The reduction in the generation of visually detectable particles can be confirmed by counting the number of particles. The size and number of particles can be measured using a light-obscuration particle counting method, a microscopic particle counting method, a flow cytometer particle image analysis method, visual inspection, or microscopic infrared spectroscopy (IR spectroscopy) after isolating particles. The measurement can be performed by R) measurement or microscopic Raman spectroscopy, and is preferably performed by a combination of visual inspection and microscopic infrared spectroscopy or microscopic Raman spectroscopy.

[0121] (2) Particles detectable by the naked eye In this specification, visually detectable particles are particles that can be visually detected at high illuminance and have a particle size of 40 μm or more. Among them, particles that can be visually detected at standard illuminance (about 2,000-3,000 lx) specified in the Pharmacopoeia are called "visible particles" or "insoluble visible particles". Visible particles generally have a particle size larger than 100 μm (Non-Patent Document 1). Particles that are smaller than visible particles and cannot be seen with the eye at standard illuminance (about 2,000-3,000 lx) specified in the Pharmacopoeia but can be visually detected by increasing the illuminance or lengthening the observation time are "particles that can be visually detected only at high illuminance" and have a particle size of 40 μm to 100 μm. Visible particles are confirmed by visual inspection with the naked eye for 5 seconds or more under illumination at standard illuminance (about 2,000-3,000 lx) by gently rotating or inverting a container in front of a black or white background. Particles that are only visually detectable under high light intensity are identified by visual inspection with the naked eye for 30 seconds or more under lighting and high light intensity (6,000 lx or greater) while gently swirling or inverting the container in front of a black background. Visible particles are also visible under high light intensity inspection.

[0122] As used herein, "protein-derived particles" refers to visually detectable particles generated from proteins, including particles consisting of only proteins and particles consisting of a complex of proteins and polydimethylsiloxane (PDMS). The fact that the particles are protein molecules can be confirmed by microscopic Raman spectroscopy. The only protein contained in the solution is the active pharmaceutical ingredient (API), and visually detectable particles are derived from the API. The number of visually detectable particles can be counted using a light obscuration particle counting method, a microscopic particle counting method, a flow cytometer particle image analysis method, visual inspection, and microscopic infrared spectroscopy (IR) after isolating the particles. The measurement can be performed by infrared spectroscopy (IR) or micro-Raman spectroscopy, and is preferably performed. or by a combination of visual inspection and microinfrared spectroscopy or microRaman spectroscopy.

[0123] As used herein, "aggregates" refer to relatively high molecular weight protein species resulting from the assembly of multiple denatured proteins and are used interchangeably with the terms "high molecular weight species" and "HMWS". Protein aggregates can generally differ in size (small (dimers) to large (microscopic or even visible particles) aggregates with diameters ranging from nanometers to micrometers), morphology (roughly spherical to fibrous), protein structure (native vs. non-native / denatured), type of intermolecular bonds (covalent vs. non-covalent), reversibility, and solubility. Soluble aggregates cover a size range of approximately 1-100 nm, while protein particles cover the microscopic (approximately 0.1-100 μm) and visible (>100 μm) ranges. All of the aforementioned types of protein aggregates are generally encompassed by the term. Thus, the term "(protein) aggregate" refers to any kind of non-native species in which two or more protein monomers are physically associated or chemically linked.

[0124] In one aspect of the present invention, the pharmaceutical preparation is stored at −30° C. to 25° C., preferably the freezing point of the solution to 25° C., more preferably 1° C. to 10° C., more preferably 2° C. to 8° C., and even more preferably 5° C., without freezing the solution in the container. The storage is carried out for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, or 96 hours. The storage may be for at least 24 hours, at least 2 days, at least 3 days, at least 4 days, at least 10 days, at least 20 days, at least 30 days, at least 40 days, at least 50 days, at least 60 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months.

[0125] 5. Container In one aspect of the invention, the pharmaceutical formulation is packaged in a container, which may be a plastic or glass syringe, cartridge, or vial.

[0126] In one aspect of the invention, the pharmaceutical formulation is loaded into a syringe, cartridge, or vial for administration to a patient. In some embodiments, the pharmaceutical formulation is loaded into the syringe, cartridge, or vial at a manufacturing filling facility. In some embodiments, the syringe, cartridge, or vial is sterilized prior to loading the composition. In some embodiments, the syringe, cartridge, or vial loaded with the pharmaceutical formulation has a shelf life of 1 day, or at least 7 days, or at least 14 days, or at least 1 month, or at least 6 months, or at least 1 year, or at least 2 years prior to administration of the pharmaceutical formulation to a patient. In some embodiments, the syringe, cartridge, or vial is exposed to storage and / or transport conditions.

[0127] In one embodiment of the present invention, the syringe, cartridge or vial is subjected to mechanical stress. Mechanical stress includes, but is not limited to, drop stress and vibration stress. In one embodiment of the present invention, the syringe, cartridge or vial is subjected to drop stress 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 25 or more, 30 or more, or 40 or more times. The stress applied to the syringe, cartridge or vial when dropped is The response varies depending on the number of drops as well as the height and direction. 38.1 cm as specified in ASTM D4169. However, the present invention is not limited to this.

[0128] The syringe may be made of PP (polypropylene) or glass, and the inner surface of the syringe may be coated with silicone oil as a lubricant. In one aspect of the invention, the silicone oil is a polydimethylsiloxane. Some exemplary polydimethylsiloxanes include, for example, Dow Corning® 360 Medical Fluid, which has a viscosity of 350 centistokes, Dow Corning® 360 Medical Fluid, which has a viscosity of 10 ... Dow Corning® 360 Medical Fluid, with a viscosity of 12,500 centistokes Examples of suitable fluids include, but are not limited to, Dow Corning® 360 Medical Fluids, including, but not limited to, Dow Corning® 360 Medical Fluids, and Dow Corning® MDX4-4159 fluid.

[0129] In one aspect of the present invention, the size (standard) of the volume of the syringe is not particularly limited. Specifically, the volume is 0.5 mL to 5.0 mL, preferably 1 mL or 2.25 mL.

[0130] In one aspect of the present invention, the size (standard) of the cartridge capacity is not particularly limited. Specifically, the capacity may be 0.5 mL to 20.0 mL, for example, 1.0 mL, 1.5 mL, 1.8 mL, 2.0 mL, 2.2 mL, 3.0 mL, 5.0 mL, 10.0 mL, 15.0 mL, or 20.0 mL, but is not limited to these amounts.

[0131] In one aspect of the present invention, the size (standard) of the vial capacity is not particularly limited. Specifically, the size may be 3 mL to 100 mL, and may be 3 mL, 5 mL, 10 mL, 15 mL, 20 mL, 30 mL, 50 mL, or 100 mL, but is not limited to these amounts.

[0132] The present invention is further illustrated by, but not limited to, the following examples. EXAMPLES

[0133] [Example 1] Component analysis of poloxamer 188 (PX188) by reversed-phase chromatography Seven types of PX188, including different manufacturers, grades, and lots, shown in Table 2, were analyzed by reversed-phase chromatography to evaluate the length of polypropylene oxide (PPO) blocks. A previous paper was used as a reference for the method of analyzing the PPO block length by reversed-phase chromatography (Non-Patent Document 1).

[0134] [Table 2]

[0135] The HPLC system used was an Alliance e2695 liquid chromatograph (Waters) equipped with a 2424 evaporative light scattering detector (ELSD) (Waters). Empower 3 software (Waters) was used for data acquisition and analysis. A PLRP-S column (1000 Å, 5 μm, 50 × 2.1 mm; Agilent Technologies) was used for separation. The column temperature was set at 65±5°C. The flow rate was fixed at 0.2 mL / min. A linear gradient was applied with ultrapure water (Milli-Q water) as mobile phase A and acetonitrile as mobile phase B. The gradient details were 58%B to 64%B (0-16.0 min), 64%B to 90%B (16.0-18.5 min), fixed at 90%B (18.5-21.5 min), 90%B to 100%B (21.5-23.5 min), fixed at 100%B (23.5-30.0 min), 100%B to 58%B (30.0-30.1 min), fixed at 58%B (30.1-40.0 min). The temperature of the ELSD drift tube was 50 ± 25 °C, the heating power level of the nebulizer was 75%, the gain value was 250, and the gas pressure was 20 psi. Each PX188 was dissolved in ultrapure water to a concentration of 0.5 mg / mL, and 20 μL of the PX188 solution was injected into the HPLC system. The elution after 17 minutes from the start was defined as "late elution product", and the ratio of the late elution product was calculated from the chromatogram of each PX188 (Figure 1) as the ratio (%) of the peak area after 17 minutes to the total peak area. Furthermore, the ratio of the peak area after 17 minutes to the total peak area excluding the peak area up to 1.5 minutes after the start was defined as "late elution product (excluding early elution product)" and calculated from the chromatogram of each PX188 (Figure 1).

[0136] Furthermore, the mass % of molecular species with a PPO block length of 34 or more, which corresponds to the late elution product, was calculated for five types of PX188. The PPO block length of each elution peak was calculated by assuming that the PPO block length of the peak first eluted after 17 minutes was 34. In addition, the molecular weight of molecular species corresponding to each elution peak was calculated by assuming that the PEO block lengths of both ends were 80. Next, these molecular weights were multiplied by the elution peak area value, and the values ​​obtained by adding up the values ​​with a PPO block length of 34 or more were taken as the numerator, and the values ​​obtained by adding up the values ​​for all peaks were taken as the denominator and multiplied by 100 to calculate the mass % of molecular species with a PPO block length of 34 or more for each PX188. The calculated ratio (mass %) of molecular species with a PPO block length of 34 or more to the total poloxamer is shown in Table 3.

[0137] [Table 3]

[0138] [Example 2] Measurement of surface tension values ​​of PX188 and PS80 The surface tension values ​​of the aqueous solutions of seven types of PX188 and one type of PS80 shown in Table 2, in which each surfactant was dissolved in ultrapure water to a concentration of 0.5 mg / mL, were measured. Using a surface tensiometer (Force Tensiometer K100C, Kruss), measurements were performed using the Wilhelmy method with a platinum plate at 20-25 degrees. The measurement parameters of the K100C were a detection speed of 6 mm / min, a detection sensitivity of 0.005 g, and an immersion depth of 2 mm, and surface tension values ​​were obtained at 60-second intervals from the start of measurement until 600 seconds (Figure 2). The glass container containing the surfactant solution in which the platinum plate was immersed was washed multiple times with isopropyl alcohol and then ultrapure water after each measurement. The platinum plate was also washed with isopropyl alcohol and then ultrapure water before being red-hot washed with an alcohol lamp after each measurement.

[0139] The surface tension values ​​of the various PX188 aqueous solutions gradually decreased after the platinum plate was immersed, and reached equilibrium 600 seconds after the start of the measurement. Therefore, the values ​​at 600 seconds were used as the surface tension values ​​of the various surfactant solutions. The surface tension value of the PS80 aqueous solution continued to decrease even after 600 seconds had passed, but for comparison with the PX188 aqueous solution, the surface tension value at 600 seconds was used.

[0140] [Example 3] Correlation between the percentage of slow extractables and surface tension value We analyzed the correlation between the ratio of slow extractables for the seven types of PX188 shown in Table 2 and the surface tension measured by the above method. The results showed that there was a high correlation between the two values ​​(Figure 3). In other words, it was shown that in PX188, the ratio of long PPO block components correlates with the surface tension value, which is an index of surface activity, and further that the surface tension value decreases when the PPO block contains a large amount of long components.

[0141] Example 4: Sample preparation for visually detectable particle evaluation To investigate the effect of various surfactants on the generation of visually detectable particles, the generation of particles in two types of mAb formulations was investigated using seven types of PX188 and one type of PS80 shown in Table 2. The mAbs used were mAb1 (emicizumab, IgG4, anti-blood coagulation factor IXa / X humanized bispecific monoclonal antibody) and mAb2 (satralizumab, IgG2, pH-dependent binding humanized anti-IL-6 receptor monoclonal antibody) manufactured and purified by Chugai Pharmaceutical. The mAb1 sample was prepared by preparing an aqueous solution containing 158 mg / mL mAb1, 20 mM histidine, 150 mM arginine, aspartic acid (appropriate amount), and 0.5 mg / mL PX188 or PS80 at pH 6.0 and filling a vial (3 mL). A sulfur-treated glass vial (Murase Glass Co., Ltd.) was filled with 1 mL of the solution. For the mAb2 sample, an aqueous solution containing 123 mg / mL mAb1, 20 mM histidine, 150 mM arginine, aspartic acid (appropriate amount), and 0.5 mg / mL PX188 or PS80 was prepared at pH 6.0, and 1 mL was filled into a syringe (1 mL ClearJect, Taisei Kako Co., Ltd.). Eight types of samples were prepared for each mAb using the eight types of surfactants shown in Table 2. The prepared samples were visually inspected, and samples containing visible foreign matter during the sample manufacturing process were excluded.

[0142] After visual inspection, samples determined to contain no visible foreign matter were left at 25°C, or left at 40°C and then visually inspected. Ten samples each of mAb1 and mAb2 were provided to each of the groups. In addition, 20 samples of mAb1 and 40 samples of mAb2 were provided to each of the groups that were left at 5°C and subjected to periodic mechanical stress before visual inspection.

[0143] How to apply regular mechanical stress The samples subjected to periodic mechanical stress were stored at 5°C except when mechanical stress was applied at room temperature. The samples were appropriately packaged to prevent damage to the container and subjected to the following combined drop and vibration tests (drop test) in accordance with ASTM D4169. The samples were subjected to a series of tests (test → vibration test → drop test). Drop stress was performed using a drop tester (PDT-56ED, Lansmont) by dropping each sample from a height of 38.1 cm on each of the four sides with the sample facing down so that the stress applied to each sample was uniform, twice for each of the four sides, which constituted one set of drop tests. One set of drop tests was performed before and after the vibration test. Vibration stress was performed using a vibration tester (D-5900, Shinken) by applying four different levels of vibration stress (Truck Low Level 40 minutes, Truck Medium Level 15 minutes, Truck High Level 5 minutes, Air Level I 120 minutes), which constituted one set of vibration tests. As shown in Figure 4, there were two cases where only one set of vibration tests was applied, and four consecutive sets were applied, and the application timing for each is shown in Figure 6.

[0144] [Example 5] Generation of particles when left standing at 25°C Visual inspection was performed on the samples after 6 months of static storage at 25 °C as described in Visual Inspection Methods 1 and 2. The composition of the particles detected by visual inspection was identified by Raman spectroscopy using an imaging Raman microscope (DXR2xi, Thermo Scientific). Identification was performed as per the composition identification method for visually detectable particles.

[0145] Visual Inspection Method 1 (Vial; mAb1) A visual inspection table (EM-M102-06, Hitachi Industrial Control Solutions) was used for visual inspection of the vials. The exterior of the vials was cleaned, and the number of vials containing visually detectable particles in the filled drug solution was counted with the naked eye against a black background and with an illumination intensity of approximately 20,000 lx.

[0146] Visual Inspection Method 2 (Syringe; mAb2) Fluorescent lighting was used for visual inspection of the syringes. The exteriors of the syringes were cleaned, and the number of syringes containing visually detectable particles in the filled drug solution was counted with the naked eye against a black background and an illumination intensity of approximately 10,000 lx.

[0147] Method for identifying particle composition using Raman spectroscopy Particles were collected on a nickel filter with a pore size of 3 mm (Tokyo Process Service) and analyzed with a 532 nm laser using an imaging Raman microscope (DXR2xi, Thermo Scientific). The Raman spectrum obtained when the particles were irradiated with 10x or 50x magnification was used to confirm that they were endogenous proteinaceous particles. The spectrum was obtained by setting the laser intensity (5.0-10.0mW), exposure time (0.05-1.0 seconds), and number of accumulations (15-35 times) within the ranges described above to obtain an appropriate spectrum that would enable the composition to be determined. Representative proteinaceous particles included particles made of simple proteins and particles made of a complex of proteins and polydimethylsiloxane (PDMS), and examples of their Raman spectra are shown in Figure 6.

[0148] The number of containers containing proteinaceous particles was counted for samples stored at 25°C for 6 months, and the results are shown in Table 4. It was shown that the particle generation rate differed depending on the type of surfactant contained in the formulation for both mAb1 and mAb2.

[0149] [Table 4]

[0150] [Example 6] Generation of particles after standing at 40°C Visual inspection was performed on the samples after 6 months of static storage at 40°C as described in Visual Inspection Methods 1 and 2. The composition of the particles detected by visual inspection was identified by Raman spectroscopy using an imaging Raman microscope (DXR2xi, Thermo Scientific). The composition of the particles was identified according to the method for identifying the composition of the particles.

[0151] The number of containers containing proteinaceous particles was counted for samples stored at 40°C for 6 months, and the results are shown in Table 5. It was shown that the particle generation rate differed greatly depending on the type of surfactant contained in the formulation for both mAb1 and mAb2.

[0152] [Table 5]

[0153] [Example 7] Generation of particles under static conditions at 5°C and periodic mechanical stress conditions Visual inspection was performed on samples stored at 5℃ and subjected to periodic drop and vibration stress (6 months for mAb1, 3 months for mAb2). The method was carried out as described in Methods 1 and 2. For particles detected by visual inspection, the composition was identified by Raman spectroscopy using an imaging Raman microscope (DXR2xi, Thermo Scientific). The composition of the particles was identified by the method described in the NMR spectroscopy.

[0154] The number of containers containing proteinaceous particles was counted for samples stored at 5°C and subjected to periodic mechanical stress, and the results are shown in Table 6.

[0155] [Table 6]

[0156] It was shown that the particle generation rate of mAb2 differed greatly depending on the type of surfactant contained in the formulation. The particle generation rate of mAb1 was not high enough, so it was not used in the subsequent analysis.

[0157] [Example 8] Correlation analysis between the rate of slow elution of PX188 and the surface tension value of the surfactant aqueous solution and the particle generation rate A correlation analysis was performed to clarify the correlation between the rate of late elution and the particle generation rate for the seven types of PX188 shown in Table 2. The particle generation rate (%) was calculated based on the percentage of particles generated in each sample. The particle generation rate (%) was calculated by dividing the number of containers that had been cooled by the total number of containers used in the test and multiplying the result by 100. In addition, since both the 25°C and 40°C static storage conditions were caused by stress due to temperature rise, and the composition of the proteinaceous particles that were generated consisted mostly of complexes of protein and polydimethylsiloxane, it was assumed that the particles were generated in accordance with the same particle formation pathway, and these were added together as heat stress conditions to calculate the particle generation rate (%). In the case of samples subjected to long-term mechanical stress, the majority of the particles were insoluble foreign matter consisting of simple proteins, which were thought to have been formed through a particle formation pathway different from that observed under heat stress conditions, so we decided to conduct a separate analysis under mechanical stress conditions.

[0158] As a result, a correlation was observed between the rate of slow elution and the rate of particle generation in mAb1 under heat stress conditions (Figure 7). A correlation was also observed between the rate of slow elution and the rate of particle generation in mAb2 under heat stress conditions and mechanical stress conditions (Figures 8 and 10, left). A weak correlation was observed in mAb1 under mechanical stress conditions (Figure 9, left).

[0159] From this correlation diagram, it was shown that the higher the value of the ratio of slow elution, that is, the longer the PPO block of PX188 species is used, the more the generation of particles in mAb1 and mAb2 formulations can be reduced. In addition, as shown in Example 3, the value of the ratio of slow elution correlates well with the value of surface tension, so in other words, it can be said that the generation of particles in mAb1 and mAb2 formulations can be reduced by using a surfactant that can lower the surface tension value. This is demonstrated to be independent of the surfactant type, since the surface tension value of the PS80 aqueous solution is low and the particle generation rate is low, even in the case of PS80, a surfactant type different from PX188. In addition, since the results obtained showed that the particle generation rate is significantly different in the surface tension value range of 50-53 mN / m, it is considered that a threshold value for the increase or decrease in particle generation in mAb1 and mAb2 formulations exists in this range.

[0160] [Example 9] Correlation analysis between the degree of unsaturation of PX188 and particle generation rate Under mechanical stress conditions, we analyzed not only the ratio of slow elution but also the correlation between the degree of unsaturation and the occurrence rate of particles. As a result, a certain degree of correlation was observed, in which the lower the degree of unsaturation, that is, the less diblock body (PEO-PPO body) in the product, the lower the occurrence rate of particles (center figure in Figure 9, center figure in Figure 10). In particular, PX(7), which shows an unsaturation degree below the lower limit (0.018mEq / g) of the current USP standard for the degree of unsaturation of PX188, completely suppressed the occurrence of proteinaceous foreign matter for both mAb1 and mAb2.

[0161] In addition, multiple regression analysis using the two parameters of the ratio of late elution products and the degree of unsaturation showed an increase in the correlation coefficient, which was particularly remarkable for mAb2 (right panel in Figure 9 and right panel in Figure 10). [Example 10] Evaluation of HMWS generated in each formulation To evaluate the effect of surfactant type on the occurrence of antibody-derived aggregates (high molecular weight species: HMWS) in the formulation, seven types of PX shown in Table 2 were used. The amount of HMWS generated in the formulations using 188 and one type of PS80 was evaluated by size exclusion chromatography. Evaluations were performed before, after 3 months, and after 6 months of storage under conditions of 5°C static storage, 25°C static storage, 40°C static storage, and 5°C static storage with periodic mechanical stress. However, for mAb2, visual evaluation was not performed at 6 months under conditions of 5°C static storage and periodic drop vibration, so the HMWS evaluation was not performed. No evaluation was carried out.

[0162] The HPLC system used was an Alliance 2695 liquid chromatograph (Waters) equipped with a 2489 UV / Visible detector (Waters), and data acquisition and analysis were performed using Empower 3 software (Waters). The column used for separation was a TSKgel G3000SWXL column (250 A column was used (A, 5 μm, 300 × 7.8 mm; Tosoh Corporation) and the column temperature was set at 25 ± 5°C. The flow rate was fixed at 0.2 mL / min, and the mobile phase consisted of 50 mM NaH2PO4 / Na2HPO4, 300 mM NaCl, and 0.5 mg / mL NaN3 (pH 7.0), with a fixed flow rate of 0.5 mL / min. Each antibody solution was diluted with the mobile phase solution to an antibody concentration of 1 mg / mL, and 60 μL was injected into the HPLC system. The value of HMWS% was defined as the ratio of the peak area of ​​HMWS appearing around 14.5 minutes to the total peak area in the peak area integration range (10-24 minutes), and was calculated from the chromatogram of each sample.

[0163] The calculated HMWS values ​​are shown in Table 7.

[0164] [Table 7]

[0165] The 5°C static condition and the 5°C static storage and periodic mechanical stress conditions showed almost the same amount of HMWS, and no significant increase in HMWS or differences between samples were observed. An increase in the amount of HMWS was observed compared to the PX188 sample group, but no significant differences were observed between samples. Under heat stress conditions, PS80 showed a slightly lower value than the PX188 sample group, but there was no significant difference in the particle generation rate compared to PX188 (e.g., PX(1) and PX(4)), which has a relatively low surface tension value. This suggests that the amount of HMWS generation is not involved in the reduction of particle generation in the mAb1 and mAb2 formulations, and that the protective ability of various surfactants from the air-liquid interface, container surface, and silicone oil (PDMS) surface stress affects the particle generation rate (Figure 11). The left side of Figure 11 shows an image of a case where the interface is sufficiently protected by the surfactant, and the right side of Figure 9 shows an image of a case where protection is insufficient.

[0166] [Example 11] Measurement of surface tension of low concentration PX188 Of the seven types of PX188 shown in Table 2, the surface tension values ​​of aqueous solutions of PX(3), PX(6), and PX(7) were measured by dissolving each surfactant in ultrapure water to a concentration of 0.01 mg / mL. Measurements were performed at 20-25°C using a surface tensiometer (Force Tensiometer K100C, Kruss) and a platinum plate by the Wilhelmy method. The measurement parameters of the K100C were a detection speed of 6 mm / min, a detection sensitivity of 0.005 g, and an immersion depth of 2 mm, and surface tension values ​​were obtained at 10-second intervals from the start of measurement until 600 seconds (Figure 12). The glass container containing the surfactant solution in which the platinum plate was immersed was washed multiple times with isopropyl alcohol and then ultrapure water for each measurement. The platinum plate was also washed with isopropyl alcohol and then ultrapure water for each measurement, and then red-hot washed with an alcohol lamp.

[0167] The surface tension values ​​of the various PX188 aqueous solutions showed similar behavior to that of the aqueous solutions in which PX188 was dissolved in ultrapure water to a concentration of 0.5 mg / mL, and therefore the values ​​at 600 seconds were used as the surface tension values ​​of the various surfactant solutions (PX(3) 55.7 mN / m, PX(6) 56.4 mN / m, PX(7) 55.3 mN / m). The magnitude of the surface tension values ​​of PX188 measured here at 600 seconds was the same as in Example 2, and the difference in the surface activity of each PX188 could be evaluated even when the PX188 concentration was lower than 0.5 mg / mL.

[0168] [Example 12] Component analysis of poloxamer 237 (PX237) by reversed-phase chromatography The results of component analysis of poloxamer 237 (PX237) by reverse phase chromatography performed in the same manner as in Example 1 are shown in Figure 13. It was shown that for PX237, all peaks appeared after 17 minutes, excluding the peak areas up to 1.5 minutes after the start of the analysis.

[0169] [Example 13] Measurement of surface tension of PX237 The surface tension of an aqueous solution of PX237 dissolved in ultrapure water (Milli-Q water) at 0.05 mg / mL was measured. Measurements were performed at 20-25°C using a Wilhelmy method with a platinum plate using a surface tensiometer (Force Tensiometer K100C, Kruss). The measurement parameters of the K100C were a detection speed of 6 mm / min, a detection sensitivity of 0.005 g, and an immersion depth of 2 mm, and surface tension values ​​were obtained at 60-second intervals from the start of measurement until 600 seconds (Figure 14). The glass container containing the surfactant solution in which the platinum plate was immersed was washed multiple times with isopropyl alcohol and then ultrapure water for each measurement. The platinum plate was also washed with isopropyl alcohol and then ultrapure water for each measurement, and then red-hot washed with an alcohol lamp.

[0170] The surface tension value of the 0.5 mg / mL PX237 aqueous solution showed the same behavior as when the surface tension value of an aqueous solution of PX188 dissolved in ultrapure water to a concentration of 0.5 mg / mL was measured. Therefore, the surface tension value of the PX237 aqueous solution was determined to be the value at 600 seconds (45.9 mN / m). The surface tension value of the 0.5 mg / mL PX188 aqueous solution and the value at 600 seconds were used. The surface tension value of the PX237 aqueous solution was lower than any of the surface tension values ​​of the PX188 aqueous solutions listed in Table 2, and it was possible to evaluate that the surface activity of PX237 is higher than that of PX188.

[0171] Example 14: Sample preparation for visually detectable particle evaluation To investigate the effect of PX237 on the generation of particles detectable by visual inspection, particle generation in one type of mAb formulation was investigated using PX237 and PX(3) of PX188 shown in Table 2 as a control. The mAb used was mAb1 (emicizumab, IgG4, anti-blood coagulation factor IXa / X humanized bispecific monoclonal antibody) manufactured and purified by Chugai Pharmaceutical. The mAb1 sample was prepared by preparing an aqueous solution containing 150 mg / mL mAb1, 20 mM histidine, 150 mM arginine, aspartic acid (appropriate amount), and 0.5 mg / mL PX188 or PX237 at pH 6.0, and filling 1 mL into a vial (3 mL sulfur-treated glass vial, Murase Glass). The prepared samples were visually inspected, and samples containing visible foreign matter during the sample manufacturing process were excluded.

[0172] After visual inspection, samples that were determined to contain no visible foreign matter were left to stand at 25°C, and 60 tubes of mAb1 were provided to each group for visual inspection.

[0173] [Example 15] Generation of particles when left standing at 25°C Visual inspection was performed on the samples after 6 months of static storage at 25 °C as described in Visual Inspection Method 1. The composition of the particles detected by visual inspection was identified by Raman spectroscopy using an imaging Raman microscope (DXR2xi, Thermo Scientific) and visual inspection by Raman spectroscopy. Identification was carried out as per the method for composition identification of more detectable particles.

[0174] The number of containers containing proteinaceous particles was counted for samples stored at 25°C for 6 months, and the results are shown in Table 8. When the surfactant contained in the formulation was PX237, it was shown that the particle generation rate was lower than that of PX(3). [Table 8]

Claims

1. A pharmaceutical formulation comprising an aqueous solution containing a monoclonal antibody selected from an antibody having an H chain of SEQ ID NO: 1 and 2 and an L chain of SEQ ID NO: 3, or an antibody having an H chain of SEQ ID NO: 4 and an L chain of SEQ ID NO: 5, and polyoxyethylene polyoxypropylene glycol (poloxamer), Poloxamers have the formula I: HO(C ２ H ４ O) ａ (C ３ H ６ O) ｂ (C ２ H ４ O) ｃ H (I) wherein a and c are independently numbers selected from 75 to 85; b is a number selected from 22 to 40; a, b, and c are average values ​​for the whole poloxamer. is expressed as A pharmaceutical formulation, wherein the poloxamer has a degree of unsaturation of less than 0.018 mEq / g.

2. 2. The pharmaceutical formulation according to claim 1, wherein b is a number selected from 22 to 33.

3. 2. The pharmaceutical formulation according to claim 1, wherein b is a number selected from 25 to 30.

4. 2. The pharmaceutical formulation according to claim 1, wherein b is a number selected from the group consisting of 35 and 40.

5. 5. The pharmaceutical formulation according to any one of claims 1 to 4, wherein the number average molecular weight of the poloxamer is in the range of 7680 to 9510.

6. 5. The pharmaceutical formulation according to claim 1, wherein the concentration of the poloxamer in the aqueous solution is 0.001 to 100 mg / mL.

7. The pharmaceutical formulation according to any one of claims 1 to 4, wherein the concentration of the antibody in the aqueous solution is 10 to 300 mg / mL.

8. 5. The pharmaceutical formulation of claim 1, wherein the aqueous solution comprises one or more pharmaceutically acceptable excipients selected from sugars, sugar alcohols, buffers, preservatives, carriers, antioxidants, chelating agents, natural polymers, synthetic polymers, cryoprotectants, bulking agents, and stabilizers.

9. 5. The pharmaceutical formulation of any one of claims 1 to 4, wherein the poloxamer is poloxamer 188 or poloxamer 237.

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