Method for preparing prefilled syringe formulation

Abstract

To provide a method and system for reducing formation of visually detectable particles in an injectable formulation in which a protein-contained solution is filled in a container.SOLUTION: Provided is a method for determining the risk of formation of particles in solution in a pharmaceutical formulation containing protein as an active ingredient in the solution, the method including: constructing a three-dimensional protein structure model from an amino acid sequence using homology modeling or antibody modeling; identifying regions of clustered hydrophobic residues and clustered charged residues on the surface of the obtained model as hydrophobic patches and charge patches, respectively, and calculating their respective areas; calculating the sum of the areas of the top five largest hydrophobic patches (X (Å2)) and the total charge patch area (Y (Å2)); and determining that a protein poses a high risk of particle formation when X+Y×1.5 is 1700 or higher, where particles have a particle size of 40 μm or larger.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a pharmaceutical preparation that contains a protein in solution as an active ingredient and is filled in a container. [Background technology]

[0002] In recent years, various antibody preparations have been developed and put to practical use, but most of the antibody preparations are used as intravenous injection preparations. On the other hand, due to the needs of the medical field, there is a high demand for the development of antibody-containing preparations as self-injectable subcutaneous injection preparations. In particular, there is a high demand for the development of solution preparations enclosed in prefilled syringes due to their convenience.

[0003] When designing an antibody-containing formulation for subcutaneous injection, it is essential to increase the concentration of the antibody in the administration solution because, while the amount of antibody administered per dose is large (approximately 80 to 200 mg), subcutaneous injections generally have limitations on the amount of injection solution.

[0004] In recent years, prefilled syringes have come to be used in medical settings as prefilled syringe preparations for self-injection, the prefilled syringes comprising a cylindrical syringe body filled with a drug, an injection needle attached to the tip of the syringe body, a syringe cap covering the detachably attached injection needle, and a plunger inserted into the syringe body and slidable in the axial direction of the syringe body.

[0005] When using a prefilled syringe, the syringe cap is removed, the needle is inserted into the injection site, and the plunger is moved forward with the plunger rod to dispense and administer the drug. In general, to ensure the sliding properties of the plunger, a lubricant such as silicone oil is applied to the inner wall and plunger of the prefilled syringe.

[0006] In antibody-containing preparations, particle formation in aqueous solutions is a problem. The particles that form are aggregates larger than multimers such as dimers and trimers, but are generally difficult to see with the naked eye. Known particles include sub-visible particles (SVPs), which are fine particles with a diameter of less than 1.5 μm to 50 μm, and visible particles (VPs, larger than 100 μm) that can be detected visually at standard illuminance (about 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 (about 2,000-3,000 lx) specified in the Pharmacopoeia, the detection sensitivity of particles with a diameter of 100 μm is about 40%, the detection sensitivity of particles with a diameter of 150 μm is about 70%, and the detection sensitivity of particles with a diameter of 200 μm is almost 100% (Non-Patent Document 1). In addition, 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 about 40 μm. In this specification, such particles with a diameter of 40 μm to 100 μm are particularly referred to as particles that can be visually detected only at high illuminance. Moreover, particles with a diameter of 40 μm or more are particles that can be visually detected at high illuminance, and are referred to as visually detectable particles.

[0007] In general, antibodies have the property of adsorbing to and aggregating at interfaces such as air-liquid interfaces and solid-liquid interfaces. The presence of these interfaces may contribute to the formation of the above-mentioned visually detectable particles. It has been reported that a significant increase in fine particles due to the presence of interfaces occurs when mechanical stress is applied to a syringe filled with an antibody solution (Non-Patent Document 2). The antibody solution filled in the syringe forms an air-liquid interface due to the presence of air bubbles, and forms a solid-liquid interface by contacting the plunger and the syringe barrel. In addition, when the plunger and barrel of the prefilled syringe are coated with silicone, the antibody solution comes into contact with the silicone on the solid phase surface and forms a new solid-liquid interface. It has also been reported that proteins that are adsorbed to and aggregated at the solid-liquid interface peel off into the liquid due to the movement of air in the prefilled syringe and appear as visible particles (Non-Patent Document 3).

[0008] One method for reducing the stress at various interfaces is to reduce the amount of air bubbles in the prefilled syringe. By reducing the amount of air bubbles, the amount of air moving through the prefilled syringe can be reduced, which is thought to suppress adsorption to the gas-liquid interface and solid-liquid interface and detachment of aggregates.

[0009] It has been reported that the amount of invisible and visible particles in a solution that does not contain a surfactant can be reduced by reducing the amount of air bubbles in the solution of a prefilled syringe containing a specific antibody; however, these results are based on molecular theory and are evaluation results under extremely unstable conditions (Patent Documents 1 and 2).

[0010] The physical properties of amino acid residues, which are the building blocks of proteins, differ depending on the functional groups contained in their side chains. The physical properties of side chains can be broadly classified into two types based on whether they have a charged functional group and how hydrophobic the side chain is.

[0011] It is well known to those skilled in the art that a three-dimensional structural model of a protein can be constructed in a computer by inputting amino acid sequence information using computational chemistry software. Among the physical properties of amino acid residues, partial charges at the atomic level can be calculated using parameters called molecular force fields. Molecular Operating Environment (MOE; Chemical Computing Group Inc. (CCG)), which is used as computational chemistry software, employs a molecular force field called Amber10:EHT, and partial charges of each atom constituting an amino acid in a protein are assigned by version ff10 of the Amber force field (Non-Patent Document 4), which has been continuously improved since its publication in 1995. As an index of hydrophobicity of amino acid residues, an index of hydrophobicity correlated with the experimentally measurable water-octanol partition coefficient logP was established in the 1990s, and MOE employs an index developed by Crippen et al. (Non-Patent Document 5).

[0012] A method for detecting a certain level of localization (patches) of charged and hydrophobic amino acid residues in the three-dimensional structure of a protein based on the charge and hydrophobicity indices of individual amino acid residues as described above was proposed in the 1990s (Non-Patent Document 6). These can be detected as charge patches and hydrophobic patches using the aforementioned computational chemistry software MOE, and it was reported in 2018 that they correlate to a certain degree with experimental data in drug discovery (Non-Patent Document 7).

[0013] As an example of the application of the computational chemistry software MOE to pharmaceutical development, it has been reported that there is a certain degree of correlation between the area of ​​hydrophobic patches of antibodies calculated by MOE and the incidence of visible particles (Non-Patent Document 8). [Prior art documents] [Patent documents]

[0014] [Patent Document 1] JP 2015-042638 A [Patent Document 2] International Publication No. 2017 / 184880 [Non-patent literature]

[0015] [Non-Patent Document 1] James A. Melchore, AAPS PharmSciTech; 2011; 12(1): 215-221. [Non-Patent Document 2] Torisu et al., J. Pharm. Sci. 106 (2017) 2966-2978. [Non-Patent Document 3] Gerhardt et al., J. Pharm. Sci. 103 (2014) 1601-1612. [Non-Patent Document 4] Cornell et al., J. Am. Chem. Soc. 1995, 117, 5179-5197. [Non-Patent Document 5] Wildman et al., J. Chem. Inf. Comput. Sci. 1999, 39, 868-873. [Non-Patent Document 6] Jones et al., J. Mol. Biol. 1997 272, 133-143. [Non-Patent Document 7] Jetha et al., MABS 2018, 10, 6, 890-900. [Non-Patent Document 8] Grapentin et al., J. Pharm. Sci. 109 (2020) 2393-2404. Summary of the Invention [Problem to be solved by the invention]

[0016] No correlation was known between parameters other than the hydrophobic patch based on the results of the three-dimensional structure model calculations and the risk of visually detectable particles forming in the solution contained in the prefilled syringe formulation. Also, better methods for suppressing particle generation are needed.

[0017] The inventors have found that, particularly for biopharmaceuticals that have numerous modifications in the molecule and have increased hydrophobicity or charge bias, it is difficult to completely suppress the formation of visually detectable particles even after the addition of an appropriate amount of surfactant. [Means for solving the problem]

[0018] Therefore, the present inventors have found that for antibodies in which the value calculated from the area of ​​the hydrophobic patch and the area of ​​the charged patch is equal to or greater than a certain value, by reducing the bubble volume, the formation of visually detectable particles in the prefilled syringe formulation, which could not be completely suppressed even after the addition of a surfactant, can be significantly suppressed. This specification includes the disclosure of the following inventions.

[0019] [1-1] A method for determining a protein having a high risk of forming particles in a solution in a pharmaceutical preparation containing a protein as an active ingredient in the solution, comprising: Creating a three-dimensional structure model of a protein from the amino acid sequence of the protein by homology modeling or antibody modeling; Identifying, on the surface of the obtained model, the parts where hydrophobic residues are accumulated in clusters and the parts where charged residues are accumulated in clusters as hydrophobic patches and charged patches, respectively, and calculating the area of ​​each of them; The sum of the areas of the top 5 hydrophobic patches in the ranking by area size (X(Å 2 )), and the total charge patch area (Y(Å 2 )), and determining that a protein having a value of X+Y×1.5 of 1700 or more is a protein having a high risk of forming particles in a solution; The process wherein the particles have a particle size of 40 μm or more.

[0020] [1-2] The method according to [1-1], wherein the charge is a positive charge. [1-3] The method according to [1-1], wherein the charge is a negative charge. [1-4] The method according to any one of [1-1] to [1-3], wherein a protein for which X+Y×1.5 is 2000 or more is determined to be a protein with a high risk of forming particles in a solution.

[0021] [1-5] The method according to any one of [1-1] to [1-4], wherein Amber10: EHT is used as a molecular force field in homology modeling or antibody modeling. [1-6] The method according to any one of [1-1] to [1-5], wherein the particles have a particle size of more than 100 μm.

[0022] [1-7] The method according to any one of [1-1] to [1-6], wherein the solution is an aqueous solution. [1-8] The method according to any one of [1-1] to [1-7], wherein the protein is a monoclonal antibody, a fusion protein, a hormone, a cytokine, an enzyme, or a vaccine.

[0023] [1-9] The method according to any one of [1-1] to [1-8], wherein the protein is a monoclonal antibody. [1-10] The method according to [1-9], wherein the monoclonal antibody is either a monospecific antibody or a bispecific antibody.

[0024] [1-11] The method according to [1-9], wherein the monoclonal antibody is any one of IgG1, IgG2 and IgG4. [1-12] The method according to any one of [1-1] to [1-11], wherein the generation of a three-dimensional structure model of a protein is carried out by antibody modeling.

[0025] [1-13] The method according to any one of [1-1] to [1-12], wherein the homology modeling or antibody modeling is performed using Molecular Operating Environment (MOE) software.

[0026] [2-1] A method for determining a protein having a high risk of forming particles in a solution in a pharmaceutical preparation containing a protein as an active ingredient in the solution, comprising: Creating a three-dimensional structure model of a protein from the amino acid sequence of the protein by homology modeling or antibody modeling; The parts of the surface of the obtained model that correspond to clusters of charged residues are identified as charge patches, and the total charge patch area (Y(Å 2 )), and determining that a protein having Y of 600 or more is a protein with a high risk of forming particles in a solution; The process wherein the particles have a particle size of 40 μm or more.

[0027] [2-2] The method according to [2-1], wherein the charge is a positive charge. [2-3] The method according to [2-1], wherein the charge is a negative charge. [2-4] The method according to any one of [2-1] to [2-3], wherein a protein having Y of 700 or more is determined to be a protein with a high risk of forming particles in a solution.

[0028] [2-5] A method according to any one of [2-1] to [2-4], in which Amber10: EHT is used as a molecular force field in homology modeling or antibody modeling. [2-6] The method according to any one of [2-1] to [2-5], wherein the particles have a particle size of more than 100 μm.

[0029] [2-7] The method according to any one of [2-1] to [2-6], wherein the solution is an aqueous solution. [2-8] The method according to any one of [2-1] to [2-7], wherein the protein is a monoclonal antibody, a fusion protein, a hormone, a cytokine, an enzyme, or a vaccine.

[0030] [2-9] The method according to any one of [2-1] to [2-8], wherein the protein is a monoclonal antibody. [2-10] The method according to [2-9], wherein the monoclonal antibody is either a monospecific antibody or a bispecific antibody.

[0031] [2-11] The method according to [2-9], wherein the monoclonal antibody is any one of IgG1, IgG2 and IgG4. [2-12] A method according to any one of [2-1] to [2-11], in which the generation of a three-dimensional structure model of a protein is carried out by antibody modeling.

[0032] [2-13] A method according to any one of [2-1] to [2-12], wherein homology modeling or antibody modeling is performed using Molecular Operating Environment (MOE) software.

[0033] [3-1] A method for reducing generation of particles in a solution in an injectable preparation in which a solution containing a protein as an active ingredient is filled in a container, comprising: The volume of the air bubbles in the container is 40 μL or less; the container is a syringe or a cartridge; A method, wherein the protein is determined to have a high risk of forming particles in a solution by a method according to any one of [1-1] to [1-13] and [2-1] to [2-13].

[0034] [3-2] The method according to [3-1], which includes keeping the volume of bubbles in the container to 10 μL or less. [3-3] The method according to [3-1] or [3-2], wherein the container is a syringe.

[0035] [3-4] The method according to [3-3], wherein the syringe is stoppered by vacuum stopper placement or mechanical stopper placement. [3-5] The method according to any one of [3-1] to [3-4], wherein the particles have a particle size of 100 μm or more.

[0036] [3-6] The method according to any one of [3-1] to [3-5], wherein the solution is an aqueous solution. [3-7] The method according to any one of [3-1] to [3-6], wherein the protein is a monoclonal antibody, a fusion protein, a hormone, a cytokine, an enzyme, or a vaccine.

[0037] [3-8] The method according to any one of [3-1] to [3-7], wherein the protein is a monoclonal antibody. [3-9] The method according to [3-8], wherein the monoclonal antibody is either a monospecific antibody or a bispecific antibody.

[0038] [3-10] The method according to [3-8], wherein the monoclonal antibody is any one of IgG1, IgG2 and IgG4. [3-11] The method described in [3-8], wherein the monoclonal antibody is selected from an antibody having an H chain of SEQ ID NO: 3 and 4 and an L chain of SEQ ID NO: 5, or an antibody having an H chain of SEQ ID NO: 6 and an L chain of SEQ ID NO: 7. [3-12] The method described in [3-8], wherein the monoclonal antibody is an antibody having a combination of an H chain of SEQ ID NO: 8 and an L chain of SEQ ID NO: 9 and a combination of an H chain of SEQ ID NO: 11 and an L chain of SEQ ID NO: 10.

[0039] [4-1] A method for preparing an injectable formulation in which a solution containing a protein as an active ingredient is filled in a container, comprising the steps of: and filling the container with the solution so that the volume of air bubbles in the container in the resulting injectable preparation is 40 μL or less; the container is a syringe or a cartridge; A method, wherein the protein is determined to have a high risk of forming particles in a solution by a method according to any one of [1-1] to [1-13] and [2-1] to [2-13].

[0040] [4-2] The method according to [4-1], which comprises filling a solution into a container so that the volume of air bubbles in the container in the resulting injectable formulation is 10 μL or less. [4-3] The method according to [4-1] or [4-2], wherein the container is a syringe.

[0041] [4-4] The method according to [4-3], in which, when filling a solution into a container, the container is stoppered by a vacuum stopper placement method or a mechanical stopper placement method. [4-5] The method according to any one of [4-1] to [4-4], wherein the particles have a particle size of 100 μm or more.

[0042] [4-6] The method according to any one of [4-1] to [4-5], wherein the solution is an aqueous solution. [4-7] The method according to any one of [4-1] to [4-6], wherein the protein is a monoclonal antibody.

[0043] [4-8] The method according to [4-7], wherein the monoclonal antibody is either a monospecific antibody or a bispecific antibody. [4-9] The method according to [4-7], wherein the monoclonal antibody is any one of IgG1, IgG2 and IgG4.

[0044] [4-10] The method described in [4-7], wherein the monoclonal antibody is selected from an antibody having an H chain of SEQ ID NO: 3 and 4 and an L chain of SEQ ID NO: 5, or an antibody having an H chain of SEQ ID NO: 6 and an L chain of SEQ ID NO: 7. [4-11] The method described in [4-7], wherein the monoclonal antibody is an antibody having a combination of an H chain of SEQ ID NO: 8 and an L chain of SEQ ID NO: 9 and a combination of an H chain of SEQ ID NO: 11 and an L chain of SEQ ID NO: 10.

[0045] [5-1] An injectable preparation in which a solution containing a protein as an active ingredient is filled in a container, The protein is determined to have a high risk of forming particles in a solution by the method according to any one of [1-1] to [1-13] and [2-1] to [2-13], the container is a syringe or a cartridge; An injectable preparation, the volume of air bubbles in the container being 40 μL or less.

[0046] [5-2] The injectable formulation described in [5-1], wherein the volume of air bubbles in the container is 10 μL or less. [5-3] An injectable preparation according to [5-1] or [5-2], wherein the container is a syringe.

[0047] [5-4] The injectable preparation according to any one of [5-1] to [5-3], wherein the concentration of the protein in the solution is 0.1 mg / mL or more. [5-5] The injectable preparation according to any one of [5-1] to [5-4], wherein the concentration of the protein in the solution is in the range of 0.1 to 300 mg / mL.

[0048] [5-6] The injectable preparation according to any one of [5-1] to [5-5], wherein the concentration of the protein in the solution is in the range of 1 to 200 mg / mL. [5-7] An injectable preparation according to any one of [5-1] to [5-6], wherein the amount of solution contained in a 1 mL syringe is in the range of 0.1 to 1.2 mL, or the amount of solution contained in a 2.25 mL syringe is in the range of 0.1 to 2.5 mL.

[0049] [5-8] An injectable preparation according to any one of [5-1] to [5-7], wherein the amount of solution contained in a 1 mL syringe is in the range of 0.2 to 1.1 mL, or the amount of solution contained in a 2.25 mL syringe is in the range of 0.3 to 2.3 mL.

[0050] [5-9] The injectable preparation according to any one of [5-1] to [5-8], wherein the injectable preparation in which a solution containing a protein as an active ingredient is filled in a container includes a syringe or cartridge containing a pharmaceutical preparation therein and a stopper, and the syringe or cartridge is made of glass or a cycloolefin-based resin.

[0051] [5-10] The injectable preparation according to [5-9], wherein the cycloolefin resin is a cycloolefin polymer (COP) or a cycloolefin copolymer (COC). [5-11] The injectable preparation according to any one of [5-1] to [5-10], wherein the particles have a particle size of 100 μm or more.

[0052] [5-12] The method according to any one of [5-1] to [5-11], wherein the solution is an aqueous solution. [5-13] The injectable preparation according to any one of [5-1] to [5-12], wherein the protein is a monoclonal antibody.

[0053] [5-14] The injectable preparation described in [5-13], wherein the monoclonal antibody is either a monospecific antibody or a bispecific antibody. [5-15] The injectable formulation described in [5-13], wherein the monoclonal antibody is any one of IgG1, IgG2, and IgG4.

[0054] [5-16] An injectable formulation according to [5-13], wherein the monoclonal antibody is selected from an antibody having an H chain of SEQ ID NO: 3 and 4 and an L chain of SEQ ID NO: 5, or an antibody having an H chain of SEQ ID NO: 6 and an L chain of SEQ ID NO: 7.

[0055] [5-17] The injectable formulation according to any one of [5-1] to [5-16], wherein the solution contains one or more pharma- ceutically acceptable excipients, including a sugar, a sugar alcohol, a buffer, a preservative, a carrier, an antioxidant, a chelating agent, a natural polymer, a synthetic polymer, a cryoprotectant, a surfactant, a bulking agent, a stabilizer, or a combination thereof.

[0056] [5-18] The injectable formulation described in [5-17], wherein the surfactant is polysorbate, poloxamer 188, sodium lauryl sulfate, polyol, poly(ethylene glycol), glycerol, propylene glycol or poly(vinyl alcohol).

[0057] [5-19] An injectable formulation according to [5-17] or [5-18], wherein the surfactant is polysorbate or poloxamer 188. [5-20] The injectable preparation according to any one of [5-17] to [5-19], wherein the concentration of the surfactant in the solution is 0.01 mg / mL or more.

[0058] [5-21] The injectable preparation according to any one of [5-17] to [5-20], wherein the concentration of the surfactant in the solution is in the range of 0.01 to 5 mg / mL. [5-22] The injectable preparation according to any one of [5-17] to [5-21], wherein the concentration of the surfactant in the solution is in the range of 0.25 to 0.75 mg / mL.

[0059] [5-23] The injectable preparation according to any one of [5-1] to [5-22], wherein the pH of the solution is in the range of 4.5 to 7.5. [5-24] The injectable preparation according to any one of [5-1] to [5-23], wherein the pH of the solution is in the range of 5.0 to 7.0.

[0060] [5-25] The injectable preparation according to any one of [5-1] to [5-24], wherein the pH of the solution is in the range of 5.5 to 6.5. [5-26] An injectable formulation according to any one of [5-1] to [5-25], wherein the average number of particles after three months of storage of an injectable formulation subjected to drop stress during storage at 25°C is reduced compared to the case where the volume of bubbles in the injectable formulation is 120 μL.

[0061] [5-27] An injectable formulation according to any one of [5-1] to [5-25], wherein the average particle number of an injectable formulation containing 0.01 mg / mL of a surfactant after storage at 5°C for 1 day is reduced compared to the case where the volume of air bubbles in the injectable formulation is 120 μL. [5-28] An injectable preparation according to any one of [5-13], [5-14], or [5-17] to [5-26], wherein the monoclonal antibody is an antibody having a combination of an H chain of SEQ ID NO: 8 and an L chain of SEQ ID NO: 9 and a combination of an H chain of SEQ ID NO: 11 and an L chain of SEQ ID NO: 10.

[0062] [6-1] A system for determining a protein that is at high risk of forming particles in a solution in a pharmaceutical formulation containing a protein as an active ingredient in the solution, comprising: A means for creating a three-dimensional structure model of a protein from the amino acid sequence of the protein by homology modeling or antibody modeling; a means for identifying, on the surface of the obtained model, a portion where hydrophobic residues are accumulated in clusters and a portion where charged residues are accumulated in clusters as hydrophobic patches and charged patches, respectively, and calculating the area of ​​each of them; The sum of the areas of the top 5 hydrophobic patches in the ranking by area size (X(Å 2 )), and the total charge patch area (Y(Å 2 )) and A means for determining that a protein having X+Y×1.5 of 1700 or more is a protein with a high risk of forming particles in a solution, The system, wherein the particles have a particle size of 40 μm or more.

[0063] [6-2] The system described in [6-1], wherein the charge is a positive charge. [6-3] The system described in [6-1], wherein the charge is a negative charge. [6-4] The system according to any one of [6-1] to [6-3], which determines that a protein for which X+Y×1.5 is 2000 or more is a protein with a high risk of forming particles in a solution.

[0064] [6-5] A system described in any one of [6-1] to [6-4], in which Amber10: EHT is used as a molecular force field in homology modeling or antibody modeling. [6-6] A system described in any of [6-1] to [6-5], wherein the particles have a particle size greater than 100 μm.

[0065] [6-7] The system according to any one of [6-1] to [6-6], wherein the solution is an aqueous solution. [6-8] A system described in any of [6-1] to [6-7], wherein the protein is a monoclonal antibody, a fusion protein, a hormone, a cytokine, an enzyme, or a vaccine.

[0066] [6-9] A system described in any of [6-1] to [6-8], wherein the protein is a monoclonal antibody. [6-10] The system described in [6-9], wherein the monoclonal antibody is either a monospecific antibody or a bispecific antibody.

[0067] [6-11] The system described in [6-9], wherein the monoclonal antibody is any of IgG1, IgG2 and IgG4. [6-12] A system described in any of [6-1] to [6-11], in which the generation of a three-dimensional structure model of a protein is performed by antibody modeling.

[0068] [6-13] A program for causing a computer to operate each of the means in the system described in any one of [6-1] to [6-12]. A storage medium characterized by storing the program described in [6-14] or [6-13].

[0069] [6-15] [6-13] An apparatus for determining proteins that have a high risk of forming particles in a solution in a pharmaceutical preparation containing a protein as an active ingredient in the solution, the apparatus having the program described in [6-13] installed therein.

[0070] [7-1] A system for determining a protein having a high risk of forming particles in a solution in a pharmaceutical formulation containing a protein as an active ingredient in the solution, comprising: A means for creating a three-dimensional structure model of a protein from the amino acid sequence of the protein by homology modeling or antibody modeling; The parts of the surface of the obtained model that correspond to clusters of charged residues are identified as charge patches, and the total charge patch area (Y(Å 2 )) and A means for determining that a protein having Y of 600 or more is a protein having a high risk of forming particles in a solution, The system, wherein the particles have a particle size of 40 μm or more.

[0071] [7-2] The system described in [7-1], wherein the charge is a positive charge. [7-3] The system described in [7-1], wherein the charge is a negative charge. [7-4] A system according to any one of [7-1] to [7-3], which determines that a protein with Y of 700 or more is a protein with a high risk of forming particles in a solution.

[0072] [7-5] A system described in any of [7-1] to [7-4], in which Amber10: EHT is used as a molecular force field in homology modeling or antibody modeling. [7-6] A system described in any of [7-1] to [7-5], wherein the particles have a particle size greater than 100 μm.

[0073] [7-7] A system according to any one of [7-1] to [7-6], wherein the solution is an aqueous solution. [7-8] A system described in any of [7-1] to [7-7], wherein the protein is a monoclonal antibody, a fusion protein, a hormone, a cytokine, an enzyme, or a vaccine.

[0074] [7-9] A system described in any of [7-1] to [7-8], wherein the protein is a monoclonal antibody. [7-10] The system described in [7-9], wherein the monoclonal antibody is either a monospecific antibody or a bispecific antibody.

[0075] [7-11] The system described in [7-9], wherein the monoclonal antibody is any of IgG1, IgG2 and IgG4. [7-12] A system described in any of [7-1] to [7-11], in which the generation of a three-dimensional structure model of a protein is performed by antibody modeling.

[0076] [7-13] A program for causing a computer to operate each of the means in the system described in any one of [7-1] to [7-12]. A storage medium characterized by storing the program described in [7-14] or [7-13].

[0077] [7-15] [7-13] An apparatus for determining proteins that have a high risk of forming particles in a solution in a pharmaceutical preparation containing a protein as an active ingredient in the solution, the apparatus having the program described in [7-13] installed therein. Effect of the Invention

[0078] According to one aspect of the present invention, it is possible to determine a protein that has a high risk of forming particles that can be detected by the naked eye in a solution of a prefilled syringe formulation. According to another aspect of the present invention, it is possible to provide a prefilled syringe formulation that minimizes the formation of particles that can be detected by the naked eye. [Brief description of the drawings]

[0079] [Figure 1] Figure 1 shows photographs of the air bubbles in the syringe when they were (a) 120 μL, (b) 40 μL, and (c) 10 μL. [Diagram 2] FIG. 2 is a schematic diagram of a cardboard box used in the drop test. [Diagram 3] FIG. 3 is a diagram showing an example of the configuration of a syringe. [Figure 4] FIG. 4 is a histogram of the sizes of the proteinaceous visually detectable particles identified in Example 3. [Diagram 5] FIG. 5 shows a process flow when a program for an apparatus for determining proteins with a high risk of forming particles in a solution based on hydrophobic patches and charge patches is executed. [Figure 6] FIG. 6 shows a process flow when a program for an apparatus for determining proteins that have a high risk of forming particles in a solution based on charge patches is executed. [Figure 7] FIG. 7 shows a schematic diagram of an apparatus for determining proteins that have a high risk of forming particles in a solution. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0080] (1) Determining the risk of forming 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 visually inspecting 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 illumination are confirmed by visual inspection with the naked eye for 30 seconds or more under illumination, at high illumination (6,000 lx or more), with the container gently swirled or inverted in front of a black background. Visible particles can also be confirmed by inspection under high illumination. Particles that are not generated from protein molecules in the solution are not considered to be "visually detectable particles" regardless of size. The fact that visually detectable particles are caused by protein molecules can be confirmed by micro-Raman spectroscopy. The only protein contained in the solution is the active pharmaceutical ingredient (API), and the visually detectable particles are caused by the API. The size and number of visually detectable particles can be measured by light-obscured particle counting method, microscopic particle counting method, flow cytometer particle image analysis method, visual inspection, or by isolating the particles and then performing micro-infrared spectroscopy (IR) or micro-Raman spectroscopy, preferably by a combination of visual inspection and micro-infrared spectroscopy or micro-Raman spectroscopy.

[0081] As used herein, "high risk of forming particles" in a pharmaceutical formulation solution refers to the tendency of protein molecules in the solution to aggregate and form particles that are visible to the naked eye. Examples of proteins that have a high risk of forming particles include proteins that form particles that are visible to the naked eye even after the addition of an appropriate amount of surfactant.

[0082] As used herein, "air bubbles" refer to the space between the liquid in a container and the wall of the container or the gas space in the liquid. It is of a size that can be seen by the naked eye or by optical microscopy. "Inside the container" includes the entire space sealed by the rigid needle shield (RNS) and the stopper, for example, in the case of a prefilled syringe with a needle, specifically the space inside the needle and the space inside the barrel. When the container is in a vertical position, it does not extend over the entire diameter of the container, and some, but not all, of the liquid is in contact with the bottom surface of the container lid (e.g., the stopper). In some embodiments, the air bubbles are spherical. In some embodiments, the air bubbles are not spherical. In some such embodiments, the air bubbles are ovoid.

[0083] In one aspect of the present invention, the volume of the bubbles may be 120 μL or less, 110 μL or less, 100 μL or less, 90 μL or less, 80 μL or less, 70 μL or less, 60 μL or less, 50 μL or less, 40 μL or less, 30 μL or less, 20 μL or less, or 10 μL or less. In one aspect of the present invention, the volume of the bubbles is measured as the volume of the bubbles when all the bubbles contained in the container are integrated. The volume of the bubbles can be measured by a method such as "discharging gas and solution from the tip of a needle into an appropriate graduated container such as a pipette that already contains a solution, in that order, to actually measure the volume of the bubbles", "obtaining an image and calculating the volume of the bubbles from the area of ​​the bubble portion", or "calculating the volume of the bubbles from the height of the bubble portion based on known barrel inner diameter information".

[0084] In one aspect of the present invention, "homology modeling" is used to determine proteins that are at high risk of forming particles in solution. "Homology modeling" is a method for predicting the three-dimensional structure of a protein having a particular sequence based on the sequence similarity to one or more proteins with known three-dimensional structures. Homology modeling for a particular amino acid sequence typically involves the following steps: 1) identifying a homologue of the known structure in the Protein Data Bank, 2) aligning the sequence of interest with the template structure, 3) building a model based on the alignment, and 4) evaluating and refining the model (Xiang, Curr Protein Pept Sci. 2006 June; 7(3):217-227).The following software programs are available for predicting three-dimensional structures with homology modeling functions: Molecular Operating Environment (MOE; Chemical Computing Group Inc. (CCG) (Canada), Web Antibody Modelling (WAM; http: / / antibody.bath.ac.uk), Rosetta (https: / / www.rosettacommons.org / software), Prime (Schrodinger), MODELLER (Eswar, et al., Comparative Protein Structure Modeling With MODELLER. Current Protocols in Bioinformatics, John Wiley & Sons, Inc., Supplement 15, 5.6.1-5.6.30, 200.), SEGMOD / ENCAD (Levitt M. J Mol Biol 1992; 226:507-533), SWISS―MODEL (Schwede T, Kopp J, Guex N, Peitsch M C. Nucleic Acids Research 2003; 31 :3381-3385.), 3D-JIGSAW (Bates et al., Proteins: Structure, Function and Genetics, Suppl 2001; 5:39-46), NEST (Xiang, Curr Protein Pept Sci. 2006 June; 7(3): 217-227), and BUILDER (Koehl and Delarue, Curr Opin Struct Biol 1996; 6(2): 222-226). A preferred software is MOE.

[0085] In one aspect of the present invention, "antibody modeling" refers to a three-dimensional structure prediction function and database specialized for monoclonal antibodies. In antibody modeling, the whole structure can be assembled based on the structures of the fragments. For example, an antibody Fab fragment can be added to an Fc fragment crystal structure, or a Fab fragment can be formed as a predicted protein structure and added to an Fc fragment crystal structure. For example, this can be performed using the functions installed in MOE.

[0086] In one aspect of the invention, a "patch" refers to a surface region of a cluster of residues that exhibits a particular physicochemical property in the three-dimensional structure of a protein or antibody. Patches include hydrophobic patches and charge patches. A hydrophobic patch is a surface region where hydrophobic residues are clustered together. The clustered together of hydrophobic residues may also contain residues other than hydrophobic residues. A charge patch is a surface region where charged residues are clustered together. The clustered together of charged residues may also contain uncharged residues.

[0087] In one aspect of the present invention, the protein properties function of MOE can calculate a feature value related to the patch area of ​​a protein. With respect to the area of ​​the patch on the surface of a specific protein, the sum of the areas of the top five hydrophobic patches in the ranking by the area of ​​the hydrophobic patch is defined as X(Å 2 ), the total charge patch area is Y (Å 2 ), the particle formation risk of a protein is determined based on X+Y×1.5. In one embodiment, a protein for which the value of X+Y×1.5 is 1700 or more is determined to have a high particle formation risk. In another embodiment, a protein for which the value of X+Y×1.5 is 2000 or more, 2500 or more, 3000 or more, 3500 or more, or 4000 or more is determined to have a high particle formation risk.

[0088] In one aspect of the present invention, the protein properties function of MOE can calculate a feature related to the patch area of ​​a protein. The total charge patch area Y (Å) can be calculated for the area of ​​the patch on the surface of a specific protein. 2 ) and the particle formation risk of the protein is judged based on the above. In one embodiment, a protein having a Y value of 600 or more is judged to have a high particle formation risk. In another embodiment, a protein having a Y value of 700 or more, 800 or more, 900 or more, 1000 or more, 1500 or more, 2000 or more, 2500 or more, 3000 or more, or 4000 or more is judged to have a high particle formation risk.

[0089] Here, the ranking by hydrophobic patch area is a list of hydrophobic patches found on the protein surface arranged in order of area, and each hydrophobic patch means a hydrophobic patch consisting of a certain size of hydrophobic residue cluster that exists independently on the protein surface. The top five hydrophobic patch areas (Å 2 ) is calculated as X. Here, the sum of the areas of the top 5 hydrophobic patches refers to the total area of ​​the top 5 hydrophobic patches. However, when the number of hydrophobic patches in a molecule is 4 or less, it refers to the total area of ​​all the hydrophobic patches that exist.

[0090] The total charge patch area is the area (Å) of all positively or negatively charged charge patches present on the protein surface. 2 ) In one aspect of the present invention, a "molecular force field" is a parameterization of the type of force acting on each atom in a molecule as a function. In molecular mechanics calculations and molecular dynamics calculations based on a molecular force field, the forces acting between atoms are expressed as numerical values ​​using parameters representing the bonds between atoms (such as bond distance and bond angle) as variables, and a potential function determined by the type and bond style of the atoms. In molecular mechanics calculations and molecular dynamics calculations based on a molecular force field, the forces acting between atoms are expressed as numerical values ​​using parameters representing the bonds between atoms (such as bond distance and bond angle) as variables, and a potential function determined by the type and bond style of the atoms.

[0091] In one aspect of the present invention, the molecular force field that can be used is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include an Amber-based molecular force field, a CHARMm-based molecular force field, and an OPLS-based molecular force field. Examples of the Amber-based molecular force field include Amber10 / 14:EHT, Amber ff99SB-ILDN, and Amber 12SB. Examples of the CHARMm-based molecular force field include CHARMm36. Among these, Amber10:EHT is preferred when using MOE.

[0092] (2) Reduce particle formation In one aspect of the present invention, "reducing particle formation" refers to preventing the formation of visually detectable particles or reducing the number of particles formed in a pharmaceutical formulation solution in which visually detectable particles are formed under certain conditions by adjusting the volume of air bubbles. The reduction in the formation of visually detectable particles can be confirmed by counting the number of particles before and after adjusting the volume of air bubbles. The size and number of particles can be measured by light-shielding particle counting, microscopic particle counting, flow site particle image analysis, visual inspection, or by isolating particles and then subjecting them to microscopic infrared spectroscopy (IR) or microscopic Raman spectroscopy, preferably by a combination of visual inspection and microscopic infrared spectroscopy or microscopic Raman spectroscopy.

[0093] (3) Pharmaceutical preparations In one aspect of the present invention, the pharmaceutical preparation is a solution containing a protein as an active ingredient. The pharmaceutical preparation may be an injectable preparation.

[0094] In one aspect of the present invention, the injectable preparation is a pharmaceutical preparation that contains a protein as an active ingredient in a solution, and is filled in an injection container to be administered by injection. In one aspect of the present invention, the "obtained injectable formulation" refers to the injectable formulation obtained as the final product after adjusting the air volume.

[0095] 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.

[0096] In one aspect of the present invention, the protein used in the solution formulation includes, but is not limited to, an antibody, a fusion protein, an enzyme, a hormone, a cytokine, and a vaccine. More specifically, the protein includes a monoclonal antibody, a granulocyte colony-stimulating factor (G-CSF), a granulocyte macrophage colony-stimulating factor (GM-CSF), an erythropoietin (EPO), an interferon, an interleukin such as IL-1 or IL-6, a tissue plasminogen activator (TPA), a thrombopoietin, an urokinase, a serum albumin, a blood coagulation factor VIII, a leptin, a stem cell factor (SCF), and the like.

[0097] In one aspect of the present invention, the protein used in the pharmaceutical preparation has substantially the same biological activity as a physiologically active protein of a mammal, particularly a human, and includes naturally occurring proteins and those obtained by recombinant gene techniques. Proteins obtained by recombinant gene techniques include those having the same amino acid sequence as a naturally occurring protein, or those having one or more amino acid sequences deleted, substituted or added and having the biological activity.

[0098] In one aspect of the present invention, the concentration of the protein in the solution may be 0.1 mg / mL or more, in the range of 0.1 to 300 mg / mL, or in the range of 1 to 200 mg / mL. In one aspect of the present invention, the antibody used is not particularly limited as long as it binds to the desired antigen, and may be a polyclonal or monoclonal antibody, with monoclonal antibodies being preferred in terms of the ability to stably produce homogeneous antibodies. In addition, in one aspect of the present invention, the antibody used may be a monospecific or bispecific antibody, or an antibody having multiple specificities having three or more antigen recognition sites in the molecule.

[0099] 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.

[0100] In addition, in one aspect of the present invention, the immunoglobulin class of the antibody used is not particularly limited, and may be any class, such as IgG, such as IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, etc., although IgG is preferred, and IgG1, IgG2, and IgG4 are particularly preferred.

[0101] Furthermore, in one aspect of the present invention, the antibodies used include not only antibodies having a constant region and a variable region (full-length antibodies), but also antibody fragments such as Fv, Fab, F(ab)2, and minibodies such as bispecific antibodies such as single-chain Fv (scFv, sc(Fv)2) and scFv dimers having one or two sites in which the variable regions of the antibodies are linked via a linker such as a peptide linker, although full-length antibodies are preferred.

[0102] In one aspect of the present invention, the antibody used can be prepared by a known method. Hybridomas producing monoclonal antibodies can be prepared basically 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. When the antigen has low immunogenicity, it may be bound to a macromolecule having immunogenicity such as albumin and then immunized.

[0103] Alternatively, a recombinant antibody can be produced by cloning an antibody gene from a hybridoma, incorporating it into a suitable vector, and introducing it into a host using recombinant gene technology (see, for example, Carl, AK Borrebaeck, James, W. Larrick, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 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 desired antibody is obtained, it is linked to DNA encoding the desired antibody constant region (C region), 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, such as an enhancer or promoter. Next, the host cell is transformed with this expression vector to express the antibody.

[0104] 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.

[0105] A humanized antibody, also called a reshaped human antibody, is a product of transplanting the complementarity determining region (CDR) of a non-human mammal, such as a mouse antibody, into the complementarity determining region of a human antibody, and a general genetic recombination method for the antibody 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 prepared so as to have overlapping portions at the ends. The resulting DNA is linked to DNA encoding the constant region of a human antibody, 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 linked via the CDR is selected from one whose complementarity determining region forms a good antigen-binding site. If necessary, amino acids in the framework region of the variable region of the antibody may be replaced so that the complementarity determining regions of the reshaped human antibody form an appropriate antigen-binding site (Sato, K. et al., Cancer Res. (1993) 53, 851-856).

[0106] 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).

[0107] 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 (Kim SJ, Park Y, Hong HJ., Antibody engineering for the development of therapeutic antibodies., Mol Cells. 2005 Aug 31;20(1):17-29. Review.) In addition, a technique for enhancing antibody-dependent cellular cytotoxicity (ADCC) activity and complement-dependent cytotoxicity (CDC) activity by performing amino acid substitution in the Fc region of an IgG antibody is known (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 such effector functions but also improves the half-life of an antibody 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).

[0108] 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 known, an appropriate expression vector containing the sequence can be constructed 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 present invention, the antibody used also includes such a human antibody.

[0109] 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 Saccharomyces, e.g., Saccharomyces cerevisiae, and filamentous fungi such as Aspergillus, e.g., Aspergillus niger. When using prokaryotic cells, there are production systems using bacterial cells. Known examples of bacterial cells include E. coli and Bacillus subtilis. Antibodies can be obtained by introducing the desired antibody gene into these cells by transformation and culturing the transformed cells in vitro.

[0110] 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 have already been established in this field.

[0111] In one aspect of the present invention, the antibody of the present disclosure 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 may contain non-human variable regions (e.g., variable regions derived from a non-human primate such as a monkey, or a mouse, rat, hamster, or rabbit, etc.) and human constant regions.

[0112] 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.

[0113] 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).

[0114] In one aspect of the invention, the human frameworks to be used for humanization may include, for example, frameworks selected using the "best-fit" method (Sims et al. J. Immunol. 151:2296 (1993)), frameworks derived from consensus sequences of human antibodies of a particular 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)), and framework regions derived from screening of FR libraries (Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618(1996)).

[0115] 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 may be prepared by administering an immunogen to a transgenic animal that has been engineered to produce fully human antibodies or complete antibodies with human variable regions in response to an antigen. 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 also, e.g., 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 2007 / 0061900, which describes VELOCIMOUSE® technology. The human variable regions from intact antibodies produced by such animals may be further modified, e.g., by combining with different human constant regions.

[0116] In another aspect of the invention, human antibodies can also be produced by 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 may include, for example, US Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (trioma technology) is 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).

[0117] 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.

[0118] 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).

[0119] In a particular phage display method in one aspect of the invention, VH and VL repertoires can be cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, which can be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). The phage display antibody fragments, e.g., scFv and 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 single-origin antibodies to a broad range of non-self or self antigens, as described in Griffiths et al., EMBO J, 12: 725-734 (1993). In yet another embodiment, the naive library 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 No. 5,750,373, US2005 / 0079574, US2005 / 0119455, US2005 / 0266000, US2007 / 0117126, US2007 / 0160598, US2007 / 0237764, US2007 / 0292936, US2009 / 0002360.

[0120] Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments. 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.

[0121] 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 knob-in-hole technology (e.g., U.S. Patent No. 5,731,168). Multispecific antibodies can be made by manipulating electrostatic steering effects to create Fc heterodimeric molecules (e.g., WO2009 / 089004A1); cross-linking two or more antibodies or antibody fragments (e.g., US 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 (1993)); (1994)); by 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).

[0122] In one aspect of the invention, an antibody or antibody fragment thereof in this disclosure may be a "dual-acting Fab" or "DAF" that contains one antigen-binding site that binds to an antigen and another distinct antigen (e.g., US2008 / 0069820).

[0123] 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).

[0124] 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. Examples of antibodies used in pharmaceutical preparations include, but are not limited to, anti-tissue factor antibodies, anti-IL-6 receptor antibodies, anti-IL-6 antibodies, anti-glypican-3 antibodies, anti-CD3 antibodies, anti-CD20 antibodies, anti-GPIIb / IIIa antibodies, anti-TNF antibodies, anti-CD25 antibodies, anti-EGFR antibodies, anti-Her2 / neu antibodies, anti-RSV antibodies, anti-CD33 antibodies, anti-CD52 antibodies, anti-IgE antibodies, anti-CD11a antibodies, anti-VEGF antibodies, anti-VLA4 antibodies, anti-HM1.24 antigen antibodies, anti-parathyroid hormone-related peptide antibodies (anti-PTHrP antibodies), anti-ganglioside GM3 antibodies, anti-TPO receptor agonist antibodies, coagulation factor VIII surrogate antibodies, anti-IL31 receptor antibodies, anti-HLA antibodies, anti-AXL antibodies, anti-CXCR4 antibodies, anti-NR10 antibodies, and bispecific antibodies of Factor IX and Factor X.

[0125] Preferred reshaped humanized antibodies for use in pharmaceutical preparations include humanized anti-interleukin 6 (IL-6) receptor antibody (tocilizumab, hPM-1 or MRA, see WO92 / 19759), humanized anti-HM1.24 antigen monoclonal antibody (see WO98 / 14580), humanized anti-parathyroid hormone-related peptide antibody (anti-PTHrP antibody) (see WO98 / 13388), humanized anti-tissue factor antibody (see WO99 / 51743), anti-glypican-3 humanized IgG1κ antibody (codrituzumab, GC33, see WO2006 / 006693), anti-NR10 humanized antibody (see WO2009 / 072604), and bispecific humanized antibody of Factor IX and Factor X (ACE910, see WO2012 / 067176).

[0126] 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, surfactants (PEG, Tween, etc.), chelating agents (EDTA, etc.), and binders. 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, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, and may be used in combination with appropriate solubilizing agents such as alcohol (ethanol, etc.), polyalcohols (propylene glycol, PEG, etc.), nonionic surfactants (polysorbate 80, polysorbate 20, poloxamer 188, HCO-50), etc.

[0127] 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.

[0128] 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.

[0129] 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.

[0130] In one aspect of the present invention, the surfactant may be a nonionic surfactant, for example, sorbitan fatty acid esters such as sorbitan monocaprylate, sorbitan monolaurate, and sorbitan monopalmitate; glycerin fatty acid esters such as glycerin monocaprylate, glycerin monomyriate, and glycerin monostearate; polyglycerin fatty acid esters such as decaglyceryl monostearate, decaglyceryl distearate, and decaglyceryl monolinoleate; polyoxyethylene sorbitan fatty acid esters such as polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate, and polyoxyethylene sorbitan tristearate; polyoxyethylene sorbitan fatty acid esters such as polyoxyethylene sorbitan tetrastearate and polyoxyethylene sorbitan tetraoleate; polyoxyethylene glycerin fatty acid esters such as polyoxyethylene glyceryl monostearate; polyethylene polyethylene glycol fatty acid esters such as glycol distearate; polyoxyethylene alkyl ethers such as polyoxyethylene lauryl ether; polyoxyethylene polyoxypropylene glycol ether, polyoxyethylene polyoxypropylene propyl ether, polyoxyethylene polyoxypropylene cetyl ether, and other polyoxyethylene polyoxypropylene alkyl ethers; polyoxyethylene alkyl phenyl ethers such as polyoxyethylene nonylphenyl ether; polyoxyethylene hydrogenated castor oils such as polyoxyethylene castor oil and polyoxyethylene hydrogenated castor oil (polyoxyethylene hydrogen castor oil); polyoxyethylene beeswax derivatives such as polyoxyethylene sorbitol beeswax; polyoxyethylene lanolin derivatives such as polyoxyethylene lanolin; polyoxyethylene fatty acid amides such as polyoxyethylene stearic acid amide, and other surfactants having an HLB of 6 to 18; anionic surfactants, for example, alkyl sulfates having an alkyl group having 10 to 18 carbon atoms, such as sodium cetyl sulfate, sodium lauryl sulfate, sodium oleyl sulfate, and other alkyl sulfates having an alkyl group having 10 to 18 carbon atoms;Typical examples include polyoxyethylene alkyl ether sulfates having an average added mole number of ethylene oxide of 2 to 4 and an alkyl group having 10 to 18 carbon atoms, such as sodium polyoxyethylene lauryl sulfate; alkyl sulfosuccinate salts having an alkyl group having 8 to 18 carbon atoms, such as sodium lauryl sulfosuccinate; natural surfactants, such as lecithin, glycerophospholipids; phingophospholipids such as sphingomyelin; and sucrose fatty acid esters of fatty acids having 12 to 18 carbon atoms. In one aspect of the present invention, one or more of these surfactants can be added to the formulation.

[0131] Preferred surfactants are polyoxyethylene sorbitan fatty acid esters and polyoxyethylene polyoxypropylene alkyl ethers, particularly preferred are polysorbates 20, 21, 40, 60, 65, 80, 81, 85 and Pluronic® type surfactants, most preferred are polysorbate 20, 80 and Pluronic® F-68 (poloxamer 188).

[0132] In one aspect of the present invention, the amount of surfactant added to an antibody formulation is generally 0.0001 to 10% (mg / mL), preferably 0.001 to 5%, and more preferably 0.005 to 3%.

[0133] 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.

[0134] Examples of cryoprotectants include sugars such as trehalose, sucrose, and sorbitol. Examples of solution aids include polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinamide, polyoxyethylene sorbitan monolaurate, macrogol, castor oil fatty acid ethyl ester, and the like.

[0135] 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.

[0136] Examples of anti-adsorption agents include human serum albumin, lecithin, dextran, ethylene oxide-propylene oxide copolymer, hydroxypropyl cellulose, methyl cellulose, polyoxyethylene hydrogenated castor oil, and polyethylene glycol.

[0137] Examples of sulfur-containing reducing agents include those having a sulfhydryl group, such as N-acetylcysteine, N-acetylhomocysteine, thioctic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and its salts, sodium thiosulfate, glutathione, and thioalkanoic acids having 1 to 7 carbon atoms.

[0138] 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.

[0139] In one aspect of the invention, the pharmaceutical preparation is for the treatment of autoimmune diseases, immune diseases, infectious diseases, inflammatory diseases, nervous system diseases, and tumor and neoplastic diseases, including cancer. In certain embodiments, the pharmaceutical use is for the treatment of congestive heart failure (CHF), ischemia-induced severe arrhythmias, hypercholesterolemia, vasculitis, rosacea, acne, eczema, myocarditis and other conditions of the myocardium, Kawasaki disease, systemic lupus erythematosus, diabetes, spondylosis, synovial fibroblasts, and bone marrow stroma; bone loss; Paget's disease, giant cell tumor of bone; breast cancer; disuse bone loss; malnutrition, periodontal disease, Gaucher's disease, Langerhans cell histiocytosis, spinal cord injury, acute myocardial infarction, and chronic myocardial infarction. Septic arthritis, osteomalacia, Cushing's syndrome, monoostotic fibrous dysplasia, polyostotic fibrous dysplasia, periodontal remodeling, and fractures; sarcoidosis; melanoma, prostate cancer, pancreatic cancer, osteolytic bone cancer, breast cancer, lung cancer, gastric cancer, renal cancer, and rectal cancer; bone metastases, bone pain management, and humoral malignant hypercalcemia, ankylosing spondylitis, and other spondyloarthropathies; transplant rejection, viral infections, hematologic neoplasms, and neoplastic-like conditions, such as Hodgkin's lymphoma;Non-Hodgkin's lymphomas (Burkitt's lymphoma, small lymphocytic lymphoma / chronic lymphocytic leukemia, mycosis fungoides, mantle cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, marginal zone lymphoma, hairy cell leukemia, and lymphoplasmacytic leukemia), neoplasms of lymphoid precursor cells, including B-cell acute lymphoblastic leukemia / lymphoma and T-cell acute lymphoblastic leukemia / lymphoma, thymoma, mature T-cell and mature N-cell leukemia, including peripheral T-cell leukemia, adult T-cell leukemia / T-cell lymphoma, and large granular lymphocytic leukemia Myeloid neoplasms such as K-cell tumors, Langerhans cell histiocytosis, AML with maturation, AML without differentiation, acute myeloid leukemia including acute promyelocytic leukemia, acute myelomonocytic leukemia, and acute monocytic leukemia, myelodysplastic syndromes, and chronic myeloproliferative disorders including chronic myeloid leukemia, tumors of the central nervous system, such as brain tumors (glioma, neuroblastoma, astrocytoma, medulloblastoma, ependymoma, and retinoblastoma), solid tumors (nasopharyngeal carcinoma, basal cell carcinoma, pancreatic cancer, cholangiocarcinoma, Kaposi's sarcoma, testicular cancer, uterine cancer, vaginal cancer, or for the treatment of cervical cancer, ovarian cancer, primary liver cancer, or endometrial cancer, as well as tumors of the vascular system (angiosarcoma and hemangiopericytoma), osteoporosis, hepatitis, HIV, AIDS, spondyloarthritis, rheumatoid arthritis, inflammatory bowel disease (IBD), sepsis and septic shock, Crohn's disease, psoriasis, scleroderma, graft-versus-host disease (GVHD), allogeneic islet graft rejection, hematological malignancies such as multiple myeloma (MM), myelodysplastic syndromes (MDS), and acute myeloid leukemia (AML), inflammation associated with tumors, peripheral nerve injury, or demyelinating diseases. In certain embodiments, the use of the medicament is for the treatment of psoriasis vulgaris, pancreatitis, ulcerative colitis, non-Hodgkin's lymphoma, breast cancer, colorectal cancer, mesothelioma, soft tissue sarcoma, juvenile idiopathic arthritis, macular degeneration, respiratory syncytial virus, Crohn's disease, rheumatoid arthritis, psoriatic arthritis, Castleman's disease, ankylosing spondylitis, osteoporosis, treatment-induced bone loss, bone metastases, multiple myeloma, Alzheimer's disease, glaucoma, Sjogren's disease, Still's disease, multiple sclerosis, hyperimmunoglobulinemia, anemia, mesangial proliferative nephritis, and asthma;

[0140] In one aspect of the invention, the antigen to which the antibody has specific binding affinity can be a ligand, such as a transmembrane molecule (e.g., a receptor) or a growth factor. Exemplary antigens include molecules such as renin; growth hormones, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin; insulin A chain; insulin B chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factors, such as factor VIIIC, factor IX, tissue factor (TF), and von Willebrand factor; anticoagulants, such as protein C; atrial natriuretic factor; pulmonary surfactant; plasminogen activators, such as urokinase or human urinary or tissue-type plasminogen activator (t-PA); bombesin; thrombin; hematopoietic growth factors; tumor necrosis factor-alpha and -beta; enkephalinase; RANTES (regulated on activation normally T-cell expressed and secreted);human macrophage inflammatory protein (MIP-1-α);serum albumin such as human serum albumin;Müllerian inhibitory substance;relaxin A chain;relaxin B chain;prorelaxin;mouse gonadotropin-related peptide;microbial proteins such as beta-lactamase;DNAse;IgE;cytotoxic T-lymphocyte-associated antigen (CTLA) such as CTLA-4;inhibin;activin;vascular endothelial growth factor (VEGF);hormone or growth factor receptors;protein A or D;rheumatoid factor;neurotrophic factors, e.g. bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5 , or -6 (NT-3, NT-4, NT-5, or NT-6), or nerve growth factors, e.g., NGF-b; platelet-derived growth factor (PDGF); fibroblast growth factors, such as aFGF and bFGF; epidermal growth factor (EGF); transforming growth factors (TGFs), such as TGF-α and TGF-β, including TGF-b1, TGF-b2, TGF-b3, TGF-b4, or TGF-b5; tumor necrosis factors (TNFs), such as TNF-α or TNF-β; insulin-like growth factors-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding protein;CD proteins such as CD3, CD4, CD8, CD19, CD20, CD22 and CD40; erythropoietin; bone morphogenetic factors; antitoxins; bone morphogenetic proteins (BMPs); interferons such as interferon-α, -β and -γ; colony stimulating factors (CSFs) such as M-CSF, GM-CSF, G-CSF; interleukins (ILs) such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9 and IL-10; superoxide dismutase; T cell receptors; surface membrane proteins; decay accelerating factors; viral antigens such as parts of the AIDS envelope; transport proteins; homing receptors; addressins; regulatory proteins; integrins such as CD11a, CD11b, CD11c, CD18, ICAM, VLA-4 and VCAM; tumor associated antigens such as the HER2, HER3 or HER4 receptor; and fragments of any of the above listed polypeptides. ;

[0141] In one aspect of the invention, exemplary molecular targets of encompassed antibodies include CD proteins, such as CD3, CD4, CD8, CD19, CD20, CD22, CD34, and CD40; members of the ErbB receptor family, such as the EGF receptor, HER2, HER3, or HER4 receptor; B cell surface antigens, such as CD20 or BR3; members of the tumor necrosis receptor superfamily, including DR5; prostate stem cell antigen (PSCA); LFA-1, Mac1, p150.95, VLA-4, ICAM-1, VCAM, alpha These include cell adhesion molecules such as αv / β3 integrin, including α4 / β7 integrin and either its α or β subunits (e.g., anti-CD11a, anti-CD18 or anti-CD11b antibodies); growth factors, such as VEGF and its receptors; tissue factor (TF); tumor necrosis factor (TNF), such as TNF-α or TNF-β, α interferon (α-IFN); interleukins such as IL-8; IgE; blood group antigens; flk2 / flk3 receptors; obesity (OB) receptor; mp1 receptor; CTLA-4; protein C, and the like.

[0142] (4) Container In one aspect of the invention, the containers filled with the pharmaceutical formulation include syringes and cartridges.

[0143] In one aspect of the present invention, "prefilled syringe" refers to a syringe in which a liquid composition is filled in the syringe as a container. In some embodiments, the prefilled syringe is filled with a pharmaceutical composition for administration to a patient. Here, the syringe may be capped with a syringe closure, such as, but not limited to, a stopper. In some embodiments, the composition is filled into the syringe at a manufacturing filling facility. In some embodiments, the syringe is sterilized before the composition is filled into the syringe. In some embodiments, the prefilled syringe 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 before administration of the composition to a patient. In some embodiments, the prefilled syringe is exposed to storage and / or transportation conditions.

[0144] In one embodiment of the present invention, the prefilled syringe is subjected to mechanical stress.Mechanical stress includes, but is not limited to, drop stress, vibration stress, and rotation stress.In one embodiment of the present invention, the prefilled syringe 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 prefilled syringe when dropped varies depending on the height, direction, etc. in addition to the number of drops.The drop height is, for example, 38.1 cm as described in American Society for Testing and Materials (ASTM) D4169, but is not limited thereto. In addition, in order to apply the same degree of drop stress with good reproducibility, the prefilled syringe may be appropriately packaged so that the orientation of the prefilled syringe does not change during the drop. For example, the packaging may be "put in a tray and stack the trays," or "pack the stacked trays in a cardboard box numbered as shown in FIG. 2 with the needle tip of the prefilled syringe facing side 2," but is not limited thereto. Preferably, the drop height and direction are such that the side that is on the bottom during the drop is changed in the order of side 1, side 2, side 3, and side 4, and the syringe is dropped from a height of 38.1 cm. This is regarded as one set, and two sets of drops are performed with one drop stress.

[0145] As shown in FIG. 3, the prefilled syringe includes a syringe for administering a drug. The prefilled syringe also includes a stopper that is inserted into the syringe. The prefilled syringe also includes an injection needle that is connected to the syringe. The prefilled syringe also includes a cap that caps the injection needle. The drug is a liquid drug, for example, a protein preparation.

[0146] The syringe includes a barrel having a distal end and a proximal end, and is generally cylindrical. The barrel is a member that contains a medicine inside. In addition to a tip end and a base end, the barrel of this embodiment has a cylindrical part that connects the tip end and the base end (see FIG. 3). The barrel also has a flange part that extends outward (diametrically outward of the cylindrical part) from the entire outer circumference of the other end of the cylindrical part in the cylindrical axis direction.

[0147] The barrel is formed, for example, from a transparent material that can withstand the internal pressure applied when administering a drug. Specifically, the barrel is made of a resin that simply contains repeated cyclic olefins such as norbornene. More specifically, the barrel is made of a transparent resin such as COP (cycloolefin polymer), which is a homopolymer of cyclic olefins, or COC (cycloolefin copolymer), which is a copolymer of cyclic olefins and ethylene or the like. The barrel may be made of PP (polypropylene) or glass.

[0148] The inner surface of the barrel (for example, the inner surface of the cylindrical portion) may be coated with silicone oil as a lubricant in order to reduce the sliding resistance of the piston against the inner surface of the barrel.

[0149] In one aspect of the invention, the silicone oil is a polydimethylsiloxane. Some exemplary polydimethylsiloxanes include, but are not limited to, Dow Corning® 360 Medical Fluids, including, but not limited to, Dow Corning® 360 Medical Fluids having a viscosity of 350 centistokes, Dow Corning® 360 Medical Fluids having a viscosity of 1000 centistokes, Dow Corning® 360 Medical Fluids having a viscosity of 12,500 centistokes, and Dow Corning® MDX4-4159 fluid.

[0150] In one aspect of the present invention, the size (standard) of the volume of the syringe is not particularly limited. Specifically, in the case of a small-sized syringe with a volume of 0.5 mL to 5.0 mL, preferably 1 mL, the advantageous effect in one aspect of the present invention is remarkable. In addition, the volume of the solution contained in the 1 mL standard syringe is in the range of 0.1 to 1.2 mL, preferably in the range of 0.2 to 1.1 mL. The volume of the solution contained in the 2.5 mL standard syringe is in the range of 0.1 to 2.5 mL, preferably in the range of 0.3 to 2.3 mL. In addition, when the pharmaceutical preparation contains an aqueous solution, the volume of the aqueous solution contained in the 1 mL standard syringe is in the range of 0.1 to 1.2 mL, preferably in the range of 0.2 to 1.1 mL. The volume of the aqueous solution contained in the 2.5 mL standard syringe is in the range of 0.1 to 2.5 mL, preferably in the range of 0.3 to 2.3 mL.

[0151] 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.

[0152] In one aspect of the invention, the cartridge used is a standard injection cartridge made of plastic or glass. The dimensions and tolerances of the injection cartridge made of glass are specified in the international standard ISO 13926-1. The stoppers and closures (caps and disks) are described in the standard international standards ISO 13926-2 and 3. The dimensions and tolerances of the ready-to-fill or prefilled syringes are specified in the international standard ISO 11040-4. In one embodiment, the cartridge used is a injection cartridge made of plastic or glass that meets one or more of the above international standards. In another aspect of the invention, the cartridge used is a injection cartridge made of plastic or glass that does not meet an international standard such as ISO.

[0153] (5) System In one aspect of the present invention, a system for determining proteins that have a high risk of forming particles in a pharmaceutical preparation containing the protein as an active ingredient in a solution is provided. The system includes a means for creating a three-dimensional protein structure model from the amino acid sequence of the protein by homology modeling or antibody modeling, a means for identifying, on the surface of the obtained model, portions where hydrophobic residues are accumulated in clusters and portions where charged residues are accumulated in clusters as hydrophobic patches and charged patches, respectively, and a means for calculating the area of ​​each of the hydrophobic patches, and a means for calculating the sum of the areas of the top five hydrophobic patches ranked by area size (X(Å 2 )), and the total charge patch area (Y(Å 2 ) and means for determining that a protein for which X+Y×1.5 is 1700 or greater is a protein with a high risk of forming particles in a solution.

[0154] In one aspect of the present invention, the system is a system for carrying out a method for determining whether a protein has a high risk of forming particles in a solution, and can be implemented by installing the program described below in an apparatus, computer, etc. for determination.

[0155] In one aspect of the present invention, the program is a program that causes a computer to operate each means in the system, and is a computer program that can be installed in a general-purpose device to be used as the determination device for determining a protein that has a high risk of forming particles in a pharmaceutical preparation containing a protein as an active ingredient in a solution. In one aspect of the present invention, the computer program does not necessarily need to be installed in the determination device, and can be provided, for example, by being stored in a recording medium. Here, the "recording medium" refers to a medium that can carry a program that cannot occupy space by itself, and includes, for example, a flexible disk, a hard disk, a CD-R, a CD-RW, an MO (magneto-optical disk), a DVD-R, a DVD-RW, a flash memory, and the like. In addition, the computer program can be transmitted from a computer that stores the computer program to another computer or device through a communication line. In one aspect of the present invention, the computer program includes a computer program stored in such a computer and a computer program being transmitted.

[0156] (A program for an instrument that determines proteins with a high risk of forming particles in solution based on hydrophobic and charge patches) In one aspect of the present invention, the present invention relates to a program stored in a recording medium or used in an apparatus for determining proteins that are at high risk of forming particles in a pharmaceutical formulation containing a protein as an active ingredient in a solution based on hydrophobic patches and charge patches.

[0157] The program includes a means for creating a three-dimensional protein structure model from the amino acid sequence of the protein by homology modeling or antibody modeling, a means for identifying, on the surface of the obtained model, the areas where hydrophobic residues are clustered and the areas where charged residues are clustered as hydrophobic patches and charged patches, respectively, and a means for calculating the area of ​​each of the hydrophobic patches, and a means for calculating the sum of the areas of the top five hydrophobic patches ranked by area size (X(Å 2 )), and the total charge patch area (Y(Å 2 ) and determining that a protein for which X+Y×1.5 is 1700 or greater is a protein with a high risk of forming particles in a solution.

[0158] In the configuration diagram of the device (10) for determining proteins with a high risk of forming particles in a solution shown in FIG. 7, the means for creating the above-mentioned model is executed by a model creation unit (12) based on an amino acid sequence input from an input unit (11). The hydrophobic patch and the charge patch are identified, the area of ​​each is calculated, and the sum of the areas of the top five hydrophobic patches ranked by area size (X(Å) is calculated. 2 )), and the total charge patch area (Y(Å 2 ) is performed by a calculation unit (13). A means for determining that a protein for which X+Y×1.5 is 1700 or more is a protein with a high risk of forming particles in a solution is performed by a determination unit (14). The determination result is output by an output unit (15). Each of these means is executed, for example, by a CPU reading a computer program stored in a HDD.

[0159] This computer program cannot occupy space by itself, but can be stored in an information recording medium and distributed. Here, the "information recording medium" refers to, for example, a flexible disk, a hard disk, a CD-ROM, a CD-R, a CD-RW, an MO (magneto-optical disk), an MD, a DVD-R, a DVD-RW, a flash memory, an IC card, etc. By connecting these information recording media to the data input / output unit of the device, the computer program can be installed in a memory such as a HDD in the device. In addition, the computer program can be transmitted to the device from another computer that stores the computer program via a communication line, and installed in the internal memory such as a HDD.

[0160] FIG. 5 shows a flow of processing performed by an apparatus for determining proteins with a high risk of forming particles in a solution when the apparatus executes a computer program for the apparatus. The CPU of the apparatus reads the computer program for the apparatus stored in a memory such as a HDD of the apparatus, and creates a three-dimensional structure model of the protein from the amino acid sequence of the protein by homology modeling or antibody modeling. Next, the CPU reads the computer program, and specifies, from the created model, portions in which hydrophobic residues are accumulated in clusters and portions in which charged residues are accumulated in clusters as hydrophobic patches and charged patches, respectively, calculates the area of ​​each, and calculates the sum of the areas of the top five hydrophobic patches ranked by area size (X(Å 2 )), and the total charge patch area (Y(Å 2 Next, the CPU loads the computer program and determines that proteins for which X+Y×1.5 is 1700 or greater are at high risk of forming particles in the solution.

[0161] (A program for an instrument that determines proteins at high risk of forming particles in solution based on charge patches) In one aspect, the present invention relates to a program stored in a recording medium or used in an apparatus for determining, based on a charge patch, a protein that has a high risk of forming particles in a pharmaceutical formulation containing a protein as an active ingredient in a solution.

[0162] The program includes a means for creating a three-dimensional protein structure model from the amino acid sequence of the protein by homology modeling or antibody modeling, and a means for identifying the areas on the surface of the resulting model where charged residues are clustered as charge patches, and calculating the total charge patch area (Y(Å 2 ) and determining that a protein with Y of 600 or more is a protein with a high risk of forming particles in a solution.

[0163] The above-mentioned means for creating the model is executed by the model creation unit. The charge patch is identified and the total charge patch area (Y(Å 2 The means for calculating Y is implemented in the calculation unit. The means for determining that a protein with Y of 600 or more is a protein with a high risk of forming particles in a solution is implemented in the determination unit. Each of these procedures is executed, for example, by the CPU reading a computer program stored in the HDD.

[0164] This computer program cannot occupy space by itself, but can be stored in an information recording medium and distributed. Here, the "information recording medium" is, for example, a flexible disk, a hard disk, a CD-ROM, a CD-R, a CD-RW, an MO (magneto-optical disk), an MD, a DVD-R, a DVD-RW, a flash memory, an IC card, etc. These information recording media can be connected to a data input / output unit of an apparatus for determining proteins with a high risk of forming particles in a solution, and the computer program can be installed in a memory such as a HDD in the apparatus. In addition, the computer program can be transmitted from another computer storing the computer program to the above-mentioned computer through a communication line, and installed in a memory such as a HDD inside the computer.

[0165] FIG. 6 shows a flow of processing performed by an apparatus for determining proteins with a high risk of forming particles in a solution when the apparatus executes a computer program for the apparatus. The CPU of the apparatus reads the computer program for the apparatus stored in a memory such as a HDD of the apparatus, and creates a three-dimensional structure model of the protein from the amino acid sequence of the protein by homology modeling or antibody modeling. Next, the CPU reads the computer program, identifies a portion in the created model where charged residues are accumulated in a cluster as a charge patch, and calculates the total charge patch area (Y(Å 2 Next, proteins with Y of 600 or more are determined to be at high risk of forming particles in the solution. EXAMPLES

[0166] Example 1 Counting of visually detectable particles Six types of antibodies, mAb1 (H chain / SEQ ID NO: 1, L chain / SEQ ID NO: 2; tocilizumab), mAb2 (H chain / SEQ ID NO: 3 and 4: common L chain: SEQ ID NO: 5), mAb3 (H chain / SEQ ID NO: 6, L chain / SEQ ID NO: 7), mAb4 (humanized bispecific antibody with blood coagulation factor VIII (FVIII) cofactor functional replacement activity), mAb5 (anti-latent myostatin sweeping humanized antibody), mAb6 (combination of H chain / SEQ ID NO: 8 and L chain / SEQ ID NO: 9, and combination of H chain / SEQ ID NO: 11 and L chain / SEQ ID NO: 10; anti-HLA-DQ2.5 humanized bispecific antibody), were analyzed by antibody-containing solutions (mAb1-6: 50 mg / mL, buffer: 20 mmol / L histidine, stabilizer: 150 mmol / L arginine and 162 mmol / L aspartic acid, surfactant: 0.01 mg / mL poloxamer 188, pH 6.0) was prepared and filtered through a 0.22 μm filter, and 1.0 mL of the solution was filled into a COP syringe (1 mL standard) with a 27G needle sterilized by radiation (25 kGy) and stoppered. Visually detectable particle evaluation 1 was performed on the filled and stoppered samples immediately after stoppering, and samples that were determined to contain visually detectable particles in the syringe were excluded. Ten samples were used for the test for each antibody, but mAb-6 was more unstable than the other antibodies and had an extremely high frequency of visually detectable particle generation immediately after filling, so three samples of each were used for the test. Based on the calibration curve created by the bubble volume measurement and setting method below, the position of the stopper of the syringe was adjusted to prepare samples with bubble volumes of 120 μL and 10 μL. Visually detectable particle evaluation 2 was performed on samples with each bubble volume after storing them at 5°C for one day.

[0167] [Visually detectable particle rating 1] The outer surface of the sample syringe container was cleaned, and the syringe was gently rotated or inverted in front of a black background at a position directly under a white light source with a brightness of approximately 10,000 lx, and visually inspected with the naked eye for approximately 30 seconds to check for the presence or absence of visually detectable particles in the solution filled in the syringe.

[0168] [How to measure and set bubble volume] A COP syringe (1 mL standard) with a 27G needle containing an antibody solution-containing syringe sample filled with 1.0 mL of antibody-containing solution and stoppered with a stopper was oriented so that the needle was facing up, and the position of the stopper was adjusted so that the distance from the flange to the stopper was 11 mm by pushing the air out from the needle tip by raising the air bubbles to the base of the needle. The solution and all the air contained in the syringe were injected into a tube with an inner diameter of 0.5 mm. The bubble volume of the syringe was calculated by measuring the length of the air layer in the tube. This measurement was repeated four times, and when the distance from the flange to the stopper was 11 mm, the average bubble volume was 120 μL. Next, three samples were prepared by removing as much air as possible from the needle tip of the syringe, and the distance from the flange to the stopper was measured. The average distance from the flange to the stopper was 15.1 mm. The actual bubble volume was 3 μL. From these measurement results, a calibration curve of the distance from the flange to the stopper and the bubble volume was created, and the bubble volume was set based on the distance from the flange to the stopper. The bubble volume conditions set are as shown in Table 1 below.

[0169] [Table 1]

[0170] [Visually detectable particle assessment 2] The outer surface of the sample syringe container was cleaned, and the syringe was gently rotated or inverted in front of a black background at a position of brightness of about 8000 lx directly under a white light source, and visually inspected for about 30 seconds to check for the presence or absence of visually detectable particles in the solution filled in the syringe. For samples in which the presence of visually detectable particles was confirmed, the sample was gently rotated or inverted in front of a black background at a position of brightness of about 8000 lx directly under a white light source, and the number of visually detectable particles in the syringe was counted with the naked eye.

[0171] [Evaluation Results] The results of the Visually Detectable Particles in the Syringe Assessment 2 after storing the samples at 5° C. for 1 day are shown in Table 2 below.

[0172] [Table 2]

[0173] Example 2 Determination of conditions with high risk of particle formation 1. Creation of a 3D structure model of an antibody In the Antibody modeling function of Molecular Operating Environment (MOE), 2019.01 (Chemical Computing Group ULC), the amino acid sequences of six types of antibodies (mAb1-6) were input, and each complementarity determining region (CDR) was matched with an individual template, and by combining them, a 3D structural model of each antibody was created in the range of the full length of IgG. The calculation conditions used were the Chains: VL, VH options for monospecific antibodies, the Chains: bispecific option for some bispecific antibodies, and Ig Immunoglobulin for the Model type. For each antibody, the template structures of the framework region and the complementarity determining region were determined to be optimal using the crystal structure information evaluated as optimal by the Antibody modeling function. A gradient limit of 0.1 kcal / mol / Å was used as the threshold for the convergence gradient value for the energy minimization of the structure. 2 was applied. Other parameters were set to the default values. Amber10: EHT, the standard for MOE 2019.01, was used as the force field.

[0174] Residues that were not included in the antibody sequences of mAb2 and mAb3 but were automatically completed by MOE were deleted, and energy minimization was performed on the residues surrounding the deleted residues. No residue deletion was performed on mAb1.

[0175] 2. Calculation of physical properties of the three-dimensional structure model of an antibody Using the 3D structural models of the six types of antibodies (mAb1-6) created in 1 as input, the feature values ​​of each antibody were comprehensively calculated using the Protein properties function of MOE 2019.01. The default values ​​were used for all parameters except for the target pH, which was changed to 6. The output feature values ​​were saved as an MDB file.

[0176] For each antibody, four types of feature quantities related to the patches among the calculated feature quantities, specifically, the sum of the top 5 areas of all hydrophobic patches in area order (Patch_hyd_5), the sum of the areas of all hydrophobic patches (Patch_hyd), the sum of the top 5 areas of all charged patches in area order (Patch_ion_5), and the sum of the areas of all charged patches (Patch_ion) were extracted from the MDB file to examine the correlation with the experimental values. At that time, the correlation with the experimental values ​​was also examined for the combination of two types of feature quantities. To examine the correlation, the Pearson product-moment correlation coefficient and the Spearman rank correlation coefficient were used when each feature quantity was used as a single variable and the average number of particles that could be visually detected per syringe of a sample with a bubble volume of 120 μL shown in Table 2 of Example 1 was used as a single variable. The unit of area for all feature quantities is Å. 2 It is.

[0177] In all calculations using the above MOE, the molecular force field used was Amber10: EHT, which is the standard for MOE 2019.01. Patch_hyd_5, Patch_hyd, Patch_ion_5, Patch_ion, and their combined values ​​for six types of antibodies (mAb1-6) (all units are in Å) 2 ) and the Pearson's product moment correlation coefficient and Spearman's rank correlation coefficient with the average number of visually detectable particles per syringe for the sample with a bubble volume of 120 μL shown in Table 2 of Example 1 are shown in Table 3.

[0178] As a result, it was found that there was a high correlation between the Patch_ion value and the average number of particles detectable by visual inspection per syringe for samples with a bubble volume of 120 μL (product-moment correlation coefficient: 0.91). Furthermore, it was found that the sum of "Patch_hyd_5" and "(Patch_ion*1.5)" had the highest correlation with the average number of particles detectable by visual inspection per syringe for samples with a bubble volume of 120 μL (product-moment correlation coefficient: 0.92).

[0179] [Table 3]

[0180] The relationship between the average number of visually detectable particles per syringe and "Patch_hyd_5+(Patch_ion*1.5)" for the sample with a bubble volume of 120 μL shown in Table 2 of Example 1 is shown in Table 4 below. By comparing the number of visually detectable particles for the bubble volumes of 120 μL and 10 μL obtained in Example 1, the degree of reduction in the average number of visually detectable particles (reduction rate of visually detectable particles) was shown.

[0181] [Table 4]

[0182] Example 3: Test to confirm the formation of visually detectable particles of mAb2 A solution containing mAb2 (mAb2: 150 mg / mL, buffer: 20 mmol / L histidine, stabilizer: 150 mmol / L arginine and approximately 162 mmol / L aspartic acid, surfactant: 0.5 mg / mL poloxamer 188, pH 6.0) was filtered through a 0.22 μm filter, and then 1.0 mL of the solution was filled into a COP syringe (1 mL standard) with a 27G needle sterilized by radiation (25 kGy) and stoppered. Visually detectable particle evaluation 1 was performed on the filled and stoppered samples immediately after stoppering, and samples that were determined to contain visually detectable particles in the syringe were excluded. Based on the calibration curve obtained in Example 1, the position of the stopper of the syringe was adjusted to the desired bubble volume shown in Table 5. Samples with each bubble volume were stored at 5°C for approximately 7 months, then changed to storage at 25°C, and then stored at 25°C for 6 weeks. During the storage period at 25°C, the mechanical stress described below was applied three times, and after 6 weeks had passed, visually detectable particle evaluation 2 was performed. For samples in which the presence of visually detectable particles was confirmed, the visually detectable particles were identified by Raman spectrum measurement using a Raman Imaging Microscope (DXR2xi), and it was confirmed that the particles were derived from mAb2.

[0183] [Visually detectable particle rating 1] The presence or absence of visually detectable particles in the solution filled in the syringe was examined in the same manner as in Example 1 for evaluation of visually detectable particles 1, except that the brightness directly below the white light source was about 8000 lx.

[0184] [Table 5]

[0185] [Mechanical stress] Referring to ASTM D4169, the following drop test and vibration test were applied in the following order: drop test, vibration test, drop test.

[0186] [Drop test] The prefilled syringes were placed in a tray, and a total of three trays were stacked. The trays were stacked from the top in the following order: empty tray, sample tray, empty tray. The cardboard boxes were numbered as shown in Figure 2. The stacked trays were packed in the cardboard box so that the needle tips of the prefilled syringes faced side 2. The samples packed in the cardboard box were dropped from a height of 38.1 cm, alternating between side 1, side 2, side 3, and side 4, with the side that was on the bottom when dropped. This constituted one set, and two sets of drops were performed in one drop test.

[0187] [Vibration test] After placing the syringe samples in a tray, the tray was packed with cardboard and the cardboard was placed so that the barrel of the syringe was parallel to the ground. Vibration stress was applied to the cardboard at the following intensities: Truck Low 40 min, Truck Middle 15 min, Truck High 5 min, and Air level I 120 min.

[0188] [Visually detectable particle assessment 2] The presence or absence of visually detectable particles in the solution filled in the syringe was examined in the same manner as in Example 1 for visually detectable particle evaluation 1, except that the brightness directly below the white light source was about 6000 lx.

[0189] [Methods for identifying visually detectable particles] For all samples in which visually detectable particles were found to exist in the evaluation 2 of visually detectable particles after mechanical stress, the entire solution was suction filtered through a nickel filter with a pore size of 3 μm. The Raman spectrum of the largest foreign matter among the particles collected on the filter was obtained, and identification was performed to confirm that the particle was derived from mAb2.

[0190] [Evaluation Results] The results of identifying particles detectable by visual inspection after mechanical stress are shown in Table 6. As shown below, even when an appropriate amount of surfactant was added, proteinaceous particles detectable by visual inspection were observed in multiple samples at a bubble volume of 120 μL, which is the normal bubble volume. It was found that by reducing the bubble volume to 69 μL or less, it is possible to reduce the number of proteinaceous particles detectable by visual inspection that could not be suppressed even by adding a surfactant.

[0191] A histogram of the sizes of the identified proteinaceous visually detectable particles is shown in Figure 4. The size range of the proteinaceous visually detectable particles was 46.0 to 279 μm.

[0192] [Table 6]

[0193] Example 4: Confirmation test of visible particle formation of mAb3 A solution containing mAb3 (mAb3: 120 mg / mL, buffer: 20 mmol / L histidine, stabilizer: 150 mmol / L arginine and approximately 162 mmol / L aspartic acid, surfactant: 0.5 mg / mL poloxamer 188, pH 6.0) was filtered through a 0.22 μm filter, and then 1.0 mL was filled into a COP syringe (1 mL standard) with a 27G needle sterilized by radiation (25 kGy) and stoppered. Visible particle evaluation 1 was performed on the filled and stoppered samples immediately after stoppering, and samples that were determined to contain visible particles in the syringe were excluded. Based on the calibration curve obtained in Example 1, the stopper position of the syringe was adjusted to adjust the target bubble volume shown in Table 7. For samples with each bubble volume, mechanical stress was applied at the start of the test, and then the samples were stored at 40 ° C for 60 days, and then visible particle evaluation 2 was performed under the same conditions as visible particle evaluation 1 in this example.

[0194] [Visible Particle Rating 1] Using the same method as in Evaluation 1 of Visually Detectable Particles in Example 1, the syringe was gently rotated or inverted in front of a black background for 11 seconds or more and in front of a white background for 5 seconds or more at a brightness of 3000-3750 lx, and observed to check for the presence or absence of visible particles in the solution filled in the syringe.

[0195] [Table 7]

[0196] [Mechanical stress] Vibration stress was applied under the following conditions in accordance with ASTM D4169. Rotational stress was then applied 200 times under the following conditions.

[0197] [Vibration stress] The syringe sample was placed in the tub and the tub was positioned so that the barrel of the syringe was perpendicular to the ground. The tub was subjected to vibration stress at the following intensities: Truck Low 40 min, Truck Middle 15 min, Truck High 5 min, and Air level II 120 min.

[0198] [Rotational Stress] The syringe sample was placed in the tub and oriented so that the barrel of the syringe was perpendicular to the ground.The syringe was manually rotated fast enough to move the air inside the syringe.

[0199] [Evaluation Results] The results of visible particle evaluation 2 after storage at 40°C for 60 days are shown in Table 8 below. As in Table 6 of Example 3, visible particles were observed in multiple samples with a bubble volume of 120 μL, even when an appropriate amount of surfactant was contained. It was found that by reducing the bubble volume to 42 μL or less, it is possible to reduce the number of visible particles that could not be suppressed even by adding a surfactant.

[0200] [Table 8]

[0201] Example 5: Test to confirm the formation of visually detectable particles of mAb3 A solution containing mAb3 (mAb3: 120 mg / mL, buffer: 20 mmol / L histidine, stabilizer: 150 mmol / L arginine and 162 mmol / L aspartic acid, surfactant: 0.5 mg / mL poloxamer 188, pH 6.0) was filtered through a 0.22 μm filter, and then 2.0 mL of the solution was filled into a COP syringe (2.25 mL standard) with a 27G needle sterilized by radiation (25 kGy) and stoppered. Immediately after stoppering, the filled and stoppered samples were subjected to visually detectable particle evaluation 1, and samples that were determined to contain visually detectable particles in the syringe were excluded. Based on the calibration curve created by the bubble volume measurement and setting method described below, the stopper position of the syringe was adjusted to the desired bubble volume shown in Table 9. For samples with each bubble volume, visually detectable particle evaluation 2 was performed after storage at 25 °C for about 3 months. During storage, mechanical stress was applied three times in total: at the start of storage, two weeks after the start of storage, and three weeks after the start of storage.

[0202] [Visually detectable particle rating 1] The presence or absence of visually detectable particles in the solution filled in the syringe was examined in the same manner as in Example 1 for evaluation of visually detectable particles 1, except that the brightness directly below the white light source was about 8000 lx.

[0203] [How to measure and set bubble volume] A COP syringe (2.25 mL standard) with a 27G needle was filled with 2.0 mL of antibody-containing solution and sealed with a stopper. The needle was oriented upwards, and the air bubbles were pushed up to the base of the needle, pushing the air out from the tip of the needle to create a sample with as little air as possible removed. The distance from the flange to the stopper was 14.2 mm.

[0204] For the samples from which as much air as possible had been removed, a needle was inserted into the rubber stopper side of the sample using another syringe, and 120 μL of air was injected. The distance from the flange to the stopper was 12 mm.

[0205] From the above measurement results, a calibration curve of the distance from the flange to the stopper and the bubble volume was created, and the bubble volume was set according to the distance from the flange to the stopper. The bubble volume conditions set are as shown in Table 9 below.

[0206] [Table 9]

[0207] [Mechanical stress] With reference to ASTM D4169, at the start of storage, the following drop test and vibration test were combined in the order of drop test, vibration test, and drop test to apply stress. Two times, two weeks after the start of storage and three weeks after the start of storage, the stress of the drop test alone was applied by repeating two sets of the drop test. The drop test and vibration test were performed in the same manner as in Example 3.

[0208] [Visually detectable particle assessment 2] The presence or absence of visually detectable particles in the solution filled in the syringe was examined in the same manner as in Example 1 for visually detectable particle evaluation 1, except that the brightness directly below the white light source was about 6000 lx.

[0209] [Evaluation Results] The results of visually detectable particle evaluation after approximately 3 months of storage at 25° C. are shown in Table 8 below. Similar to Example 3, Table 6, visually detectable particles were observed in multiple samples at a bubble volume of 120 μL, even when the appropriate amount of surfactant was contained.

[0210] It was found that by reducing the bubble volume to 10 μL or less, it was possible to reduce the number of visually detectable particles, which could not be suppressed even by adding a surfactant.

[0211] [Table 10]

Claims

1. A method for determining a protein having a high risk of forming particles in a pharmaceutical preparation containing a protein as an active ingredient in a solution, comprising: Creating a three-dimensional structure model of a protein from the amino acid sequence of the protein by homology modeling or antibody modeling; Identifying the portions of the surface of the obtained model that correspond to clusters of hydrophobic residues and clusters of charged residues as hydrophobic patches and charged patches, respectively, and calculating the areas of each; The sum of the areas of the top five hydrophobic patches in the ranking by area size (X(Å ２ )), and the total charge patch area (Y(Å ２ ) and determining that a protein having a value of X+Y×1.5 of 1700 or more is a protein having a high risk of forming particles in a solution; The process wherein the particles have a particle size of 40 μm or more.

2. The method according to claim 1, wherein a protein for which X+Y×1.5 is 2000 or more is determined to be a protein with a high risk of forming particles in a solution.

3. A method for determining a protein having a high risk of forming particles in a pharmaceutical preparation containing a protein as an active ingredient in a solution, comprising: Creating a three-dimensional structure model of a protein from the amino acid sequence of the protein by homology modeling or antibody modeling; The parts of the surface of the obtained model that correspond to clusters of charged residues are identified as charge patches, and the total charge patch area (Y(Å ２ ) and determining that a protein having Y of 600 or more is a protein having a high risk of forming particles in a solution; The process wherein the particles have a particle size of 40 μm or more.

4. The method according to any one of claims 1 to 3, wherein the solution is an aqueous solution.

5. The method according to any one of claims 1 to 4, wherein the protein is a monoclonal antibody.

6. The method of claim 5 , wherein the monoclonal antibody is either a monospecific antibody or a bispecific antibody.

7. The method of any one of claims 1 to 6, wherein the homology modelling or antibody modelling is carried out using Molecular Operating Environment (MOE) software.

8. A method for reducing generation of particles in a solution in an injectable preparation in which a solution containing a protein as an active ingredient is filled in a container, comprising: The volume of the air bubbles in the container is 40 μL or less; the container is a syringe or a cartridge; A method, wherein the protein is a protein determined to have a high risk of forming particles in solution by the method according to any one of claims 1 to 7.

9. 9. The method of claim 8, comprising limiting the volume of air bubbles in the container to 10 μL or less.

10. A method for preparing an injectable formulation in which a solution containing a protein as an active ingredient is filled in a container, comprising the steps of: and filling the container with the solution so that the volume of air bubbles in the container in the resulting injectable preparation is 40 μL or less; the container is a syringe or a cartridge; A method, wherein the protein is a protein determined to have a high risk of forming particles in solution by the method according to any one of claims 1 to 7.

11. 10. The method of claim 9, comprising filling the container with the solution such that the volume of air bubbles in the container in the resulting injectable formulation is 10 μL or less.

12. 12. The method of claim 10 or 11, wherein the solution is an aqueous solution.

13. An injectable preparation in which a solution containing a protein as an active ingredient is filled in a container, The protein is determined to have a high risk of forming particles in a solution by the method according to any one of claims 1 to 6, the container is a syringe or a cartridge; An injectable formulation, wherein the volume of air bubbles in the container is 40 μL or less.

14. The injectable formulation according to claim 13, wherein the volume of air bubbles in the container is 10 μL or less.

15. 15. The injectable formulation according to claim 13 or 14, wherein the solution is an aqueous solution.

16. A system for determining a protein having a high risk of forming particles in a pharmaceutical preparation containing a protein as an active ingredient in a solution, comprising: A means for creating a three-dimensional structure model of a protein from the amino acid sequence of the protein by homology modeling or antibody modeling; a means for identifying, on the surface of the obtained model, a portion where hydrophobic residues are accumulated in clusters and a portion where charged residues are accumulated in clusters as hydrophobic patches and charged patches, respectively, and calculating the area of ​​each of them; The sum of the areas of the top five hydrophobic patches in the ranking by area size (X(Å ２ )), and the total charge patch area (Y(Å ２ ) and A means for determining that a protein having a value of X+Y×1.5 of 1700 or more is a protein having a high risk of forming particles in a solution, The system, wherein the particles have a particle size of 40 μm or more.

17. A system for determining a protein having a high risk of forming particles in a pharmaceutical preparation containing a protein as an active ingredient in a solution, comprising: A means for creating a three-dimensional structure model of a protein from the amino acid sequence of the protein by homology modeling or antibody modeling; The parts of the surface of the obtained model that correspond to clusters of charged residues are identified as charge patches, and the total charge patch area (Y(Å ２ ) and a means for determining that a protein having Y of 600 or more is a protein having a high risk of forming particles in a solution; The system, wherein the particles have a particle size of 40 μm or more.

Images