Stable pharmaceutical preparation

Abstract

A stable pharmaceutical preparation according to the present invention comprises: (A) an antibody binding to programmed cell death protein 1 (CD279, PD-1) or an antigen-binding fragment thereof; (B) a surfactant; (C) a stabilizer; (D) a buffering agent; and (E) an antioxidant. The stable pharmaceutical preparation according to the present invention is stable even while comprising an antibody binding to programmed cell death protein 1 (CD279, PD-1), has excellent long-term storage stability on the basis of excellent stability, particularly under accelerated and severe conditions, and can be administered intravenously or subcutaneously.

Description

Stable pharmaceutical preparations

[0001] The present invention relates to a stable pharmaceutical preparation comprising an antibody or an antigen-binding fragment thereof that binds to PD-1 (CD279, Programmed cell death protein 1).

[0002] PD-1 (CD279, Programmed cell death protein 1) is a surface protein of activated T cells, and PD-L1 (CD274, Programmed death-ligand 1) and PD-L2 (CD273, Programmed cell death-ligand 2), which are surface proteins of cancer cells, can bind to PD-1 and neutralize T cells. By blocking the binding between PD-1 and PD-L1 and PD-L2 through immunotherapy, the evasion function of cancer cells can be suppressed, and this can prevent the worsening of symptoms of PD-1-related diseases.

[0003] Pembrolizumab, which may be a component of the above immunotherapy, is a humanized monoclonal antibody that selectively binds to and neutralizes PD-1 (CD279, Programmed cell death protein 1), and was approved for use by the European Medicines Agency (EMA) and the US Food and Drug Administration (FDA) in July 2015 and October 2016, respectively.

[0004] However, antibodies that bind to PD-1 are relatively unstable proteins and can undergo physical or chemical changes due to factors such as heat and light stress. Therefore, long-term storage of the product may impair its stability and / or activity.

[0005] International Patent Publication No. 2012-135408 discloses a lyophilized pharmaceutical formulation containing an anti-human PD-1 antibody. However, this formulation requires reconstitution with a diluent before use, which increases the risk of pharmaceutical contamination during the reconstitution step.

[0006] Therefore, there is a need for the development of a stable pharmaceutical formulation that does not require a reconstitution step after lyophilization and has excellent long-term storage stability while containing antibodies.

[0007] The problem to be solved by the present invention is to provide a stable pharmaceutical preparation comprising an antibody that binds to PD-1 (CD279, Programmed cell death protein 1).

[0008] Another problem to be solved by the present invention is to provide a vial filled with the pharmaceutical preparation.

[0009] Another problem to be solved by the present invention is to provide a cartridge filled with the pharmaceutical preparation.

[0010] Another problem to be solved by the present invention is to provide a pre-filled syringe filled with the pharmaceutical preparation.

[0011] Another problem to be solved by the present invention is to provide an auto-injector having the pre-filled syringe included therein.

[0012] To solve the above problem, the present invention provides a stable pharmaceutical formulation comprising (A) an antibody or antigen-binding fragment thereof that binds to PD-1 (CD279, Programmed cell death protein 1); (B) a surfactant; (C) a stabilizer; (D) a buffer; and (E) an antioxidant.

[0013] In one embodiment of the present invention, the (A) antibody or antigen-binding fragment thereof may include a light chain variable region comprising a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 1, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 2, and a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3; and a heavy chain variable region comprising a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 4, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 6.

[0014] In one embodiment of the present invention, the (A) antibody or antigen-binding fragment thereof may include a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7; and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8.

[0015] In one embodiment of the present invention, the (A) antibody or antigen-binding fragment thereof may include a light chain comprising the amino acid sequence of SEQ ID NO: 9; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 10.

[0016] In one embodiment of the present invention, the (A) antibody or antigen-binding fragment thereof may be Pembrolizumab.

[0017] In one embodiment of the present invention, the pharmaceutical preparation may be in liquid form.

[0018] In one embodiment of the present invention, the pharmaceutical preparation may be used for treating a disease related to PD-1 (CD279, Programmed cell death protein 1).

[0019] In one embodiment of the present invention, the concentration of the (A) antibody or antigen-binding fragment thereof may be 1 to 300 mg / ml.

[0020] In one embodiment of the present invention, the (B) surfactant may include polysorbate, poloxamer or a mixture thereof.

[0021] In one embodiment of the present invention, the polysorbate may include at least one selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

[0022] In one embodiment of the present invention, the polysorbate may include polysorbate 80.

[0023] In one embodiment of the present invention, the concentration of the (B) surfactant may be 0.001 to 1% (w / v).

[0024] In one embodiment of the present invention, the (C) stabilizer may include at least one selected from the group consisting of amino acids or amino acid derivatives; and sugars or sugar alcohols.

[0025] In one embodiment of the present invention, the amino acid or amino acid derivative may include at least one selected from the group consisting of threonine, arginine, proline, leucine, glycine, phenylalanine, tryptophan, glutamine, aspartate, glutamate, alanine, asparagine, serine, and tyrosine.

[0026] In one embodiment of the present invention, the sugar or sugar alcohol may include at least one selected from the group consisting of trehalose, mannitol, sorbitol, glucose, fructose, galactose, xylose, maltose, lactose, xylitol, D-maltitol, inositol, lactitol, and isomalt.

[0027] In one embodiment of the present invention, the concentration of the amino acid or amino acid derivative may be 10 to 450 mM.

[0028] In one embodiment of the present invention, the concentration of the sugar or sugar alcohol may be 0.1 to 30% (w / v).

[0029] In one embodiment of the present invention, the (C) stabilizer may include sorbitol.

[0030] In one embodiment of the present invention, the (D) buffer may include at least one selected from the group consisting of acetic acid or a salt thereof, phosphoric acid or a salt thereof, citric acid or a salt thereof, succinic acid or a salt thereof, glutamic acid or a salt thereof, 2-(N-morpholino)ethanesulfonic acid, and tris or a salt thereof.

[0031] In one embodiment of the present invention, the concentration of the (D) buffer may be 0.1 to 50 mM.

[0032] In one embodiment of the present invention, the (D) buffer may include acetic acid or a salt thereof.

[0033] In one embodiment of the present invention, the (E) antioxidant may include at least one selected from the group consisting of methionine, ascorbic acid or a salt thereof, and taurine.

[0034] In one embodiment of the present invention, the (E) antioxidant may include methionine.

[0035] In one embodiment of the present invention, the concentration of the (E) antioxidant may be 0.1 to 100 mM.

[0036] In one embodiment of the present invention, the pH of the pharmaceutical preparation may be 4 to 7.

[0037] In one embodiment of the present invention, the pharmaceutical preparation may not contain sucrose.

[0038] In one embodiment of the present invention, the pharmaceutical preparation may not contain histidine.

[0039] In one embodiment of the present invention, the pharmaceutical preparation may be for intravenous administration or subcutaneous administration.

[0040] In addition, the present invention provides a stable pharmaceutical formulation comprising: (A) 1 to 300 mg / ml of an antibody or antigen-binding fragment thereof, comprising a light chain variable region comprising a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 1, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 2, and a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3; and a heavy chain variable region comprising a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 4, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 6; (B) 0.001 to 1% (w / v) of a surfactant; (C) 0.1 to 30% (w / v) of a sugar or sugar alcohol; (D) 0.1 to 50 mM of a buffer; and (E) 0.1 to 100 mM of an antioxidant.

[0041] In one embodiment of the present invention, the (C) sugar or sugar alcohol may include sorbitol, the (D) buffer may include acetic acid or a salt thereof, and the (E) antioxidant may include methionine.

[0042] In addition, the present invention provides a vial filled with a stable pharmaceutical preparation according to the present invention.

[0043] In addition, the present invention provides a cartridge filled with a stable pharmaceutical preparation according to the present invention.

[0044] In addition, the present invention provides a pre-filled syringe filled with a stable pharmaceutical preparation according to the present invention.

[0045] In addition, the present invention provides an auto-injector having a pre-filled syringe according to the present invention included therein.

[0046] The stable pharmaceutical preparation according to the present invention has excellent long-term storage stability based on stability under accelerated and harsh conditions, even though it contains an antibody that binds to PD-1 (CD279, Programmed cell death protein 1), and can be administered intravenously or subcutaneously.

[0047] The present inventors conducted repeated research to overcome the above-described problems and developed a stable pharmaceutical preparation containing an antibody that binds to PD-1 (CD279, Programmed cell death protein 1).

[0048] The present invention provides a stable pharmaceutical formulation comprising (A) an antibody or antigen-binding fragment thereof that binds to PD-1 (CD279, Programmed cell death protein 1); (B) a surfactant; (C) a stabilizer; (D) a buffer; and (E) an antioxidant.

[0049] In one embodiment of the present invention, the pharmaceutical preparation may be in a liquid form, but is not limited thereto.

[0050]

[0051] (A) Antibody or antigen-binding fragment thereof

[0052] In the present invention, the term "antibody" refers to an immunoglobulin molecule composed of four polypeptide chains, two heavy chains and two light chains, which are connected to each other by disulfide bonds, and may include other naturally occurring antibodies with altered structures, such as camelid antibodies.

[0053] The heavy chain is composed of a heavy chain variable region and a heavy chain constant region. The heavy chain constant region is composed of three domains (CH1, CH2, and CH3), and the light chain is composed of a light chain variable region and a light chain constant region.

[0054] The light chain constant region is composed of a single domain (CL), and the heavy chain variable region and light chain variable region can be further divided into more conserved regions called framework regions (FR) and hypervariable regions called complementarity determining regions (CDRs) arranged together. Each heavy chain variable region and light chain variable region is composed of three CDRs and four FRs, which are arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0055] In one embodiment of the present invention, the antibody or antigen-binding fragment thereof according to the present invention may be a polyclonal antibody, a monoclonal antibody, a recombinant antibody, a single-chain antibody, a hybrid antibody, a chimeric antibody, a humanized antibody or a fragment thereof (antigen-binding fragment), and preferably a monoclonal antibody or an antigen-binding fragment thereof.

[0056] The above monoclonal antibody or antigen-binding fragment thereof may be a humanized monoclonal antibody or a fragment thereof, preferably a humanized immunoglobulin (Immunoglobulin G, IgG) monoclonal antibody, and may be prepared by a method known in the art.

[0057] In the present invention, the antigen-binding fragment includes any naturally occurring, enzymatically obtainable, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex, and examples thereof include, but are not limited to, Fab, F(ab'), F(ab')2, Fv, dAb, Fd, single-chain antibody fragment (scFv), scFv-Fc, complementarity determining region (CDR) fragment, bivalent single-chain antibody fragment, single-chain phage antibody fragment, diabody, triabody, tetrabody or nanobodies.

[0058] In the present invention, the humanized antibody is also called a reshaped human antibody, and may be produced by grafting the complementarity determining region (CDR) of a mammal other than a human, for example, a mouse antibody, onto the complementarity determining region of a human antibody, or may be produced by any other generally known genetic recombination technique.

[0059] In the present invention, the antibody or antigen-binding fragment thereof may include a light chain variable region comprising a CDR1 domain comprising an amino acid sequence of SEQ ID NO: 1, a CDR2 domain comprising an amino acid sequence of SEQ ID NO: 2, and a CDR3 domain comprising an amino acid sequence of SEQ ID NO: 3; and a heavy chain variable region comprising a CDR1 domain comprising an amino acid sequence of SEQ ID NO: 4, a CDR2 domain comprising an amino acid sequence of SEQ ID NO: 5, and a CDR3 domain comprising an amino acid sequence of SEQ ID NO: 6.

[0060] In the present invention, the antibody or antigen-binding fragment thereof may include a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7; and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8.

[0061] In the present invention, the antibody or antigen-binding fragment thereof may include a light chain comprising the amino acid sequence of SEQ ID NO: 9; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 10.

[0062] In the present invention, (A) the antibody may be Pembrolizumab, Nibolumab or a mixture thereof, preferably Pembrolizumab.

[0063] In the present invention, the 'Pembrolizumab' may be an antibody against human programmed death receptor PD-1 described in International Patent Publication No. 2012-135408, an original drug substance well known in the art, or a biosimilar thereof.

[0064] In the present invention, the 'Nibolumab' is Opdivo ® It may be a PD-1 (CD279, Programmed cell death protein 1) binding antibody or a biosimilar thereof, also known as a PD-1 inhibitor.

[0065] In one embodiment of the present invention, the concentration of (A) the antibody or antigen-binding fragment thereof may be 1 to 300 mg / mL, preferably 5 to 200, more preferably 10 to 100, and most preferably 15 to 35 mg / mL. When the concentration of the antibody or antigen-binding fragment thereof is within the above-described range, the degree of freedom in administration dosage and administration cycle may be increased, and long-term stability may be excellent.

[0066]

[0067] (B) Surfactant

[0068] In the present invention, the term "surfactant" means a substance that can be used to significantly increase the water solubility of a hydrophobic or oily substance or to increase the miscibility of two substances having different hydrophobicities.

[0069] In the present invention, the surfactant is a polyoxyethylene sorbitan fatty acid ester (e.g., polysorbate, etc.), a polyoxyethylene alkyl ether (e.g., Brij ®etc.), alkylphenylpolyoxyethylene ethers (e.g., Triton-X etc.), polyoxyethylene-polyoxypropylene copolymers (e.g., poloxamers, Pluronic ® etc.), sodium dodecyl sulfate (SDS), etc., but are not limited thereto.

[0070] In one embodiment of the present invention, the surfactant may include polysorbate, poloxamer or a mixture thereof, and preferably may include polysorbate.

[0071] In the present invention, the polysorbate may include at least one selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80, and preferably may include polysorbate 80.

[0072] In one embodiment of the present invention, the concentration of the surfactant can be freely adjusted within a range that does not adversely affect the stability and viscosity of the stable pharmaceutical preparation according to the present invention.

[0073] In one embodiment of the present invention, the concentration of the (B) surfactant may be 0.001 to 1% (w / v), preferably 0.005 to 0.5% (w / v), more preferably 0.01 to 0.1% (w / v), and most preferably 0.015 to 0.05% (w / v). When the concentration of the surfactant is within the above-described range, it may be excellent in terms of long-term stability.

[0074]

[0075] (C) Stabilizer

[0076] In the present invention, the term "stabilizer" refers to a substance that is physiologically acceptable and imparts stability to the formulation.

[0077] In one embodiment of the present invention, (C) the stabilizer may include at least one selected from the group consisting of i) an amino acid or an amino acid derivative; and ii) a sugar or a sugar alcohol.

[0078] In one embodiment of the present invention, the i) amino acid or amino acid derivative (different from the component included as a buffer or antioxidant according to the present invention) may include at least one selected from the group consisting of threonine, arginine, proline, leucine, glycine, phenylalanine, tryptophan, glutamine, aspartate, glutamate, alanine, asparagine, serine, and tyrosine, and may preferably include proline, but is not limited thereto.

[0079] In one embodiment of the present invention, the ii) sugar or sugar alcohol may include at least one selected from the group consisting of trehalose, sorbitol, glucose, fructose, galactose, xylose, maltose, lactose, xylitol, mannitol, D-maltitol, inositol, lactitol, and isomalt, and may preferably include sorbitol, but is not limited thereto.

[0080] In one embodiment of the present invention, the pharmaceutical preparation of the present invention may not include sucrose as a stabilizer in terms of long-term stability.

[0081] In the present invention, the concentration of the stabilizer can be freely adjusted within a range that does not substantially adversely affect the stability and viscosity of the pharmaceutical preparation according to the present invention.

[0082] In one embodiment of the present invention, when the stabilizer comprises i) an amino acid or amino acid derivative, the concentration of the amino acid or amino acid derivative may be 10 to 450 mM, preferably 100 to 350 mM, more preferably 150 to 300 mM, and most preferably 180 to 270 mM.

[0083] In one embodiment of the present invention, when the stabilizer comprises ii) sugar or sugar alcohol, the concentration of the sugar or sugar alcohol may be 0.1 to 30% (w / v), preferably 0.5 to 20% (w / v), more preferably 1 to 15% (w / v), and most preferably 2 to 10% (w / v). When the concentration of the stabilizer is within the above-described range, it may be excellent in terms of long-term stability.

[0084]

[0085] (D) Buffer

[0086] In the present invention, the term "buffer" refers to a neutralizing substance that minimizes changes in pH caused by acids or alkalis.

[0087] In one embodiment of the present invention, the (D) buffer may include at least one selected from the group consisting of acetic acid or a salt thereof, phosphoric acid or a salt thereof, citric acid or a salt thereof, succinic acid or a salt thereof, glutamic acid or a salt thereof, 2-(N-morholino) ethanesulfonic acid (MES), tromethamine (Tris) or a salt thereof, and may preferably include acetic acid or a salt thereof, but is not limited thereto.

[0088] The above acetate may be, for example, sodium acetate, zinc acetate, aluminum acetate, ammonium acetate, potassium acetate, etc., but is not limited thereto.

[0089] The above phosphate may be, for example, potassium phosphate, sodium phosphate, ammonium phosphate, calcium phosphate, magnesium phosphate, etc., but is not limited thereto.

[0090] The above citrate may be, for example, sodium citrate, calcium citrate, potassium citrate, etc., but is not limited thereto.

[0091] The above succinate may be, for example, sodium succinate, calcium succinate, potassium succinate, sodium sulfosuccinate, potassium sulfosuccinate, calcium sulfosuccinate, etc., but is not limited thereto.

[0092] The above glutamate may be, for example, sodium glutamate, potassium glutamate, ammonium glutamate, etc., but is not limited thereto.

[0093] The above 2-(N-morpholino)ethanesulfonic acid (MES) salt may be, but is not limited to, MES chlorite, MES sodium, etc.

[0094] The above tris salt may be, for example, tris chloride, tris acetate, tris borate, etc., but is not limited thereto.

[0095] In one embodiment of the present invention, the pharmaceutical preparation of the present invention may not contain histidine or a salt thereof as a buffer in terms of long-term stability.

[0096] In the present invention, the concentration of the buffer can be freely adjusted within a range that does not substantially adversely affect the stability and viscosity of the pharmaceutical preparation according to the present invention.

[0097] In one embodiment of the present invention, the concentration of the (D) buffer may be 0.1 to 50 mM, preferably 1 to 50 mM, more preferably 1 to 30 mM, and most preferably 5 to 15 mM. When the concentration of the buffer is within the above-described range, it may be excellent in terms of long-term stability.

[0098]

[0099] (E) Antioxidant

[0100] In the present invention, the term "antioxidant" refers to a substance that inhibits the activity of antibodies and the deterioration of the stability of pharmaceutical preparations due to oxidation.

[0101] In one embodiment of the present invention, the (E) antioxidant may include at least one selected from the group consisting of methionine, ascorbic acid or a salt thereof, and taurine, and may preferably include methionine, but is not limited thereto.

[0102] The above ascorbate may be, for example, sodium ascorbate, calcium ascorbate, potassium ascorbate, magnesium ascorbate, etc., but is not limited thereto.

[0103] In one embodiment of the present invention, the concentration of the antioxidant may be 0.1 to 100 mM, preferably 10 to 80 mM, more preferably 35 to 70 mM, and most preferably 55 to 65 mM. When the concentration of the antioxidant is within the above-described range, it may be excellent in terms of long-term stability.

[0104]

[0105] (F) pH adjuster

[0106] In the present invention, the term "pH adjusting agent" refers to a substance used to adjust the pH of a stable pharmaceutical preparation.

[0107] The pH of the stable pharmaceutical formulation according to the present invention can be adjusted to a range that optimizes therapeutic efficacy using a regulator. In the present invention, the pH of the stable pharmaceutical formulation may be 4 to 7, preferably 5 to 6, more preferably 5.2 to 5.5, and most preferably 5.2.

[0108] In one embodiment of the present invention, when a buffer is included in a predetermined amount, the pH within the above range can be achieved without a separate pH adjuster. In one embodiment of the present invention, a separate pH adjuster (acid or base) may be additionally included within a range that does not impair the stability of the pharmaceutical preparation and antibody.

[0109] In the present invention, the content of the pH regulator can be freely adjusted within a range that does not substantially adversely affect the stability and viscosity of the pharmaceutical preparation according to the present invention.

[0110] In one embodiment of the present invention, the (F) pH adjusting agent may be hydrochloric acid, acetic acid, phosphoric acid, sodium hydroxide, sodium bicarbonate, or a mixture thereof.

[0111]

[0112] (G) Other ingredients

[0113] In the present invention, the stable pharmaceutical preparation according to the present invention may not contain a preservative. The preservative may be, for example, octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol, benzyl alcohol, alkyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, m-cresol, etc. If a preservative is included, it may have a negative effect on improving stability.

[0114] As used herein, the terms "does not comprise" or "does not comprise" mean not comprising any component at all. Furthermore, the terms mean substantially not comprising any component, i.e., comprising any component to an extent that does not affect the activity of the antibody, the stability, or the viscosity of the pharmaceutical formulation. For example, it means comprising any component in an amount of 1 ppm (w / v) or less or 1 ppb (w / v) or less based on the total weight of the pharmaceutical formulation.

[0115] In the present invention, the stable pharmaceutical preparation of the present invention may further include an additive known in the art within a range that does not substantially adversely affect the stability and viscosity of the preparation. The additive may be, for example, an aqueous carrier, an antioxidant, or a mixture thereof. In the present invention, the aqueous carrier may be a carrier useful for the manufacture of a pharmaceutical preparation that is safe and non-toxic when administered to humans. Examples of the aqueous carrier include, but are not limited to, sterile water for injection (SWFI), bacteristetic water for injection (BWFI), sterile saline solution, Ringer's solution, dextrose, and the like.

[0116]

[0117] (H) "Stable" pharmaceutical preparations

[0118] In the present invention, the term "stable" or "stabilization" means that a component according to the present invention, or a composition or formulation containing the same, exhibits physical stability, chemical stability, and / or biological activity during the manufacturing process and / or during storage or preservation. Various analytical techniques for measuring stability in the present invention are readily available in the art.

[0119] In the present invention, the physical stability can be assessed by methods known in the art, such as measuring the apparent attenuation of light (absorbance or optical density) in a sample. This attenuation measurement is related to the turbidity of the formulation. Furthermore, physical stability can be assessed by measuring the content of high-molecular-weight components, the content of low-molecular-weight components, and the amount of intact protein.

[0120] In one embodiment of the present invention, the term “stable” pharmaceutical formulation means a pharmaceutical formulation that satisfies one or more of the following (H)-1 to (H)-7.

[0121]

[0122] (H)-1 HIC-HPLC main peak

[0123] - Main peak measured by HIC-HPLC after storage at 5±3℃ for 3 days, 7 days, 2 weeks or 4 weeks is 91% to 100%;

[0124] - The main peak measured by HIC-HPLC after storage for 18 months at a temperature of 5±3℃ was 92% to 100%;

[0125] - The main peak measured by HIC-HPLC is 91% to 100% after storage for 3 days, 7 days, 2 weeks, or 4 weeks under the conditions of temperature 40±2℃ and relative humidity 75±5%;

[0126] - The main peak measured by HIC-HPLC after storage for 3 or 7 days at 45℃ is 91% to 100%;

[0127] - The main peak measured by HIC-HPLC was 89% to 100% after storage for 72 hours under the conditions of 12,000 Lux light irradiation, temperature 25±2℃, and relative humidity 60±5%; and

[0128] - A pharmaceutical preparation having a main peak of 92% to 100% as measured by HIC-HPLC after storage for 5 hours under 1,000 RPM stirring conditions.

[0129]

[0130] (H)-2 HIC-HPLC pre-peak (peak with a retention time earlier than the main peak)

[0131] - Free peak of 0 to 5% measured by HIC-HPLC after storage at a temperature of 5±3℃ for 3 days, 7 days, 2 weeks or 4 weeks;

[0132] - Free peak of 0% to 5% measured by HIC-HPLC after storage for 18 months at a temperature of 5±3℃;

[0133] - Free peak of 0 to 5% measured by HIC-HPLC after storage for 3 days, 7 days, 2 weeks or 4 weeks under conditions of temperature 40±2℃ and relative humidity 75±5%;

[0134] - Free peak of 0 to 5% measured by HIC-HPLC after storage for 3 or 7 days at 45℃;

[0135] - Free peak of 0 to 8% measured by HIC-HPLC after storage for 72 hours under conditions of 12,000 Lux light irradiation, temperature 25±2℃, and relative humidity 60±5%; and

[0136] - A pharmaceutical preparation having a free peak of 0 to 5% as measured by HIC-HPLC after storage for 5 hours under 1,000 RPM stirring conditions.

[0137]

[0138] (H)-3 HIC-HPLC post-peak (peak with a retention time later than the main peak)

[0139] - Post peak of 0 to 4% measured by HIC-HPLC after storage at 5±3℃ for 3 days, 7 days, 2 weeks or 4 weeks;

[0140] - Post peak measured by HIC-HPLC after storage for 18 months at a temperature of 5±3℃ is 0% to 4%;

[0141] - Post peak of 0 to 5% measured by HIC-HPLC after storage for 3 days, 7 days, 2 weeks or 4 weeks under conditions of temperature 40±2℃ and relative humidity 75±5%;

[0142] - Post peak measured by HIC-HPLC after storage for 3 or 7 days at 45℃ is 0 to 5%;

[0143] - After storage for 72 hours under conditions of 12,000 Lux light irradiation, temperature 25±2℃, and relative humidity 60±5%, the post peak measured by HIC-HPLC was 0 to 4%; and

[0144] - A pharmaceutical formulation having a post-peak of 0 to 4% as measured by HIC-HPLC after storage for 5 hours under 1,000 RPM stirring conditions.

[0145]

[0146] (H)-4 SEC-HPLC monomer (main peak)

[0147] - 99% to 100% of monomer content measured by SEC-HPLC after storage at a temperature of 5±3℃ for 3 days, 7 days, 2 weeks or 4 weeks;

[0148] - Monomer content of 99% to 100% as measured by SEC-HPLC after storage for 18 months at a temperature of 5±3℃;

[0149] - Monomer content of 98% to 100% measured by SEC-HPLC after storage for 3 days, 7 days, 2 weeks, or 4 weeks under conditions of temperature 40±2℃ and relative humidity 75±5%;

[0150] - 98% to 100% of monomer content measured by SEC-HPLC after storage for 3 or 7 days at 45℃;

[0151] - Monomer content of 98% to 100% measured by SEC-HPLC after storage for 72 hours under conditions of 12,000 Lux light irradiation, temperature 25±2℃, and relative humidity 60±5%; and

[0152] - A pharmaceutical preparation having a monomer content of 99% to 100% as measured by SEC-HPLC after storage for 5 hours under 1,000 RPM stirring conditions.

[0153]

[0154] (H)-5 SEC-HPLC high molecular weight component (peak with earlier retention time than main peak)

[0155] - 0 to 1% high molecular weight components measured by SEC-HPLC after storage at a temperature of 5±3℃ for 3 days, 7 days, 2 weeks or 4 weeks;

[0156] - High molecular weight components of 0% to 1% as measured by SEC-HPLC after storage for 18 months at a temperature of 5±3℃;

[0157] - High molecular weight components of 0 to 2% measured by SEC-HPLC after storage for 3 days, 7 days, 2 weeks, or 4 weeks under conditions of temperature 40±2℃ and relative humidity 75±5%;

[0158] - High molecular weight components of 0 to 2% measured by SEC-HPLC after storage for 3 or 7 days at 45℃;

[0159] - High molecular weight components of 0 to 2% measured by SEC-HPLC after storage for 72 hours under conditions of 12,000 Lux light irradiation, temperature 25±2℃, and relative humidity 60±5%; and

[0160] - A pharmaceutical formulation having 0 to 1% high molecular weight components as measured by SEC-HPLC after storage for 5 hours under 1,000 RPM stirring conditions.

[0161]

[0162] (H)-6 SEC-HPLC low molecular weight component (peak with a retention time later than the main peak)

[0163] - 0 to 1% of low molecular weight components measured by SEC-HPLC after storage at a temperature of 5±3℃ for 3 days, 7 days, 2 weeks or 4 weeks;

[0164] - 0% to 1% of low molecular weight components measured by SEC-HPLC after storage for 18 months at a temperature of 5±3℃;

[0165] - Low molecular weight components of 0 to 1% measured by SEC-HPLC after storage for 3 days, 7 days, 2 weeks, or 4 weeks under conditions of temperature 40±2℃ and relative humidity 75±5%;

[0166] - 0 to 1% of low molecular weight components measured by SEC-HPLC after storage for 3 or 7 days at 45℃;

[0167] - Low molecular weight components of 0 to 1% measured by SEC-HPLC after storage for 72 hours under conditions of 12,000 Lux light irradiation, temperature 25±2℃, and relative humidity 60±5%; and

[0168] - A pharmaceutical formulation having 0 to 1% of low molecular weight components as measured by SEC-HPLC after storage for 5 hours under 1,000 RPM stirring conditions.

[0169]

[0170] (H)-7 methionine oxidation rate

[0171] - After storage at a temperature of 5±3℃ for 3 days, 7 days or 4 weeks, the oxidation rate of heavy chain Met105 was 0 to 2% and the oxidation rate of heavy chain Met252 was 0 to 5% as measured by LC / MS;

[0172] - After storage for 18 months at a temperature of 5±3℃, the oxidation rate of heavy chain Met105 was 0 to 2%, and the oxidation rate of heavy chain Met252 was 0 to 5%, as measured by LC / MS;

[0173] - After storage for 3 days, 7 days, or 4 weeks under conditions of 40±2℃ and 75±5% relative humidity, the oxidation rate of heavy chain Met105 measured by LC / MS was 0 to 2%, and the oxidation rate of heavy chain Met252 was 0 to 5%;

[0174] - After storage for 3 or 7 days at a temperature of 45℃, the oxidation rate of heavy chain Met105 measured by LC / MS was 0 to 2%, and the oxidation rate of heavy chain Met252 was 0 to 5%;

[0175] - After storage for 72 hours under conditions of 12,000 Lux light irradiation, temperature 25±2℃, and relative humidity 60±5%, the oxidation rate of heavy chain Met105 was 0 to 2% and the oxidation rate of heavy chain Met252 was 0 to 10% as measured by LC / MS; and

[0176] - A pharmaceutical formulation having a heavy chain Met105 oxidation rate of 0 to 1% and a heavy chain Met252 oxidation rate of 0 to 4%, as measured by LC / MS, after storage for 5 hours under 1,000 RPM stirring conditions.

[0177]

[0178] [product]

[0179] The present invention provides a product comprising a stable pharmaceutical preparation according to the present invention; and a container containing the stable pharmaceutical preparation in a sealed state.

[0180] The above stable pharmaceutical preparation is as described herein.

[0181] In the present invention, the container may be formed from a material such as glass, polymer (e.g., plastic), metal, etc., but is not limited thereto.

[0182] For example, the container according to the present invention may be, but is not limited to, a bottle, a vial, a cartridge, a syringe (e.g., a pre-filled syringe, an auto-injector, etc.) or a tube, preferably a glass or polymer vial, or a glass or polymer pre-filled syringe.

[0183] In one embodiment of the present invention, the present invention provides a vial filled with a stable pharmaceutical preparation according to the present invention, a cartridge filled with the stable pharmaceutical preparation, a pre-filled syringe filled with the stable pharmaceutical preparation, or an auto-injector containing the pre-filled syringe therein.

[0184] The specific product forms of the vials, cartridges, pre-filled syringes, automatic syringes, etc. and the method of filling the stable pharmaceutical preparation into the vials, cartridges, pre-filled syringes, automatic syringes, etc. can be easily obtained or practiced by a person having ordinary skill in the art to which the present invention pertains. For example, U.S. Patent Nos. 4,861,335 and 6,331,174 disclose specific product forms and filling methods for pre-filled syringes. For example, U.S. Patent Nos. 5,085,642 and 5,681,291 disclose specific product forms and assembly methods for automatic syringes. In addition, the commercialized products such as the vials, cartridges, pre-filled syringes, automatic syringes, etc. can be used as is, or a separately custom-made product can be used in consideration of the physical properties, administration site, administration amount, etc. of the stable pharmaceutical preparation.

[0185] In the present invention, the container may be a single-use container.

[0186] In the present invention, the product according to the present invention may further include instructions for use, storage, or both of the stable pharmaceutical formulation. These instructions may include treatment methods, administration routes, dosages, or timing of administration for PD-1-related diseases.

[0187] In the present invention, the product may include other tools necessary from a commercial and user standpoint, such as needles, syringes, etc.

[0188]

[0189] [Method for manufacturing a stable pharmaceutical preparation]

[0190] The stable pharmaceutical formulation of the present invention can be manufactured using known methods and is not limited to a specific manufacturing method. For example, a pharmaceutical formulation can be manufactured by adjusting the pH of a solution containing a surfactant, a stabilizer, and an antioxidant by adding a buffer, and then adding an antibody to the mixed solution. In another example, a pharmaceutical formulation can be manufactured by preparing a solution containing an antibody, a buffer, a stabilizer, and an antioxidant in the final stage of a purification process, and then adding a surfactant to the solution.

[0191]

[0192] [How to use stable pharmaceutical preparations]

[0193] In the present invention, the stable pharmaceutical preparation according to the present invention can exhibit a therapeutic effect on a disease related to PD-1 (CD279, Programmed cell death protein 1).

[0194] In the present invention, the stable pharmaceutical preparation according to the present invention can be used for intravenous (IV) administration or subcutaneous (SC) administration.

[0195] In one embodiment of the present invention, the stable pharmaceutical preparation may not undergo a reconstitution step prior to use.

[0196] The concentrations of other components, including antibodies, in the pharmaceutical preparation are as described above, and the total volume of the pharmaceutical preparation according to the present invention may be 0.5 to 20.0 mL.

[0197] The dosage or timing of administration of the above pharmaceutical preparation may vary depending on the type of disease, severity and progress of the disease, the patient's health and response to treatment, or the judgment of the treating physician, and is not limited to a specific dosage or timing of administration.

[0198]

[0199] [Treatment and stabilization methods]

[0200] In one embodiment of the present invention, the present invention provides a method for treating a disease, comprising administering to a patient having a disease in which inhibition of binding between PD-1 (CD279, Programmed cell death protein 1) and PD-L1 (CD274, Programmed cell death-ligand 1) and PD-L2 (CD273, Programmed cell death-ligand 2) is effective in alleviating the disease, a stable pharmaceutical formulation comprising (A) an antibody or an antigen-binding fragment thereof that binds to PD-1 (CD279, Programmed cell death protein 1), (B) a surfactant, (C) a stabilizer, (D) a buffer, and (E) an antioxidant.

[0201] In another embodiment of the present invention, the present invention provides a method for stabilizing an antibody in a pharmaceutical formulation, comprising preparing a stable pharmaceutical formulation comprising (A) an antibody or antigen-binding fragment thereof that binds to PD-1 (CD279, Programmed cell death protein 1), (B) a surfactant, (C) a stabilizer, (D) a buffer, and (E) an antioxidant.

[0202] The above stable pharmaceutical preparation is as described herein.

[0203] The treatment method or stabilization method according to the present invention applies the description described above in this specification.

[0204]

[0205] Each of the above features described herein may be used in combination, and the fact that each of the above features is described in different dependent claims of the patent claims does not indicate that they cannot be used in combination.

[0206] Hereinafter, the present invention will be described in detail through examples. The following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The references cited herein are incorporated by reference into the specification of the present invention.

[0207]

[0208] In the following examples and comparative examples, pembrolizumab cultured and purified at the Celltrion Research Institute was used. The amino acid sequence of pembrolizumab used in the examples and comparative examples herein is as shown in SEQ ID NOs: 1 to 10 attached herein.

[0209] In addition, the following methods were used to measure the physical stability, chemical stability, and biological activity of the pharmaceutical preparations described below.

[0210]

[0211] - HIC-HPLC main peak

[0212] The main peak (%) was measured using Hydrophobic Interaction High Performance Liquid Chromatography (HIC-HPLC).

[0213] - HIC-HPLC free peak

[0214] The pre-peak (%) was measured using Hydrophobic Interaction High Performance Liquid Chromatography (HIC-HPLC).

[0215] - HIC-HPLC post-peak

[0216] Post-peak (%) was measured using Hydrophobic Interaction High Performance Liquid Chromatography (HIC-HPLC).

[0217] - SEC-HPLC main component

[0218] Monomer (%) was measured using Size Exclusion High Performance Liquid Chromatography (SEC-HPLC).

[0219] - SEC-HPLC high molecular weight components

[0220] High molecular weight components (HMW; %) were measured using size exclusion high performance liquid chromatography (SEC-HPLC).

[0221] - SEC-HPLC low molecular weight components

[0222] Low molecular weight components (LMW; %) were measured using size exclusion high performance liquid chromatography (SEC-HPLC).

[0223] - Methionine oxidation rate

[0224] The oxidation rates (%) of heavy chain Met105 and Met252 of pembrolizumab were measured by peptide mapping using liquid chromatography-mass spectrometry (LC-MS).

[0225]

[0226] Examples 1 to 5 and Comparative Examples 1 to 3: Preparation of pharmaceutical preparations comprising antibodies binding to PD-1 (CD279, Programmed cell death protein 1)

[0227] Examples 1 to 5 in Table 1 below were prepared by preparing each buffer at a different pH, adding a stabilizer and antioxidant, then adding an antibody and then a surfactant. Comparative Examples 1 to 3 were prepared by applying the same formulation as the original drug to the same antibody as in the Examples, and were analyzed for comparison with each Example. The specific composition of each component is as described in Table 1 below.

[0228]

[0229] Antibody Surface active agent Stabilizer Antioxidant Buffer pH Container Example 125 mg / mL Polysorbate 80 0.02% (w / v) Sorbitol 4.5% Methionine 35 mM Acetate 10 mM 5.5 Vial Example 225 mg / mL Polysorbate 80 0.02% (w / v) Sorbitol 4.5% Methionine 35 mM Acetate 10 mM 5.2 Vial Example 325 mg / mL Polysorbate 80 0.02% (w / v) Sorbitol 4% Methionine 60 mM Acetate 10 mM 5.5 Vial Example 425 mg / mL Polysorbate 80 0.02% (w / v) Sorbitol 4% Methionine 60 mM Acetate 10 mM5.2 VialExample 525 mg / mLPolysorbate 80 0.02% (w / v)Proline 217 mMMethionine 35 mMAcetate 10 mM5.2 VialComparative Example 125 mg / mLPolysorbate 80 0.02% (w / v)Sorbitol 5%-Acetate 10 mM5.5 VialComparative Example 225 mg / mLPolysorbate 80 0.02% (w / v)Sorbitol 5%-Acetate 10 mM5.2 VialComparative Example 325 mg / mLPolysorbate 80 0.02% (w / v)Sucrose 7%-Histidine 10 mM5.5 Vial

[0230]

[0231] Experimental Example: Stability Evaluation

[0232] The above-mentioned manufactured Examples 1 to 5 and Comparative Examples 1 to 3 were stored under the following conditions: temperature 5±3°C; temperature 40±2°C, relative humidity 75±5%; temperature 45°C; 12,000 Lux light irradiation, temperature 25±2°C, relative humidity 60±5%, 72 hours; and 1,000 RPM stirring for 5 hours. The stability was evaluated using the above-described method for each condition, and the results are shown in Table 2 below.

[0233] In addition, the stability of the manufactured batches of Examples 1-1 and 1-2 was measured at 5±3℃ for 0 months, 6 months, 12 months, and 18 months, and the results are shown in Table 3 below.

[0234]

[0235] ClassificationPreliminary Comparative Example 12345123 Antioxidant: Methionine (mM) 3535606035---Antibody: Pembrolizumab (mg / ml) 252525252525252525 Buffer Acetate (mM) 10101010101010-Histidine (mM)-------10 Surfactant: Polysorbate 80 (%, w / v) 0.020.020.020.020.020.020.020.020.02 Stabilizer Sorbitol (%, w / v) 4.54.544-55-Proline (mM)----217---Sucrose (%, w / v)-------7pH5.55.25.55.25.25.55.25.5Temperature 5±3℃, 3rd dayHIC-HPLC main peak (%)92.7292.8492.8592.7992.8592.7092.3792.63HIC-HPLC pre-peak (%)3.843.843.893.853.844.034.214.05HIC-HPLC post-peak (%)3.443.323.263.363.313.273.413.32SEC-HPLC monomer (%)99.5699.6199.5099.5499.5499.5199.4999.52SEC-HPLC high molecular weight Ingredients (%) 0.39 0.36 0.44 0.43 0.40 0.45 0.45 0.46 SEC-HPLC low molecular weight components (%) 0.05 0.03 0.06 0.04 0.05 0.04 0.06 0.02 Medium chain Met105 oxidation rate (%) 1.9 1.4 1.9 1.8 1.8 1.9 2.0 1.9 Medium chain Met252 oxidation rate (%) 4.3 3.5 3.5 3.9 4.0 4.7 4.7 3.7 Temperature 5±3℃, 7th day HIC-HPLC main peak (%) 9 1.9 4 9 2.00 9 2.1 1 9 2.01 9 2.26 9 1.20 9 1.15 9 ​​0.97 HIC-HPLC free peak (%)4.114.374.244.214.074.774.844.72HIC-HPLC Post Peak (%)3.943.503.653.793.674.034.014.31SEC-HPLC Monomer (%)99.5599.5799.5599.6299.5999.4199.4699.54SEC-HPLC High Molecular Weight Component (%)0.420.410.430.360.390.550.520.44SEC-HPLC Low Molecular Weight Component (%)0.030.020.020.010.020.030.030.02Medium chain Met105 oxidation rate (%)1.41.41.311.21.71.51.4Medium chain Met252 oxidation rate (%)3.73.84.03.93.84.13.93.9Temperature 5±3℃, 2nd weekHIC-HPLC main peak (%)92.9693.0892.9492.9492.9492.8892.7691.97HIC-HPLC pre-peak (%)4.013.914.023.954.034.284.325.06HIC-HPLC post-peak (%)3.033.013.043.113.042.842.922.96SEC-HPLC monomer (%)99.4299.4799.4799.5599.4799.4199.4699.48SEC-HPLC High molecular weight component(%)0.510.500.490.410.480.550.470.48SEC-HPLC Low molecular weight component(%)0.070.030.050.040.060.040.070.03Medium chain Met105 oxidation rate(%)--------Medium chain Met252 oxidation rate(%)--------Temperature 5±3℃, 4th weekHIC-HPLC Main peak(%)92.7492.7992.7492.9692.9092.2592.6090.95HIC-HPLC Free peak (%)3.843.863.843.793.894.354.215.42HIC-HPLC Post Peak (%)3.413.353.423.243.213.403.193.63SEC-HPLC Monomer (%)99.5099.6299.4299.5799.5999.4699.6199.49SEC-HPLC High Molecular Weight Component (%)0.470.350.480.370.370.50-0.340.46SEC-HPLC Low Molecular Weight Component (%)0.030.030.100.060.030.040.050.05Medium Chain Met105 Oxidation Rate (%)1.91.91.81.81.92.32.22.6Medium chain Met252 oxidation rate (%)4.44.54.44.44.44.64.64.7Temperature 40±2℃, relative humidity 75±5%, 3rd dayHIC-HPLC main peak (%)92.7092.7592.7892.9092.8591.7892.1191.22HIC-HPLC free peak (%)3.943.923.813.813.824.634.605.22HIC-HPLC Post Peak (%)3.363.343.413.293.333.603.283.56SEC-HPLC Monomer (%)99.4799.4799.5099.5399.5099.3999.3999.53SEC-HPLC High Molecular Weight Component (%)0.500.480.440.420.470.580.550.42SEC-HPLC Low Molecular Weight Component (%)0.030.050.060.050.030.030.060.05Medium Chain Met105 Oxidation Rate (%)1.71.71.71.31.11.91.81.8Medium Chain Met252 Oxidation Rate (%)3.73.53.74.12.63.94.04.4Temperature 40±2℃, relative humidity 75±5%, 7th dayHIC-HPLC main peak (%)91.3791.4591.8691.8391.4790.4390.4790.11HIC-HPLC pre-peak (%)4.564.314.324.334.525.475.685.91HIC-HPLC post-peak (%)4.074.243.833.844.014.113.863.98SEC-HPLC monomer (%)99.2799.3599.3099.3299.2699.0499.2299.29SEC-HPLC high molecular weight Ingredients (%) 0.69 0.61 0.66 0.63 0.70 0.92 0.75 0.68 SEC-HPLC low molecular weight components (%) 0.04 0.04 0.03 0.05 0.04 0.04 0.04 0.04 Medium chain Met105 oxidation rate (%) 1.3 1.2 1.01 01 01 8 1.9 1.8 Medium chain Met252 oxidation rate (%) 3.9 3.9 3.7 3.9 3.9 4.4 4.3 4.3 Temperature 40±2℃, relative humidity 75±5%, 2nd week HIC-HPLC main peak (%) 92.89 92.76 93.06 92.90 92.97 91.67 91.34 90.30 HIC-HPLC free peak (%)3.963.963.843.833.915.435.496.68HIC-HPLC Post Peak (%)3.143.283.103.273.122.903.163.02SEC-HPLC Monomer (%)99.0199.1298.9999.1599.1298.5498.9499.04SEC-HPLC High Molecular Weight Component (%)0.910.820.970.790.821.381.000.90SEC-HPLC low molecular weight components (%)0.080.060.040.070.060.080.050.06Medium chain Met105 oxidation rate (%)--------Medium chain Met252 oxidation rate (%)--------Temperature 40±2℃, relative humidity 75±5%, 4th weekHIC-HPLC main peak (%)91.8192.0292.0992.0792.0589.4289.6886.56HIC-HPLC pre-peak (%)4.054.054.033.944.086.296.219.51HIC-HPLC post-peak (%)4.143.943.883.983.864.304.113.93SEC-HPLC Monomer (%)98.7298.7498.7998.9398.7998.1998.5598.71SEC-HPLC High molecular weight component (%)1.171.151.100.981.131.711.361.24SEC-HPLC Low molecular weight component (%)0.110.110.110.090.080.100.090.05Medium chain Met105 oxidation rate (%)1.71.71.51.41.52.82.83.9Medium chain Met252 oxidation rate (%)4.34.34.24.34.35.14.95.6Temperature 45℃, 3rd day HIC-HPLC main peak (%) 92.79 92.81 92.90 93.04 92.63 91.72 91.83 91.31 HIC-HPLC pre-peak (%) 3.82 3.80 3.87 3.73 3.90 4.86 4.75 5.20 HIC-HPLC post-peak (%) 3.39 3.40 3.24 3.23 3.47 3.42 3.40 3.50 SEC-HPLC monomer (%) 99.02 99.03 98.95 99.17 99.06 98.91 98.85 99.08 SEC-HPLC high molecular weight component (%) 0.91 0.86 0.96 0.74 0.90 1.07 1.05 0.86 SEC-HPLC low molecular weight Ingredients (%) 0.07 0.100 100 09 0.04 0.02 0.100 05 Oxidation rate of medium chain Met105 (%) 1.5 1.4 1.4 1.0 1.3 1.8 1.4 1.8 Oxidation rate of medium chain Met252 (%) 4.1 4.1 4.3 4.2 4.4 4.1 4.14 14.4 Temperature 45℃, Day 7 HIC-HPLC Main Peak (%) 9 1.4 7 9 1.8 8 9 1.2 4 9 2.3391.6290.3089.3488.83HIC-HPLC Pre-peak (%)4.504.374.284.034.295.686.006.62HIC-HPLC Post-peak (%)4.023.754.483.644.104.024.664.54SEC-HPLC Monomer (%)98.5398.5998.6998.7298.5798.4398.5898.69SEC-HPLC High molecular weight component (%)1.411.351.251.221.351.511.361.26SEC-HPLC Low molecular weight component (%)0.070.050.060.060.070.060.060.05Medium chain Met105 oxidation rate (%) 1.11.01.11.11.01.11.51.7 Heavy chain Met252 oxidation rate (%) 3.33.93.63.63.50.04.24.412,000 Lux light irradiation, temperature 25±2℃, relative humidity 60±5%, 72 hours HIC-HPLC main peak (%) 89.6089.7090.3890.4289.2882.3481.4680.78 HIC-HPLC pre-peak (%) 6.746.756.086.057.0514.0315.5216.13 HIC-HPLC post-peak (%) 3.673.543.553.543.683.633.033.08 SEC-HPLC monomer (%)98.6398.7498.7198.8798.6597.5998.2898.22SEC-HPLC high molecular weight component(%)1.321.191.261.061.302.341.661.72SEC-HPLC low molecular weight component(%)0.040.070.030.070.060.070.060.06Medium chain Met105 oxidation rate(%)1.31.60.90.81.54.25.04.4Medium chain Met252 oxidation rate(%)8.79.35.96.18.317.018.113.41,000 RPM stirring, 5 hoursHIC-HPLC main peak (%)92.3992.3292.3792.6892.1489.6092.2092.26HIC-HPLC pre-peak (%)4.074.113.993.954.134.834.444.33HIC-HPLC post-peak (%)3.553.563.643.383.735.563.363.41SEC-HPLC Monomer (%)99.5299.5999.5299.6299.5899.4699.5399.54SEC-HPLC High molecular weight component (%)0.440.380.440.350.400.510.450.42SEC-HPLC Low molecular weight component (%)0.040.030.030.030.020.030.020.03Medium chain Met105 oxidation rate (%)0.80.90.80.70.91.21.21.0Medium chain Met252 oxidation rate (%)3.73.23.83.63.52.93.73.5.

[0236]

[0237] ClassificationExample 1-1Example 1-2Antioxidant: Methionine (mM) 60Antibody: Pembrolizumab (mg / ml) 25Buffer: Acetate (mM) 10Surfactant: Polysorbate 80 (%, w / v) 0.02Stabilizer: Sorbitol (%, w / v) 4pH 5.2Temperature 5±3℃, 0 months laterHIC-HPLC Main Peak (%) 93.4293.82HIC-HPLC Pre-peak (%) 3.773.50HIC-HPLC Post-peak (%) 2.822.68SEC-HPLC Monomer (%) 99.8099.83SEC-HPLC High Molecular Weight Component (%) 0.200.17SEC-HPLC Low Molecular Weight Component (%) 0.000.00Heavy Chain Met105 Oxidation Rate (%)1.61.6Medium-chain Met252 oxidation rate (%)3.03.2Temperature 5±3℃, after 6 monthsHIC-HPLC main peak (%)93.1193.31HIC-HPLC pre-peak (%)3.913.97HIC-HPLC post-peak (%)2.982.71SEC-HPLC monomer (%)99.7499.75SEC-HPLC high molecular weight component (%)0.260.25SEC-HPLC low molecular weight component (%)0.000.00Medium-chain Met105 oxidation rate (%)1.61.4Medium-chain Met252 oxidation rate (%)2.22.6Temperature 5±3℃, after 12 monthsHIC-HPLC main peak (%)93.1393.37HIC-HPLC pre-peak (%)3.553.61HIC-HPLC post-peak (%)3.323.02SEC-HPLC Monomer (%)99.7299.72SEC-HPLC High molecular weight component (%)0.280.28SEC-HPLC Low molecular weight component (%)0.000.00Medium chain Met105 oxidation rate (%)1.91.8Medium chain Met252 oxidation rate (%)2.93.0Temperature 5±3℃, after 18 monthsHIC-HPLC Main peak (%)93.2493.40HIC-HPLC Pre-peak (%)3.783.81HIC-HPLC Post-peak (%)2.972.80SEC-HPLC Monomer (%)99.7099.69SEC-HPLC High molecular weight component (%)0.300.31SEC-HPLC Low molecular weight component (%)0.000.00Medium chain Met105 oxidation rate (%)2.01.9-chain Met252 oxidation rate (%)2.82.8.

[0238]

[0239] (1) Comparison of stability according to antioxidant content in pH 5.5 formulation

[0240] From the results in Table 2 above, it was found that under the temperature condition of 5±3℃, both Examples 1 and 3 had HIC-HPLC main peak of 91% or more, HIC-HPLC pre-peak of 5% or less, HIC-HPLC post-peak of 4% or less, SEC-HPLC monomer component of 99% or more, SEC-HPLC high molecular weight component of 1% or less, SEC-HPLC low molecular weight component of 1% or less, heavy chain Met105 oxidation rate of 2% or less, and heavy chain Met252 oxidation rate of 5% or less, making them pharmaceutically acceptable stable examples.

[0241] In addition, under the conditions of temperature 40±2℃ and relative humidity 75±5%, both Examples 1 and 3 had HIC-HPLC main peaks of 91% or more, HIC-HPLC pre-peaks of 5% or less, HIC-HPLC post-peaks of 5% or less, SEC-HPLC monomer components of 98% or more, SEC-HPLC high molecular weight components of 2% or less, SEC-HPLC low molecular weight components of 1% or less, heavy chain Met105 oxidation rates of 2% or less, and heavy chain Met252 oxidation rates of 5% or less, indicating that they were pharmaceutically acceptable stable examples.

[0242] In addition, under the temperature condition of 45℃, both Examples 1 and 3 had HIC-HPLC main peak of 91% or more, HIC-HPLC pre-peak of 5% or less, HIC-HPLC post-peak of 5% or less, SEC-HPLC monomer component of 98% or more, SEC-HPLC high molecular weight component of 2% or less, SEC-HPLC low molecular weight component of 1% or less, heavy chain Met105 oxidation rate of 2% or less, and heavy chain Met252 oxidation rate of 5% or less, indicating that they were pharmaceutically acceptable stable examples.

[0243] In addition, under the conditions of 12,000 Lux light irradiation, temperature 25±2℃, relative humidity 60±5%, and storage for 72 hours, both Examples 1 and 3 had HIC-HPLC main peaks of 89% or more, HIC-HPLC pre-peaks of 8% or less, HIC-HPLC post-peaks of 4% or less, SEC-HPLC monomer components of 98% or more, SEC-HPLC high molecular weight components of 2% or less, SEC-HPLC low molecular weight components of 1% or less, heavy chain Met105 oxidation rates of 2% or less, and heavy chain Met252 oxidation rates of 10% or less, indicating that they were pharmaceutically acceptable stable examples.

[0244] In addition, under the conditions of 1,000 RPM stirring and 5-hour storage, both Examples 1 and 3 had HIC-HPLC main peaks of 92% or more, HIC-HPLC pre-peaks of 5% or less, HIC-HPLC post-peaks of 4% or less, SEC-HPLC monomer components of 99% or more, SEC-HPLC high molecular weight components of 1% or less, SEC-HPLC low molecular weight components of 1% or less, heavy chain Met105 oxidation rates of 1% or less, and heavy chain Met252 oxidation rates of 4% or less, indicating that they were pharmaceutically acceptable and stable examples.

[0245] In addition, through comparison of Examples 1 and 3 and Comparative Example 1, it was confirmed that as the content of methionine increases, the HIC-HPLC main peak is higher, the HIC-HPLC pre-peak is lower, the SEC-HPLC monomer component is higher, the SEC-HPLC high molecular weight component is lower, and the methionine oxidation rate is lower. From this, it can be confirmed that the stability tends to improve as the content of methionine, an antioxidant, increases in the pH 5.5 formulation.

[0246]

[0247] (2) Comparison of stability according to antioxidant content in pH 5.2 formulation

[0248] From the results in Table 2 above, it was found that under the temperature condition of 5±3℃, both Examples 2 and 4 had HIC-HPLC main peak of 92% or more, HIC-HPLC pre-peak of 4% or less, HIC-HPLC post-peak of 4% or less, SEC-HPLC monomer component of 99% or more, SEC-HPLC high molecular weight component of 1% or less, SEC-HPLC low molecular weight component of 1% or less, heavy chain Met105 oxidation rate of 2% or less, and heavy chain Met252 oxidation rate of 5% or less, making them pharmaceutically acceptable stable examples.

[0249] In addition, under the conditions of temperature 40±2℃ and relative humidity 75±5%, both Examples 2 and 4 had HIC-HPLC main peak of 91% or more, HIC-HPLC pre-peak of 5% or less, HIC-HPLC post-peak of 5% or less, SEC-HPLC monomer component of 98% or more, SEC-HPLC high molecular weight component of 2% or less, SEC-HPLC low molecular weight component of 1% or less, heavy chain Met105 oxidation rate of 2% or less, and heavy chain Met252 oxidation rate of 5% or less, indicating that they were pharmaceutically acceptable stable examples.

[0250] In addition, under the condition of a temperature of 45°C, both Examples 2 and 4 had HIC-HPLC main peak of 91% or more, HIC-HPLC pre-peak of 5% or less, HIC-HPLC post-peak of 4% or less, SEC-HPLC monomer component of 98% or more, SEC-HPLC high molecular weight component of 2% or less, SEC-HPLC low molecular weight component of 1% or less, heavy chain Met105 oxidation rate of 2% or less, and heavy chain Met252 oxidation rate of 5% or less, indicating that they were pharmaceutically acceptable stable examples.

[0251] In addition, under the conditions of 12,000 Lux light irradiation, temperature 25±2℃, relative humidity 60±5%, and storage for 72 hours, both Examples 2 and 4 had HIC-HPLC main peaks of 89% or more, HIC-HPLC pre-peaks of 7% or less, HIC-HPLC post-peaks of 4% or less, SEC-HPLC monomer components of 98% or more, SEC-HPLC high molecular weight components of 2% or less, SEC-HPLC low molecular weight components of 1% or less, heavy chain Met105 oxidation rates of 2% or less, and heavy chain Met252 oxidation rates of 10% or less, indicating that they were pharmaceutically acceptable stable examples.

[0252] In addition, under the conditions of 1,000 RPM stirring and 5-hour storage, both Examples 2 and 4 had HIC-HPLC main peaks of 92% or more, HIC-HPLC pre-peaks of 5% or less, HIC-HPLC post-peaks of 4% or less, SEC-HPLC monomer components of 99% or more, SEC-HPLC high molecular weight components of 1% or less, SEC-HPLC low molecular weight components of 1% or less, heavy chain Met105 oxidation rates of 1% or less, and heavy chain Met252 oxidation rates of 4% or less, indicating that they were pharmaceutically acceptable and stable examples.

[0253] Through comparison of Examples 2 and 4 and Comparative Example 2, it was confirmed that as the content of methionine increases, the HIC-HPLC main peak is higher, the HIC-HPLC pre-peak is lower, the SEC-HPLC monomer component is higher, the SEC-HPLC high molecular weight component is lower, and the methionine oxidation rate is lower. From this, it can be confirmed that the stability tends to improve as the content of methionine, an antioxidant, increases in the pH 5.2 formulation.

[0254]

[0255] (3) Comparison of stability according to pH of the formulation

[0256] From the results in Table 2 above, it was confirmed that the formulation at pH 5.2 had a higher SEC-HPLC monomer component and a lower SEC-HPLC high molecular weight component than the formulation at pH 5.5 through comparison of Examples 1 to 4 and Comparative Examples 1 to 2, and from this, it can be confirmed that the formulation at pH 5.2 exhibited the best stability.

[0257]

[0258] (4) Comparison of stability according to the type of stabilizer

[0259] From the results in Table 2 above, it was found that under the temperature condition of 5±3℃, both Examples 2 and 5 had HIC-HPLC main peak of 92% or more, HIC-HPLC pre-peak of 4% or less, HIC-HPLC post-peak of 4% or less, SEC-HPLC monomer component of 99% or more, SEC-HPLC high molecular weight component of 1% or less, SEC-HPLC low molecular weight component of 1% or less, heavy chain Met105 oxidation rate of 2% or less, and heavy chain Met252 oxidation rate of 5% or less, making them pharmaceutically acceptable stable examples.

[0260] In addition, under the conditions of temperature 40±2℃ and relative humidity 75±5%, both Examples 2 and 5 had HIC-HPLC main peak of 91% or more, HIC-HPLC pre-peak of 5% or less, HIC-HPLC post-peak of 5% or less, SEC-HPLC monomer component of 98% or more, SEC-HPLC high molecular weight component of 2% or less, SEC-HPLC low molecular weight component of 1% or less, heavy chain Met105 oxidation rate of 2% or less, and heavy chain Met252 oxidation rate of 5% or less, indicating that they were pharmaceutically acceptable stable examples.

[0261] In addition, under the temperature condition of 45℃, both Examples 2 and 5 had HIC-HPLC main peak of 91% or more, HIC-HPLC pre-peak of 5% or less, HIC-HPLC post-peak of 4% or less, SEC-HPLC monomer component of 98% or more, SEC-HPLC high molecular weight component of 2% or less, SEC-HPLC low molecular weight component of 1% or less, heavy chain Met105 oxidation rate of 2% or less, and heavy chain Met252 oxidation rate of 5% or less, indicating that they were pharmaceutically acceptable stable examples.

[0262] In addition, under the conditions of 12,000 Lux light irradiation, temperature 25±2℃, relative humidity 60±5%, and storage for 72 hours, both Examples 2 and 5 had HIC-HPLC main peaks of 89% or more, HIC-HPLC pre-peaks of 7% or less, HIC-HPLC post-peaks of 4% or less, SEC-HPLC monomer components of 98% or more, SEC-HPLC high molecular weight components of 2% or less, SEC-HPLC low molecular weight components of 1% or less, heavy chain Met105 oxidation rates of 2% or less, and heavy chain Met252 oxidation rates of 10% or less, indicating that they were pharmaceutically acceptable stable examples.

[0263] In addition, under the conditions of 1,000 RPM stirring and 5-hour storage, both Examples 2 and 5 had HIC-HPLC main peaks of 92% or more, HIC-HPLC pre-peaks of 5% or less, HIC-HPLC post-peaks of 4% or less, SEC-HPLC monomer components of 99% or more, SEC-HPLC high molecular weight components of 1% or less, SEC-HPLC low molecular weight components of 1% or less, heavy chain Met105 oxidation rates of 1% or less, and heavy chain Met252 oxidation rates of 4% or less, indicating that they were pharmaceutically acceptable and stable examples.

[0264] Through comparison of Examples 2, 5 and Comparative Example 3, it was confirmed that when sorbitol or proline was used as a stabilizer, the HIC-HPLC main peak was higher, the HIC-HPLC pre-peak was lower, the SEC-HPLC monomer component was higher, the SEC-HPLC high molecular weight component was lower, and the methionine oxidation rate was lower than when sucrose was used. From this, it can be confirmed that when sorbitol or proline was used as a stabilizer, it exhibits better stability.

[0265]

[0266] (5) Long-term stability

[0267] From the results in Table 3 above, it was found that both Examples 1-1 and 1-2 had HIC-HPLC main peaks of 92% or more, HIC-HPLC pre-peaks of 5% or less, HIC-HPLC post-peaks of 4% or less, SEC-HPLC monomer components of 99% or more, SEC-HPLC high molecular weight components of 1% or less, SEC-HPLC low molecular weight components of 1% or less, LC / MS heavy chain Met105 oxidation rates of 2% or less, and heavy chain Met252 oxidation rates of 5% or less, making them pharmaceutically acceptable and stable examples even when stored for a very long period of time.

[0268] The stable pharmaceutical preparation according to the present invention has excellent long-term storage stability based on stability under accelerated and harsh conditions, even though it contains an antibody that binds to PD-1 (CD279, Programmed cell death protein 1), and can be administered intravenously or subcutaneously.

Claims

1. (A) An antibody or an antigen-binding fragment thereof that binds to PD-1 (CD279, Programmed cell death protein 1); (B) Surfactant; (C) stabilizer; (D) buffer; and (E) A stable pharmaceutical preparation containing an antioxidant.

2. In claim 1, A stable pharmaceutical formulation, wherein the (A) antibody or antigen-binding fragment thereof comprises a light chain variable region comprising a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 1, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 2, and a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3; and a heavy chain variable region comprising a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 4, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a CDR3 domain comprising the amino acid sequence of SEQ ID NO:

6.

3. In claim 1, A stable pharmaceutical formulation, wherein the above (A) antibody or antigen-binding fragment thereof comprises a light chain variable region comprising an amino acid sequence of SEQ ID NO: 7; and a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:

8.

4. In claim 1, A stable pharmaceutical formulation comprising the above (A) antibody or antigen-binding fragment thereof, a light chain comprising the amino acid sequence of SEQ ID NO: 9; and a heavy chain comprising the amino acid sequence of SEQ ID NO:

10.

5. In claim 1, A stable pharmaceutical formulation wherein the above (A) antibody or antigen-binding fragment thereof is Pembrolizumab.

6. In claim 1, The above pharmaceutical preparation is a liquid, stable pharmaceutical preparation.

7. In claim 1, The above pharmaceutical preparation is a stable pharmaceutical preparation for the treatment of diseases related to PD-1 (CD279, Programmed cell death protein 1).

8. In claim 1, A stable pharmaceutical formulation having a concentration of the above (A) antibody or antigen-binding fragment thereof of 1 to 300 mg / ml.

9. In claim 1, A stable pharmaceutical preparation comprising the surfactant (B) comprising polysorbate, poloxamer or a mixture thereof.

10. In claim 9, A stable pharmaceutical preparation comprising at least one polysorbate selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60 and polysorbate 80.

11. In claim 9, The above polysorbate is a stable pharmaceutical preparation comprising polysorbate 80.

12. In claim 1, A stable pharmaceutical preparation having a concentration of the surfactant (B) of 0.001 to 1% (w / v).

13. In claim 1, A stable pharmaceutical preparation comprising at least one stabilizer selected from the group consisting of an amino acid or an amino acid derivative; and a sugar or a sugar alcohol.

14. In claim 13, A stable pharmaceutical preparation, wherein the amino acid or amino acid derivative comprises at least one selected from the group consisting of threonine, arginine, proline, leucine, glycine, phenylalanine, tryptophan, glutamine, aspartate, glutamate, alanine, asparagine, serine, and tyrosine.

15. In claim 13, A stable pharmaceutical preparation comprising at least one sugar or sugar alcohol selected from the group consisting of trehalose, mannitol, sorbitol, glucose, fructose, galactose, xylose, maltose, lactose, xylitol, D-maltitol, inositol, lactitol, and isomalt.

16. In claim 13, A stable pharmaceutical preparation wherein the concentration of the amino acid or amino acid derivative is 10 to 450 mM.

17. In claim 13, A stable pharmaceutical preparation having a concentration of the sugar or sugar alcohol of 0.1 to 30% (w / v).

18. In claim 1, A stable pharmaceutical preparation comprising the stabilizer (C) comprising sorbitol.

19. In claim 1, A stable pharmaceutical preparation, wherein the above (D) buffer comprises at least one selected from the group consisting of acetic acid or a salt thereof, phosphoric acid or a salt thereof, citric acid or a salt thereof, succinic acid or a salt thereof, glutamic acid or a salt thereof, 2-(N-morpholino)ethanesulfonic acid (MES), and tromethamine (Tris) or a salt thereof.

20. In claim 1, A stable pharmaceutical preparation wherein the concentration of the above (D) buffer is 0.1 to 50 mM.

21. In claim 1, A stable pharmaceutical preparation, wherein the buffer (D) comprises acetic acid or a salt thereof.

22. In claim 1, A stable pharmaceutical preparation, wherein the above (E) antioxidant comprises at least one selected from the group consisting of methionine, ascorbic acid or a salt thereof, and taurine.

23. In claim 1, A stable pharmaceutical preparation comprising the antioxidant (E) above, methionine.

24. In claim 1, A stable pharmaceutical preparation wherein the concentration of the antioxidant (E) is 0.1 to 100 mM.

25. In claim 1, A stable pharmaceutical preparation having a pH of 4 to 7.

26. In claim 1, The above pharmaceutical preparation is a stable pharmaceutical preparation that does not contain sucrose.

27. In claim 1, The above pharmaceutical preparation is a stable pharmaceutical preparation that does not contain histidine.

28. In claim 1, The above pharmaceutical preparation is a stable pharmaceutical preparation for intravenous or subcutaneous administration. 29.(A) 1 to 300 mg / ml of an antibody or antigen-binding fragment thereof, comprising a light chain variable region comprising a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 1, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 2, and a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3; and a heavy chain variable region comprising a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 4, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 6; (B) 0.001 to 1 % (w / v) surfactant; (C) 0.1 to 30% (w / v) of sugar or sugar alcohol; (D) 0.1 to 50 mM buffer; and (E) A stable pharmaceutical preparation comprising 0.1 to 100 mM of an antioxidant.

30. In claim 29, The above (C) sugar or sugar alcohol contains sorbitol, The above (D) buffer comprises acetic acid or a salt thereof, The above (E) antioxidant is a stable pharmaceutical preparation containing methionine.

31. A vial filled with a stable pharmaceutical preparation as claimed in any one of claims 1 to 30.

32. A cartridge filled with a stable pharmaceutical preparation according to any one of claims 1 to 30.

33. A pre-filled syringe filled with a stable pharmaceutical formulation as claimed in any one of claims 1 to 30.

34. An auto-injector comprising a pre-filled syringe as described in claim 33.