Stable anti-PD-1 antibody pharmaceutical formulations

Abstract

The present invention relates to a stable, buffer-free pharmaceutical formulation of an anti-PD-1 antibody, which comprises an anti-PD-1 antibody or an antigen-binding fragment thereof and a stabilizer, and a method for producing the same.

Description

[Technical Field]

[0001] The present invention relates to a stable anti-PD-1 antibody pharmaceutical formulation and a method for producing the same. [Background technology]

[0002] There is much discussion about the need to develop stable anti-PD-1 antibody pharmaceutical formulations to enhance drug stability. Anti-PD-1 antibody pharmaceutical formulations contain buffers, stabilizers, and surfactants to ensure drug stability.

[0003] However, some buffers are prone to autodegradation under stress conditions, affecting the stability of antibody drugs. Furthermore, in the case of phosphate, it cannot maintain pH in a freezing environment, which also affects antibody stability.

[0004] Therefore, there is still a need for the development of anti-PD-1 antibody drug formulations that do not contain buffers that could affect the stability of the antibody protein and can stabilize the antibody protein. [Overview of the project] [Problems that the invention aims to solve]

[0005] One embodiment provides a stable anti-PD-1 antibody pharmaceutical formulation comprising (a) an anti-PD-1 antibody or its antigen-binding fragment, and (b) a stabilizer, without a buffer, and having a pH of approximately 4.5 to approximately 6.5.

[0006] Another embodiment provides a method for treating cancer in an individual, comprising the step of administering the pharmaceutical formulation to the individual.

[0007] Another embodiment provides a method for producing the aforementioned pharmaceutical preparation. [Means for solving the problem]

[0008] All technical terms used herein, unless otherwise defined, are used in the sense generally understood by those skilled in the art in the relevant field of the present invention. While preferred methods and samples are described herein, similar or equivalent methods are also included within the scope of the present invention. Furthermore, numerical values ​​described herein, unless explicitly stated, are considered to include an "approximate" meaning. The contents of all publications cited herein as references are incorporated herein by reference as a whole.

[0009] One embodiment provides a stable anti-PD-1 antibody pharmaceutical formulation comprising (a) an anti-PD-1 antibody or its antigen-binding fragment, and (b) a stabilizer, without a buffer, and having a pH of approximately 4.5 to approximately 6.5.

[0010] Unlike commonly known antibody-containing pharmaceutical preparations, the aforementioned pharmaceutical preparation does not contain a buffer, but can maintain a pH of 4.5 to 6.5, which is a desirable environment for storing anti-PD-1 antibodies, and a more desirable environment of approximately 5.0 to 5.5. The pH of the aforementioned pharmaceutical preparation may be, for example, pH 4.5 to 6.3, pH 4.8 to 6.3, pH 5 to 6.3, pH 5.2 to 6.3, pH 4.5 to 6.0, pH 4.8 to 6.0, pH 5.0 to 6.0, pH 5.2 to 6.0, pH 4.5 to 5.8, pH 4.8 to 5.8, pH 5.0 to 5.8, pH 5.2 to 5.8, pH 4.5 to 5.6, pH 4.8 to 5.6, pH 5.0 to 5.6, pH 5.2 to 5.6, pH 4.9 to 5.5, pH 4.9, pH 5.0, pH 5.1, pH 5.2, pH 5.3, pH 5.4, or pH 5.5. In one specific example, the pH of the aforementioned pharmaceutical preparation may be approximately pH 4.5 to approximately pH 5.5. In other specific examples, the pH of the pharmaceutical preparation is approximately pH 5.0 to approximately pH 5.5.

[0011] The pH of the aforementioned pharmaceutical preparation can be adjusted by common methods known in the art. For example, the pH of the aforementioned pharmaceutical preparation can be adjusted by adding an acid (e.g., HCl) or a base (e.g., NaOH), but is not limited to these.

[0012] The term "antibody" refers to any form of antibody that has the activity to specifically bind to PD-1. The antibody includes monoclonal antibodies, polyclonal antibodies, humanized antibodies, human antibodies, and chimeric antibodies. The antibody is also pembrolizumab.

[0013] The term "antigen-binding fragment" refers to a fragment in an anti-PD-1 antibody that can bind to the PD-1 antigen, and includes, but is not limited to, the Fab fragment, F(ab')2 fragment, Fc fragment, or scFv fragment. "Pembrolizumab antibody" is a humanized antibody used in cancer immunotherapy and is marketed under the name KEYDTRUDA(R) as one of several trade names. Pembrolizumab is also used to treat melanoma, lung cancer including non-small cell lung cancer (NSCLC), head and neck cancer including head and neck squamous cell cancer (HNSCC), Hodgkin lymphoma including classic Hodgkin lymphoma (cHL), urothelial carcinoma, renal cell carcinoma, gastric cancer, microsatellite instability-high cancer (MSI-H), mismatch repair deficient (dMMR) solid tumors, cervical cancer, liver cancer, and Merkel cell carcinoma (MCC). The pembrolizumab also includes biosimilars or biobetters of active pembrolizumab present in commercially available Keytruda. The pembrolizumab also includes a heavy chain variable region containing one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 3, and a light chain variable region containing one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 4 to 6.The pembrolizumab also includes a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1 (NYYMY), a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2 (GINPSNGGTNFNEKFK), a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3 (RDYRFDMGFDY), and a light chain CDR1 having the amino acid sequence of SEQ ID NO: 4 (RASKGVSTSGYSYLH), a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5 (LASYLES), and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6 (QHSRDLPLT). The pembrolizumab is sequence number 7 (VQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTL It also includes a heavy chain variable region having the amino acid sequence PPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK) and a light chain variable region having the amino acid sequence SEQ ID NO: 8 (IVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC).

[0014] The term "biosimilar" is also called an equivalent biopharmaceutical. Because biopharmaceuticals are produced through cells rather than synthesized chemical products, it is not possible to replicate the original drug in the same way as a generic drug. Therefore, a replicated drug related to biopharmaceuticals is called a biosimilar because it is similar to the original drug, although it is not identical.

[0015] The term "biobetter" refers to a drug that has been improved upon an original biopharmaceutical in terms of efficacy, safety, and convenience. It is called a biobetter because it is superior to existing biopharmaceuticals. These biobetters can be manufactured using biotechnology, such as genetic engineering and cell culture.

[0016] Pembrolizumab targets the programmed cell death protein 1 (PD-1) receptor on lymphocytes. It is an IgG4 isotype antibody that blocks the protective mechanisms of cancer cells, allowing the immune system to destroy them. Pembrolizumab was approved for medical use in the United States in 2014. In 2017, it was approved for use in unresectable or metastatic solid tumors with specific genetic anomalies, such as mismatch repair deficiency (dMMR) or microsatellite instability. Pembrolizumab can be used alone or in combination with other chemotherapy agents. It is also administered intravenously or subcutaneously.

[0017] Pembrolizumab can also be produced by common methods known in the art. For example, US9,834,605 ​​and WO2008 / 156712A1 describe methods that can be used by those skilled in the art to produce pembrolizumab. For example, pembrolizumab can also be produced by recombinant expression of immunoglobulin light chain genes and immunoglobulin heavy chain genes in host cells.

[0018] The anti-PD-1 antibody, for example, pembrolizumab or its antigen-binding fragment, has a buffering function for pH in aqueous solution. The concentration of the anti-PD-1 antibody, for example, pembrolizumab or its antigen-binding fragment, is also an amount suitable for providing a buffering function corresponding to pH 4.5 to pH 6.5. At the same time, the concentration of the anti-PD-1 antibody, for example, pembrolizumab or its antigen-binding fragment, is also an amount effective for the treatment of cancer. Furthermore, the concentration of the anti-PD-1 antibody, for example, pembrolizumab or its antigen-binding fragment, is selected considering the stability and viscosity of the pharmaceutical formulation. The concentration of the anti-PD-1 antibody, for example, pembrolizumab or its antigen-binding fragment, may be, for example, 5 to 300 mg / mL, 5 to 250 mg / mL, 5 to 200 mg / mL, 10 to 200 mg / mL, 10 to 165 mg / mL, 15 to 160 mg / mL, 5 to 45 mg / mL, 10 to 40 mg / mL, 15 to 35 mg / mL, 20 to 30 mg / mL, 130 to 250 mg / mL, 135 to 200 mg / mL, 135 to 170 mg / mL, 140 to 160 mg / mL, 145 to 155 mg / mL, approximately 25 mg / mL, approximately 150 mg / mL, approximately 200 mg / mL, or approximately 250 mg / mL. The aforementioned anti-PD-1 antibody, for example, pembrolizumab or its antigen-binding fragment, at the aforementioned concentration, can contribute to the stability of the pharmaceutical formulation, the viscosity of the pharmaceutical formulation suitable for administration, and the maintenance of pH.

[0019] The term "stabilizer" refers to a substance added to prevent changes in state or chemical changes when a substance is left standing or stored. The stabilizer is one or more selected from the group consisting of polyols, amino acids, and metal salts.

[0020] In this specification, the term "polyol" refers to an excipient having multiple hydroxyl groups. The polyol may also include sugars, sugar alcohols, and sugar acids. The sugar refers to a soluble carbohydrate. The sugar may also be a monosaccharide, disaccharide, oligosaccharide, or polysaccharide. The sugar alcohol is an organic compound derived from a sugar, where each carbon atom has one hydroxyl group. The sugar acid refers to a sugar having a carboxyl group on one or both of its chains. The polyol may also be one or more selected from glucose, fructose, mannose, galactose, sucrose, lactose, maltose, trehalose, mannitol, sorbitol, and polyethylene glycol. The polyol may also be sorbitol, sucrose, trehalose, mannose, maltose, mannitol, or a mixture thereof. The polyol may also include an anhydride of the polyol. For example, trehalose may include not only trehalose but also trehalose dihydrate. The concentration of the polyol may be freely adjusted within a range desirable to maintain the stability of the anti-PD-1 antibody, such as pembrolizumab or its antigen-binding fragment, and the viscosity of the liquid pharmaceutical formulation, and may vary individually depending on the specific polyol, sugar alcohol, or sugar acid.

[0021] The concentration of the sugar is 1.0 to 15.0% (w / v), 3.0 to 15.0% (w / v), 5.0 to 15.0% (w / v), 7.0 to 15.0% (w / v), 1.0 to 10.0% (w / v), 3.0 to 10.0% (w / v), 4.0 to 10.0% (w / v), 5.0 to 10.0% (w / v), 7.0 to 10.0% (w / v), 1.0 to 7.0% (w / v), 2.0 to 7.0% (w / v), 3.0 to 7.0% (w / v), 4.0 to 7.0% (w / v), 1.0 to 5.0% (w / v), 2.0 to 5.0% (w / v), 3.0 to 5.0% (w / v), 4.0 to 5.0% (w / v), 4.5 to 5.0% (w / v) (for example, about 4.7% (w / v)), 6.5 to 8.5% (w / v) (for example, about 6.8% (w / v), about 7.0% (w / v), about 7.2% (w / v)), or 7.8 to 8.2% (w / v) (for example, about 7.8% (w / v), about 7.9% (w / v), about 8.0% (w / v), about 8.1% (w / v), or about 8.2% (w / v)). The sugar is, for example, sucrose, glucose, galactose, maltose, lactose, trehalose, or a mixture thereof. In one specific example, the sugar is also 1.0 to 15.0% (w / v), 4.0 to 10.0% (w / v), 6.0 to 8.0% (w / v), or about 7.0% (w / v) of sucrose. In another specific example, the sugar is also 1.0 to 15.0% (w / v), 4.0 to 10.0% (w / v), 7.0 to 8.0% (w / v), or about 7.6% (w / v) of trehalose.

[0022] The concentration of the sugar alcohol is 1.0 to 20.0% (w / v), for example, 1.0 to 15.0% (w / v), 1.0 to 10.0% (w / v), 2.5 to 10.0% (w / v), 3.0 to 10.0% (w / v), 3.5 to 10.0% (w / v), 4.0 to 10.0% (w / v), 1.0 to 8.0% (w / v), 2.5 to 8.0% (w / v), 3.0 to 8.0% (w / v), 3.5 to 8.0% (w / v), 4.0 to 8.0% (w / v), 1.0 to 6.0% (w / v), 2.5 to 6.0% (w / v), 3.0 to 6.0% (w / v), 3.5 to 6.0% (w / v), 4.0 to 6.0% (w / v), 4.0 to 5.5% (w / v), or 4.0 to 5.0% (w / v). The sugar alcohol is, for example, sorbitol, mannitol, its hydrate, or a mixture thereof. In one specific example, the sugar alcohol is also about 4.0% (w / v) sorbitol.

[0023] The amino acid is glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, serine, threonine, cysteine, methionine, asparagine, glutamine, lysine, arginine, histidine, aspartic acid, glutamic acid, its pharmaceutically acceptable salt, or a mixture thereof. The amino acid is, for example, glycine, proline, phenylalanine, tyrosine, tryptophan, lysine, arginine, its pharmaceutically acceptable salt, or a mixture thereof. In one specific example, the amino acid is arginine, its pharmaceutically acceptable salt, or a mixture thereof. In another specific example, the amino acid is proline, its pharmaceutically acceptable salt, or a mixture thereof. In yet another specific example, the amino acid is lysine, its pharmaceutically acceptable salt, or a mixture thereof. In still another specific example, the amino acid is arginine, lysine, its pharmaceutically acceptable salt, or a mixture thereof.

[0024] As the aforementioned stabilizer, the amino acid concentrations are 0.1 to 300.0 mM, 0.5 to 300.0 mM, 1.0 to 300.0 mM, 5.0 to 300.0 mM, 10.0 to 300.0 mM, 25.0 to 300.0 mM, 30.0 to 300.0 mM, 50.0 to 300.0 mM, 80.0 to 300.0 mM, 100.0 to 300.0 mM, 120.0 to 300.0 mM, 0.1 to 250.0 mM, 0.5 to 250.0 mM, 1.0 to 250.0 mM, 5.0 to 250.0 mM, and 10.0 to 250.0 mM. mM, 25.0 to 250.0 mM, 30.0 to 250.0 mM, 50.0 to 250.0 mM, 80.0 to 250.0 mM, 100.0 to 250.0 mM, 120.0 to 250.0 mM, 0.1 to 200.0 mM, 0.5 to 200.0 mM, 1.0 to 200.0 mM, 5.0 to 200.0 mM, 10.0 to 200.0 mM, 25.0 to 200.0 mM, 30.0 to 200.0 mM, 50.0 to 200.0 mM, 80.0 to 200.0 mM, 100.0 to 200.0 mM, 120.0 to 200.0 mM, 0.1 to 160.0 mM, 0.5 to 160.0 mM, 1.0 to 160.0 mM, 5.0 to 160.0 mM, 10.0 to 160.0 mM, 25.0 to 160.0 mM, 30.0 to 160.0 mM, 50.0 to 160.0 mM, 80.0 to 160.0 mM, 100.0 to 160.0 mM, 120.0 to 160.0 mM, 130.0 to 150.0 mM, 0.1 to 100.0 mM, 0.5 to 100.0 mM, 1.0 to 100.0 mM, 5.0 to 100.0 mM, 10.0 to 100.0 mM, 25.0 to 100.0 mM, 30.0 to 100.0 mM, 50.0 to 100.0 mM, 80.0 to 100.0 mM, 0.1 to 50.0 mM, 0.5 to 50.0 mM, 1.0 to 50.0 mM, 5.0 to 50.0 mM, 10.0 to 50.0 mM, 25.0 to 50.0 mM, or 30.0 to 50.0 mM, 0.1 to 40.0 mM, 0.5 to 40.0 mM, 1.0 to 40.0 mM, 5.0 to 40.0 mM, 10.0 to 40.0 mM, 25.0 to 40.0 mM, or 30.It is also 0 to 40.0 mM, 0.1 to 30.0 mM, 0.5 to 30.0 mM, 1.0 to 30.0 mM, 5.0 to 30.0 mM, 10.0 to 30.0 mM, 25.0 to 30.0 mM, 0.1 to 20.0 mM, 0.5 to 20.0 mM, 1.0 to 20.0 mM, 5.0 to 20.0 mM, 10.0 to 20.0 mM, 0.1 to 10.0 mM, 0.5 to 10.0 mM, 1.0 to 10.0 mM, or 5.0 to 10.0 mM. In one specific example, the amino acid may be 1.0 to 10.0 mM, 1.0 to 5.0 mM, 2.0 to 10.0 mM, 2.0 to 5.0 mM, 2.0 to 4.0 mM, or approximately 3.3 mM of arginine, a pharmaceutically acceptable salt thereof, or a mixture thereof. In another specific example, the amino acid may be 24 to 29 mM of glycine, a pharmaceutically acceptable salt thereof, or a mixture thereof. The concentration of the amino acid may be freely adjusted and may vary individually depending on the specific amino acid, within a range that does not affect the target pH of the pharmaceutical formulation and does not affect the stability of the anti-PD-1 antibody, such as pembrolizumab or its antigen-binding fragment.

[0025] The metal salt may also be NaCl, KCl, NaF, KBr, NaBr, Na2SO4, NaSCN, CaCl2, MgCl2, or K2SO4. For example, the metal salt may also be NaCl or Na2SO4. The concentrations of the metal salt may be 1.0 to 300.0 mM, 5.0 to 150.0 mM, 10.0 to 150.0 mM, 30.0 to 150.0 mM, 50.0 to 150.0 mM, 80.0 to 150.0 mM, 100.0 to 150.0 mM, 120.0 to 150.0 mM, 5.0 to 125.0 mM, 10.0 to 125.0 mM, 30.0 to 125.0 mM, or 50.0 to 125. The concentrations may also be 0 mM, 80.0 to 125.0 mM, 100.0 to 125.0 mM, 120.0 to 125.0 mM, 5.0 to 100.0 mM, 10.0 to 100.0 mM, 30.0 to 100.0 mM, 50.0 to 100.0 mM, 80.0 to 100.0 mM, 5 to 80.0 mM, 10.0 to 80.0 mM, 30.0 to 80.0 mM, or 50.0 to 80.0 mM. In one specific example, the metal salt may also be sodium chloride in concentrations of 5.0 to 150.0 mM, 20.0 to 140.0 mM, or approximately 100.0 mM. The concentration of the metal salt in the pharmaceutical formulation of the present invention can be freely adjusted within a range that maintains the stability of the anti-PD-1 antibody, such as pembrolizumab or its antigen-binding fragment, without causing precipitation, and may vary individually depending on the specific metal salt. The stabilizer is also a mixture of a polyol and an amino acid. When a mixture of a polyol and an amino acid is used, the amino acid can perform one or more roles among a stabilizer and a viscosity reducer.

[0026] In this specification, the phrase "component A-free" may mean that the pharmaceutical preparation does not contain or substantially contains component A. "Substantially component A-free" may also be interpreted to include the complete absence of component A, or the presence of component A in trace amounts that do not substantially affect the properties of the pharmaceutical preparation, or in undetectable amounts.

[0027] The pharmaceutical formulation contains no buffer other than an anti-PD-1 antibody, such as pembrolizumab or its antigen-binding fragment. In this specification, the term "buffer" refers to a composition to which the pharmaceutical formulation is added to resist pH changes. The buffer can maintain the pH of the formulation within an acceptable range. Generally, the buffer makes the formulation resistant to pH changes through the action of its acid-base conjugated components. Where a buffer concentration is mentioned in this specification, the mentioned concentration refers to the molar concentration of the free acid or free base form of the buffer. In this specification, the phrase "buffer-free" indicates that the pharmaceutical formulation does not contain enough buffer to resist pH changes. This can also be interpreted as including an amount that cannot function as the intended buffer within the pharmaceutical formulation. The buffer may also be histidine, phosphoric acid (sodium phosphate or potassium phosphate), malic acid, tartaric acid, succinic acid (succinate), citric acid (citrate), acetic acid (acetate), carbonic acid, a pharmaceutically acceptable salt thereof, or a mixture thereof. In one specific example, the buffer may also be histidine, a pharmaceutically acceptable salt thereof, or a mixture thereof. In another specific example, the buffer may also be phosphoric acid, a pharmaceutically acceptable salt thereof, or a mixture thereof.

[0028] The pharmaceutical formulation may be surfactant-free or further contain a surfactant. The surfactant may also be a nonionic surfactant. The nonionic surfactant may also be a polysorbate, poloxamer, sorbitan ester of another fatty acid, or a mixture thereof. The polysorbate may also be polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, or a mixture thereof. The poloxamer may also be poloxamer 188. If the pharmaceutical formulation contains a surfactant, the concentration of the surfactant may be 0.001 to 1% (w / v), 0.001 to 0.5% (w / v), 0.001 to 0.1% (w / v), 0.001 to 0.05% (w / v), 0.001 to 0.02% (w / v), or 0.005 to 1% (w / v), based on the total volume of the pharmaceutical formulation or the reconstituted formulation. , 0.005 to 0.5% (w / v), 0.005 to 0.1% (w / v), 0.005 to 0.05% (w / v), 0.005 to 0.02% (w / v), 0.008 to 1% (w / v), 0.008 to 0.5% (w / v), 0.008 to 0.1% (w / v), 0.008 to 0.05% (w / v), or 0.008 to 0.02% (w / v). In one specific example, the pharmaceutical preparation contains 0.02 to 0.04% (w / v) of polysorbate 20 or polysorbate 80.

[0029] The aforementioned pharmaceutical preparation also further contains an antioxidant. The term "antioxidant" means a substance that prevents oxidation. The antioxidant may be an amino acid, a vitamin, a coenzyme, glutathione, methylsulfonyl sorbate, or a mixture thereof. The amino acid as an antioxidant may be methionine, L-cysteine, L-carnitine, or a mixture thereof. The vitamin as an antioxidant may be vitamin A, vitamin C, vitamin E, or a mixture thereof. The coenzyme may also be coenzyme Q10. In one specific example, the antioxidant may also be methionine.

[0030] The concentration of the antioxidant in the pharmaceutical formulation can be freely adjusted within a range desirable to maintain the stability of the anti-PD-1 antibody, such as pembrolizumab or its antigen-binding fragment, and may vary individually depending on the type of antioxidant. If the pharmaceutical formulation contains an antioxidant, the concentration of the antioxidant may be 1 to 70 mM, 1 to 50 mM, 1 to 30 mM, 5 to 50 mM, 5 to 30 mM, 1 to 20 mM, 1 to 15 mM, 1 to 10 mM, 1 to 5 mM, 3 to 20 mM, 3 to 15 mM, 3 to 10 mM, 3 to 7 mM, 20 to 60 mM, 30 to 50 mM, about 5 mM, about 20 mM, about 30 mM, about 40 mM, or about 50 mM. In one specific example, the pharmaceutical formulation contains about 5 mM methionine. In another specific example, the pharmaceutical preparation contains approximately 30 mM methionine. In yet another specific example, the pharmaceutical preparation contains approximately 50 mM methionine.

[0031] The pharmaceutical preparation may also be a liquid. The pharmaceutical preparation may also be for subcutaneous or intravenous injection. The pharmaceutical preparation may further include a suitable aqueous carrier for injection. The aqueous carrier may be pharmaceutically acceptable, safe and non-toxic when administered to humans, and may be, for example, water, saline solution, drip solution, dextrose, or a mixture thereof.

[0032] The aforementioned pharmaceutical formulations also have an appropriate osmotic pressure range for subcutaneous or intravenous injection. This osmotic pressure may be, for example, 200 to 400 mOsm / kg, 200 to 350 mOsm / kg, 250 to 300 mOsm / kg, 250 to 290 mOsm / kg, 270 to 328 mOsm / kg, 250 to 269 mOsm / kg, or 328 to 350 mOsm / kg. The osmotic pressure may be appropriately adjusted to minimize any pain that may occur during administration.

[0033] The pharmaceutical formulation may have a viscosity within an appropriate range when administered by subcutaneous or intravenous injection. When measured at room temperature (25°C ± 3°C), the viscosity may be, for example, 0.5 to 100 cp, 0.5 to 90 cp, 0.5 to 80 cp, 0.5 to 70 cp, 0.5 to 60 cp, 0.5 to 50 cp, 0.5 to 40 cp, 0.5 to 30 cp, 0.5 to 20 cp, 0.5 to 15 cp, or 0.5 to 10 cp. The viscosity may be appropriately adjusted to minimize pain that may occur during administration.

[0034] In one specific example, in the pharmaceutical preparation, the concentration of the anti-PD-1 antibody, for example, pembrolizumab or its antigen-binding fragment, is 5 to 300 mg / mL, 5 to 250 mg / mL, 5 to 200 mg / mL, 15 to 30 mg / mL, or 150 to 250 mg / mL; the stabilizer is sucrose, glucose, galactose, maltose, practose, trehalose, sorbitol, mannitol, arginine, lysine, proline, glycine, phenylalanine, tyrosine, tryptophan, its hydrate, its pharmaceutically acceptable salt, a mixture thereof, or a metal salt; and the pH of the pharmaceutical preparation is pH 4.5 to pH 6.5 or pH 5.0 to pH 5.5.

[0035] In the above specific example, the stabilizer is sucrose, glucose, galactose, maltose, practose, trehalose, its hydrate, or a mixture thereof, and the concentration of the stabilizer is 1.0 to 15.0% (w / v), 3.0 to 15.0% (w / v), 5.0 to 15.0% (w / v), 7.0 to 15.0% (w / v), 1.0 to 10.0% (w / v), 3.0 to 10.0% (w / v), 5.0 to 10.0% (w / v), 7.0 to 10.0% (w / v), 1.0 to 7.0% (w / v), 2.0 to 7.0% (w / v), 3.0 to 7.0% (w / v), 4.0 to 7.0% (w / v), 1.0 to 5.0% (w / v). It is also 0.2 to 5.0% (w / v), 3.0 to 5.0% (w / v), 4.0 to 5.0% (w / v), 4.5 to 5.0% (w / v) (for example, about 4.7% (w / v)), or 7.8 to 8.2% (w / v) (for example, about 7.8% (w / v), about 7.9% (w / v), about 8% (w / v), about 8.1% (w / v), or about 8.2% (w / v)). The stabilizer is sorbitol, mannitol, its hydrate, or a mixture thereof, and the concentration of the stabilizer is 1.0 to 20.0% (w / v), for example, 1.0 to 15.0% (w / v), 1.0 to 10.0% (w / v), 2.5 to 10.0% (w / v), 3.0 to 10.0% (w / v), 3.5 to 10.0% (w / v), 4.0 to 10.0% (w / v), 1.0 to 8.0% (w / v). It is also 2.5 to 8.0% (w / v), 3.0 to 8.0% (w / v), 3.5 to 8.0% (w / v), 4.0 to 8.0% (w / v), 1.0 to 6.0% (w / v), 2.5 to 6.0% (w / v), 3.0 to 6.0% (w / v), 3.5 to 6.0% (w / v), 4.0 to 6.0% (w / v), 4.0 to 5.5% (w / v), or 4.0 to 5.0% (w / v). The stabilizer is arginine, lysine, proline, glycine, phenylalanine, tyrosine, tryptophan, its hydrate, its pharmaceutically acceptable salt, or a mixture thereof, and the concentration of the stabilizer is 0.1 to 300.0 mM, 0.5 to 300.0 mM, 1.0 to 300.0 mM, 5.0 to 300.0 mM, 10.0 to 300 mM.0 mM, 25.0 to 300.0 mM, 30.0 to 300.0 mM, 50.0 to 300.0 mM, 80.0 to 300.0 mM, 100.0 to 300.0 mM, 120.0 to 300.0 mM, 0.1 to 250.0 mM, 0.5 to 250.0 mM, 1.0 to 250.0 mM, 5.0 to 250.0 mM, 10.0 to 250.0 mM, 25.0 to 250.0 mM, 30.0 to 250.0 mM, 50.0 to 250.0 mM, 80.0 to 250.0 mM, 100.0 to 250.0 mM, 120.0 or 250.0 mM, 0.1 to 200.0 mM, 0.5 to 200.0 mM, 1.0 to 200.0 mM, 5.0 to 200.0 mM, 10.0 to 200.0 mM, 25.0 to 200.0 mM, 30.0 to 200.0 mM, 50.0 to 200.0 mM, 80.0 to 200.0 mM, 100.0 to 200.0 mM, 120.0 to 200.0 mM, 0.1 to 160.0 mM, 0.5 to 160.0 mM, 1.0 to 160.0 mM, 5.0 to 160.0 mM, 10.0 to 160.0 mM, 25.0 or 160.0 mM, 30.0 or 160.0 mM, 50.0 or 160.0 mM, 80.0 or 160.0 mM, 100.0 or 160.0 mM, 120.0 or 160.0 mM, 130.0 or 150.0 mM, 0.1 or 100.0 mM, 0.5 or 100.0 mM, 1.0 or 100.0 mM, 5.0 or 100.0 mM, 10.0 or 100.0 mM, 25.0 or 100.0 mM, 30.0 or 100.0 mM, 50.0 or 100.0 mM, 80.0 or 100.0 mM, 0.1 or 50.0 mM, 0 0.5 to 50.0 mM, 1.0 to 50.0 mM, 5.0 to 50.0 mM, 10.0 to 50.0 mM, 25.0 to 50.0 mM, or 30.0 to 50.0 mM, 0.1 to 40.0 mM, 0.5 to 40.0 mM, 1.0 to 40.0 mM, 5.0 to 40.0 mM, 10.0 to 40.0 mM, 25.0 to 40.0 mM, or 30.0 to 40.0 mM, 0.1 to 30.0 mM, 0.5 to 30.0 mM, 1.0 to 30.0 mM, 5.0 to 30.0 mM, 10.0 to 30.0 mM, 25.The stabilizer may also be 0 to 30.0 mM, 0.1 to 20.0 mM, 0.5 to 20.0 mM, 1.0 to 20.0 mM, 5.0 to 20.0 mM, 10.0 to 20.0 mM, 0.1 to 10.0 mM, 0.5 to 10.0 mM, 1.0 to 10.0 mM, or 5.0 to 10.0 mM. The stabilizer may also be a mixture of about 1.0 to about 15.0% (w / v) of sucrose, trehalose, their hydrates, or mixtures thereof, and about 0.1 to about 300.0 mM of arginine, lysine, proline, glycine, phenylalanine, tyrosine, tryptophan, their pharmaceutically acceptable salts, or mixtures thereof. The aforementioned stabilizer is also a mixture of approximately 1.0 to approximately 20.0% (w / v) of sorbitol, mannitol, its hydrate, or a mixture thereof, and approximately 0.1 to approximately 300.0 mM of arginine, lysine, proline, glycine, phenylalanine, tyrosine, tryptophan, their pharmaceutically acceptable salts, or a mixture thereof.

[0036] In the above specific example, the pharmaceutical preparation also further contains a surfactant. The surfactant is also polysorbate or poloxamer. The polysorbate is also polysorbate 20 or polysorbate 80. The concentration of the surfactant is 0.001 to 1% (w / v), 0.001 to 0.5% (w / v), 0.001 to 0.1% (w / v), 0.001 to 0.05% (w / v), 0.001 to 0.02% (w / v), 0.005 to 1% (w / v), 0.005 to 0.5% (w / v), 0.005 to 0.1% (w / v), It is also 0.005 to 0.05% (w / v), 0.005 to 0.02% (w / v), 0.008 to 1% (w / v), 0.008 to 0.5% (w / v), 0.008 to 0.1% (w / v), 0.008 to 0.05% (w / v), 0.008 to 0.02% (w / v), or 0.02 to 0.04% (w / v).

[0037] In the above specific example, the pharmaceutical preparation also further contains an antioxidant. The antioxidant is methionine. The concentration of the methionine can be 1 to 70 mM, 1 to 50 mM, 1 to 30 mM, 5 to 50 mM, 5 to 30 mM, 1 to 20 mM, 1 to 15 mM, 1 to 10 mM, 1 to 5 mM, 3 to 20 mM, 3 to 15 mM, 3 to 10 mM, 3 to 7 mM, 20 to 60 mM, 30 to 50 mM, about 5 mM, about 20 mM, about 30 mM, about 40 mM, or about 50 mM.

[0038] In the pharmaceutical formulation of the present invention, the anti-PD-1 antibody, for example, pembrolizumab or its antigen-binding fragment, can be stabilized. The term "stabilization" means that the anti-PD-1 antibody, for example, pembrolizumab or its antigen-binding fragment substantially retains its physical stability, chemical stability, and / or biological activity before and after administration, during additional manufacturing processes, during storage, or during preservation. The physical stability, chemical stability, and / or biological activity can be evaluated by generally known methods.

[0039] In this specification, the stability of the anti-PD-1 antibody or its antigen-binding fragment also satisfies one or more of the following conditions.

[0040] Stability can be measured at a selected temperature for a selected period of time. For example, in one specific case, a stable formulation is one in which no significant changes are observed at 2°C to 8°C for 12 months or longer. In another specific case, a stable formulation is one in which no significant changes are observed at 2°C to 8°C for 18 months or longer. In yet another specific case, a stable formulation is one in which no significant changes are observed at 23°C to 27°C for 3 months or longer. In yet another specific case, a stable formulation is one in which no significant changes are observed at 23°C to 27°C for 6 months or longer. In yet another specific case, a stable formulation is one in which no significant changes are observed at 23°C to 27°C for 12 months or longer. In yet another specific case, a stable formulation is one in which no significant changes are observed at 23°C to 27°C for 18 months or longer. The stability criteria for antibody formulations are as follows: When measured by SE-HPLC, less than 10% of the antibody monomer is degraded, for example, less than 5% or less, or less than 2.5%. The antibody potency is within 60% to 140%, or 80% to 120%, of the control group or standard antibody. For example, when measuring low molecular weight species (LMW) by SE-HPLC, there may be a change of less than 10%, less than 5%, or less than 2.5% of the antibody. For example, when measuring high molecular weight species (HMW) by SE-HPLC, there may be a change of less than 10%, less than 5%, or less than 2.5% of the antibody. In addition, the concentration and pH of the formulation may have a change of less than ±10%, less than ±5%, or less than ±2.5%.

[0041] When the aforementioned pharmaceutical preparation is placed in a stability incubator at a temperature of 40±2°C and a humidity of 75±5% for 4 weeks, the HMW or pH changes within the range of 10% or less, 5% or less, or 2.5% or less.

[0042] When 0.3 to 1 mL of the aforementioned pharmaceutical preparation is placed in a 2 cc vial made of Type I glass material (manufactured by Schott), and the vial is mounted on a stirrer (manufactured by Heidolph) and stirred at 400 rpm at room temperature for 72 hours, the HMW or pH changes within the range of 10% or less, 5% or less, or 2.5% or less.

[0043] The pembrolizumab or its antigen-binding fragment possesses chemical stability within the pharmaceutical formulation if it remains chemically stable for a predetermined time during which it is expected to still retain biological activity. This chemical stability is also assessed by detecting and quantifying chemically altered forms of pembrolizumab. Chemical alterations include size alterations or charge changes. The charge changes are, for example, those resulting from deamidation. Size alterations are also assessed using size exclusion chromatography (SE-HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) analysis, and matrix-assisted laser desorption / ionization / time-of-flight mass spectrometry (MALDI / TOF MS). Furthermore, the charge change can also be evaluated by ion exchange chromatography (IEC) and imaged capillary isoelectric focusing (icIEF). The activity can be determined by PD-1 ligand binding analysis. This PD-1 ligand binding analysis can also be performed by enzyme-linked immunosorbent assay (ELISA). This ELISA analysis measures the percentage of pembrolizumab that binds to the PD-1 ligand. The degree of binding y after reacting a pembrolizumab sample with the PD-1 ligand on an ELISA substrate can be determined by measuring the absorbance at 450 nm, and the relative binding rate (%) can be obtained.

[0044] The anti-PD-1 antibody, for example, pembrolizumab or its antigen-binding fragment, possesses biological activity within the pharmaceutical formulation. This biological activity is defined, for example, as being within approximately 30%, 20%, or 10% (or within the margin of error) of the biological activity of the anti-PD-1 antibody or its antigen-binding fragment within the pharmaceutical formulation compared to the biological activity shown at the time of manufacturing of the pharmaceutical formulation. This biological activity can also be determined, for example, by antigen-binding analysis.

[0045] Stabilization can be evaluated by applying temperature stress, for example, 1 to 4 weeks at 40°C, freeze-thaw stress, for example, 5 repetitions of a cycle of freezing at -70°C and thawing at room temperature, or stirring stress, for example, applying a rotational force of 400 rpm in a stirrer for 72 hours, and measuring %HMW, % monomer, and / or %LMW using size-exclusion high-performance liquid chromatography (SE-HPLC). In one specific example, the pharmaceutical formulation of the present invention may have Δ%HMW, Δ%LMW, or Δ% monomer values ​​equivalent to or lower than Keytruda(R). Stabilization can also be evaluated by applying temperature stress, freeze-thaw stress, or stirring stress and measuring the % acidic change using image capillary isoelectric focusing (icIEF). In one specific example, the pharmaceutical formulation of the present invention may have Δ% acidic values ​​equivalent to or lower than Keytruda(R).

[0046] The stable pharmaceutical formulations provided herein, when the antibody content is 25 mg / ml (pH 5.5 or pH 5.0), are obtained by placing 0.3 to 1 mL of the formulation in a 2 cc vial of type I glass material (Schott), storing it at 40°C for 4 weeks, and measuring the change in %HMW, i.e., the %HMW value at 4 weeks minus the %HMW value at week 0, with an antibody content of 25 mg / ml.

[0047] The stable pharmaceutical formulations provided herein, when the antibody content is 25 mg / ml (pH 5.5 or pH 5.0), are also such that the change in % acidity, i.e., the % acidity after 4 weeks - % acidity value at 0 weeks, measured using icIEF, when the formulation is placed in a polypropylene microtube and stored at 40°C for 4 weeks, is ≤20.0%, ≤15.0%, or ≤10.0%.

[0048] In one specific example, the pharmaceutical preparation comprises (i) an anti-PD-1 antibody or its antigen-binding fragment, (ii) a stabilizer, and (iii) a histidine-free buffer, having a pH of approximately 4.5 to approximately 6.5. The histidine-free buffer may be succinate, citrate, acetate, phosphate, or a combination thereof.

[0049] Another embodiment provides a method for treating cancer with the aforementioned pharmaceutical preparation, which is as described above. The method for treating cancer also includes the step of administering the pharmaceutical preparation to an individual in such a manner that it is effective in treating cancer. The individual is a human being.

[0050] In the above method, the term "cancer" refers to or describes a pathological condition in mammals typically characterized by upward-regulated cell growth. The cancer includes, but is not limited to, carcinomas, lymphomas, leukemias, blastomas, and sarcomas. The aforementioned cancers also include, for example, squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, Hodgkin lymphoma, non-Hodgkin lymphoma, gastrointestinal cancer or gastrointestinal tract cancer, kidney cancer, ovarian cancer, liver cancer, lymphoplastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, pleoplastic glioma, cervical cancer, brain cancer, gastric cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon carcinoma, Merkel cell carcinoma, high-frequency microsatellite instability cancer, esophageal cancer, urothelial carcinoma, primary mediastinal giant B-cell lymphoma, and head and neck cancer.

[0051] Another embodiment provides a method for producing a stable pharmaceutical formulation, comprising the steps of: adding a stabilizer to a solvent to produce a mixed solution; adding an anti-PD-1 antibody or its antigen-binding fragment to the mixed solution; or adding an anti-PD-1 antibody or its antigen-binding fragment to a solvent to produce an anti-PD-1 antibody or its antigen-binding fragment solution; and adding a stabilizer to the solution, wherein the production steps are carried out without the addition of a buffer.

[0052] In the above method, the solvent may be an aqueous solvent, such as water or saline. The method may further include the step of adding a surfactant to the mixed solution to which the anti-PD-1 antibody or its antigen-binding fragment is added. The method may further include the step of adding an antioxidant to the mixed solution to which the anti-PD-1 antibody or its antigen-binding fragment is added. The method may further include the step of adjusting the pH of the formulation to about pH 4.5 to about pH 5.5, about pH 5.5, or about pH 5.0.

[0053] Any terms or elements mentioned in the aforementioned pharmaceutical preparation, as referred to in the description relating to the method of manufacture of the requested pharmaceutical preparation, shall be understood to be the same as those referred to in the description relating to the requested pharmaceutical preparation as stated above. [Effects of the Invention]

[0054] According to one embodiment of a stable anti-PD-1 antibody pharmaceutical formulation, the anti-PD-1 antibody can be stably maintained even without a buffer.

[0055] According to one embodiment of a method for treating cancer in an individual, cancer can be treated efficiently in that individual.

[0056] According to one embodiment of a method for producing a stable anti-PD-1 antibody pharmaceutical formulation, a stable anti-PD-1 antibody pharmaceutical formulation can be efficiently produced. [Brief explanation of the drawing]

[0057] [Figure 1] This figure shows the Δ%HMW of formulations with different buffers after being subjected to temperature stress for 4 weeks. [Figure 2] This figure shows the Tag values ​​for formulations with different buffer-free or histidine buffer and surfactant content. [Figure 3] This figure shows the ratio (348 / 332) of changes in denaturant concentration measured for buffer-free and 20 mM histidine-containing formulations. [Figure 4] This figure shows the Δ%HMW of formulations with different buffer and PS80 content after being subjected to temperature stress for 4 weeks. [Modes for carrying out the invention]

[0058] The present invention will be described in more detail below with reference to the following examples. However, the following examples are for illustrative purposes only and do not limit the scope of the present invention.

[0059] Materials and methods 1. Size exclusion chromatography (SEC) The purity of the sample was examined by size exclusion chromatography. Size exclusion chromatography determines the percentages of antibody monomers, high molecular weight species (HMW), and low molecular weight species (LMW). In size exclusion chromatography, LMW elutes more slowly than HMW. The presence of HMW indicates protein aggregation, while the presence of LMW indicates protein fragmentation.

[0060] 2.Dynamic light scattering (DLS) measurement method Aggregates in the samples were examined using dynamic light scattering measurement. Specifically, each sample was diluted with the respective buffer and loaded into the wells of a 96-well plate. The plate was then subjected to DynoPro (R) Plate ReaderTM The sample was loaded into a Wyatt Technology II instrument. The aggregates in the pharmaceutical formulation were measured via dynamic light scattering (DLS) while the temperature was increased at a rate of 0.15°C / min in the range of 25°C to 70°C. Stability was evaluated by measuring the temperature at which the aggregate size changed. agg A higher value indicates greater stability.

[0061] 3.HUNKY (ΔG, AggPath) measurement method Aggregates in the sample were examined using the HUNKY measurement method. The HUNKY device (HUNKY, Unchained Labs.) is a device that can confirm protein stability through chemical denaturation, and the device uses a denaturant and a sample to perform C 1 / 2 ΔG, AggPath and ΔG trend You can check the value of C. 1 / 2 ΔG ranks the stability. ΔG quantifies stability. AggPath predicts aggregation. trend This section provides a detailed explanation of aggregation.

[0062] 4. Imaging capillary isoelectric focusing (icIEF) The % acidity of the samples was measured using icIEF. Specifically, each sample was placed in a 1.5 mL polypropylene microtube, and the tubes were exposed to a stable incubator under temperature stress conditions of 40°C for 4 weeks. The samples were then introduced into the iCE3 system (Protein Simple, USA), and the protein charge variation values ​​were measured using iCECFR software. % acidity indicates the protein charge variation value. These measurements confirm the presence of protein charge variants. When a protein is subjected to stress, it will either aggregate or its charge will change. Therefore, protein charge variation can also be a factor in confirming its stability.

[0063] 5. Manufacturing of pharmaceutical preparations Each pharmaceutical preparation listed in Table 1 was manufactured as described below and used in the following examples.

[0064] First, buffer solutions were prepared by adding pembrolizumab and the surfactant (excluding the buffer) and stabilizer to sterile distilled water, according to the composition of each pharmaceutical formulation listed in Table 1. Pembrolizumab was placed in a dialysis cassette (Slide-A-Lyzer cassette, Thermo Fisher Scientific), and then placed in beakers containing the solutions of each composition listed in Table 1 to allow dialysis to be performed, and the existing pembrolizumab solution was replaced with the buffer solution. Finally, the surfactant PS80 from the prepared buffer solution was added to the pembrolizumab solution to a concentration of 1x. Subsequently, the concentration of pembrolizumab was adjusted to a final concentration of 25 mg / mL using each solution.

[0065] [Table 1]

[0066] Example 1: Buffer's effect on the stability of pembrolizumab-containing pharmaceutical formulations. In this example, the effect of the buffer on the stability of pembrolizumab-containing pharmaceutical formulations was investigated. Specifically, the purity of the samples was measured after applying temperature stress to formulations 1A, 2I, 3A, 4A, and 5Ano.

[0067] The aforementioned temperature stress was performed by placing 0.3 mL of each formulation into a 2 cc vial made of Type I glass material, and exposing the vial to a temperature stress condition of 40°C in a stable incubator for 4 weeks. Specifically, the vial was placed in the stable incubator and left under conditions of a temperature of 40 ± 2°C and a relative humidity of 75 ± 5% for 4 weeks. SEC analysis was performed on the formulations stored for 4 weeks as described above.

[0068] The results are shown in Table 2 and Figure 1. Table 2 is a table showing the purity of formulations with different buffers and formulations subjected to 4-week temperature stress in terms of the HMW content. Figure 1 is a drawing showing the Δ%HMW of the formulations after applying 4-week temperature stress to the formulations with different buffers.

[0069]

Table 2

[0070] As shown in Table 2 and Figure 1, at 4 weeks, the average Δ%HMW values of the buffer-containing formulations and the Δ%HMW values of the buffer-free formulations are 0.64% and 0.39% respectively, and the buffer-free formulations are more stable than the buffer-containing formulations.

[0071] Example 2: Effect of buffer and surfactant content on the stability of pembrolizumab-containing pharmaceutical formulations In this example, the effects of the contents of the buffer and surfactant on the stability of the pembrolizumab-containing pharmaceutical formulations were confirmed. Specifically, for the formulations of 1A, 1B, 1C, 1D, 2A, 2B, 2C, 2D, 2E, 2F, 2G and 2H, the T agg values were measured by the DLS measurement method.

[0072] The results are shown in Table 3 and Figure 2. Table 3 is a table showing the composition of the formulations with different buffer-free or histidine buffer and PS80 surfactant contents, and the related T agg measurement values. Figure 2 is a drawing showing the T agg values related to the formulations with different buffer-free or histidine buffer and surfactant contents.

[0073]

Table 3

[0074] As shown in Table 3 and Figure 2, for the formulations that do not contain PS80 surfactant or contain 0.02% of it, the T aggThe value is T for formulations containing 0.10% or 0.20% of the surfactant PS80. agg The values ​​were higher than expected. This indicates that formulations containing a low PS80 content of less than 0.10%, namely 1A, 1B, 2A, 2B, 2E, and 2F, undergo aggregation at higher temperatures compared to formulations containing a high PS80 content of 0.10% or more, namely 1C, 1D, 2C, 2D, 2G, and 2H. In other words, formulations containing a low PS80 content of less than 0.10% are more stable than formulations containing a high PS80 content of 0.10% or more.

[0075] On the other hand, when the PS80 concentration is less than 0.10% for 2A, 2B, 2E, and 2F, the histidine content is T agg It has not had a significant impact on the changes.

[0076] Furthermore, among the formulations containing 20 mM histidine buffer, formulations containing PS80, namely 2B, 2C, and 2D T agg Compared to the value, formulations that do not contain PS80, i.e., 2A T agg The values ​​were higher than those. On the other hand, among the formulations containing 40 mM histidine buffer, the formulation containing 0.02% PS80, i.e., 2F T agg The values ​​are T for 2E, 2G, and 2H. agg It was higher than the stated value.

[0077] Example 3: Effect of buffer on the stability of pembrolizumab-containing pharmaceutical formulations In this example, the effects of buffer-free and 20 mM histidine buffer on the stability of pembrolizumab-containing pharmaceutical formulations were investigated. Specifically, the ΔG values ​​were measured for formulations 1B and 2B using the HUNKY assay method.

[0078] Unlike the DLS method, which measures protein aggregation due to temperature rise, the HUNKY measurement method measures stability against chemical stress mediated by guanidine-HCl (Gdn-HCl). Specifically, the HUNKY method measures the ratio of absorbance intensities at 348 nm and 332 nm, i.e., the ratio (348 / 332), in a concentration gradient of Gdn-HCl from 0 M to 5.5 M. As the protein denatures, the aforementioned ratio increases.

[0079] The results are shown in Table 4 and Figure 3. Table 4 shows the composition of the buffer-free and 20 mM histidine formulations, and the measured ΔG values ​​for them. Figure 3 shows the ratio (348 / 332) of the denaturing agent concentration changes measured for the buffer-free and 20 mM histidine formulations.

[0080] [Table 4]

[0081] As shown in Table 4 and Figure 3, the ΔG values ​​for 1B and 2B were at a similar level. This indicates that the presence of histidine buffer does not significantly affect the stability of pembrolizumab-containing pharmaceutical formulations.

[0082] Example 4: Effect of buffer and surfactant content on the stability of pembrolizumab-containing pharmaceutical formulations In this example, the effect of buffer and surfactant content on the stability of pembrolizumab-containing pharmaceutical formulations was investigated. Specifically, formulations 1A, 1B, 1C, 1D, 2A, 2B, 2C, 2D, 2E, 2F, 2G, and 2H were subjected to temperature stress, and then the purity of the samples was measured via SEC.

[0083] The aforementioned temperature stress was performed by placing 0.3 mL of each formulation into a 2 cc vial made of Type I glass material, and exposing the vial to a temperature stress condition of 40°C in a stable incubator for 4 weeks. Specifically, the vial was placed in the stable incubator and left under conditions of a temperature of 40 ± 2°C and a relative humidity of 75 ± 5% for 4 weeks. SEC analysis was performed on the formulations stored for 4 weeks as described above.

[0084] The results are shown in Table 5 and Figure 4. Table 5 shows the composition of formulations with different buffer and PS80 content, and the purity of the formulations subjected to 4 weeks of temperature stress, expressed as HMW content. Figure 4 shows the Δ%HMW of formulations with different buffer and PS80 content after being subjected to 4 weeks of temperature stress.

[0085] [Table 5]

[0086] As shown in Table 5 and Figure 4, at 4 weeks, the average Δ%HMW values ​​for the 20 mM histidine buffer-containing formulation and the formulation without the buffer were 0.51% and 0.42%, respectively, indicating that the formulation without the buffer was even more stable than the formulation containing the 20 mM histidine buffer. On the other hand, the average Δ%HMW value for the 40 mM histidine buffer-containing formulation was 0.80%, showing the lowest stability.

[0087] Furthermore, among formulations containing 20 mM histidine buffer, the Δ%HMW value of formulation 2A, which does not contain PS80, was the lowest compared to formulations containing PS80, i.e., formulations 2B, 2C, and 2D. Similarly, among formulations containing 40 mM histidine buffer, the Δ%HMW value of formulation 3A, which does not contain PS80, was the lowest compared to formulations containing PS80, i.e., formulations 3B, 3C, and 3D. This indicates that formulations free of buffer and PS80 are more stable than formulations containing both buffer and PS80.

[0088] Furthermore, the pH of the formulations stored under stress conditions for four weeks was measured. Table 6 shows the results of pH measurements for samples left at 40°C under temperature stress conditions for four weeks.

[0089] [Table 6]

[0090] As shown in Table 6, the pH change in the 20 mM histidine buffer group, the 40 mM histidine buffer group, and the buffer-free dosage form compared to the initial values ​​under 40°C / 4-week stress conditions did not show a significant difference. This confirmed that the pH buffering effect was promoted in the buffer-free dosage form as well as in the buffer-containing dosage form.

[0091] Example 5: Effects of changes in buffer and pH on the stability of pembrolizumab-containing pharmaceutical formulations In this example, the effects of the presence or absence of buffer and changes in pH on the stability of pembrolizumab-containing pharmaceutical formulations were investigated.

[0092] First, each of the pharmaceutical preparations listed in Table 7 was manufactured using the same method as described above.

[0093] [Table 7]

[0094] Each of the formulations in Table 7 was subjected to temperature stress using the same method as in Example 1, and then SEC analysis was performed to measure the purity of the samples.

[0095] The results are shown in Table 8. Table 8 shows the purity of formulations with and without buffer, and with different pH levels, expressed as HMW content.

[0096] [Table 8]

[0097] As shown in Table 8, buffer-free formulations had lower %HMW values ​​than buffer-containing formulations. Furthermore, formulations at pH 5.0 had lower %HMW values ​​than formulations at pH 5.5. In particular, the buffer-free formulation at pH 5.0 had the lowest %HMW value. Therefore, it was confirmed that buffer-free formulations are more stable than buffer-containing formulations, formulations at pH 5.0 are more stable than formulations at pH 5.5, and the buffer-free formulation at pH 5.0 exhibits the best stability.

[0098] Furthermore, after applying temperature stress to each of the formulations in Table 7, the % acidity value was measured using icIEF as described above.

[0099] The results are shown in Table 9. Table 9 shows the % acidity values ​​of formulations with and without buffer, and with different pH levels.

[0100] [Table 9]

[0101] As shown in Table 9, buffer-free formulations had lower % acidity values ​​than buffer-containing formulations. Furthermore, formulations with a pH of 5.0 had lower % acidity values ​​than formulations with a pH of 5.5. In particular, the buffer-free formulation with a pH of 5.0 had the lowest % acidity value. Therefore, it was confirmed that buffer-free formulations are more stable than buffer-containing formulations, formulations with a pH of 5.0 are more stable than formulations with a pH of 5.5, and the buffer-free formulation with a pH of 5.0 exhibits the best stability.

[0102] Example 6: Expansion of stabilizers In this embodiment, the effects of changing the type of stabilizer and adding antioxidants on the stability of pembrolizumab-containing pharmaceutical formulations were investigated.

[0103] First, each of the pharmaceutical preparations listed in Table 10 was manufactured using the same method as described above.

[0104] [Table 10]

[0105] Each of the formulations in Table 10 was subjected to temperature stress using the same method as in Example 1, and then SEC analysis was performed to measure the purity of the samples.

[0106] The results are shown in Table 11. Table 11 shows the purity of formulations with different types of stabilizers and added antioxidants, expressed as HMW content.

[0107] [Table 11]

[0108] As shown in Table 11, formulations 7A, 7B, and 7C, which contained an antioxidant without a buffer and had a pH of 5.0, had lower Δ%HMW values ​​compared to formulation 6A, which contained a buffer but did not contain an antioxidant and had a pH of 5.5. In other words, formulations containing an antioxidant without a buffer and having a pH of 5.0 are more stable than formulations containing a buffer but not containing an antioxidant and having a pH of 5.5. Furthermore, formulations containing an antioxidant without a buffer and having a pH of 5.0 showed excellent stability even when the type of stabilization was varied, including sucrose, sorbitol, trehalose, arginine, or combinations thereof.

[0109] Furthermore, after applying temperature stress to each of the formulations in Table 10, the % acidity value was measured using icIEF as described above.

[0110] The results are shown in Table 12. Table 12 shows the % acidity values ​​of formulations with different types of stabilizers and added antioxidants.

[0111] [Table 12]

[0112] As shown in Table 12, formulations 7A, 7B, and 7C, which contained an antioxidant without a buffer and had a pH of 5.0, showed similar or lower % acidity changes compared to formulation 6A, which contained a buffer but did not contain an antioxidant and had a pH of 5.5. In particular, 7A showed the best stability as it had the lowest Δ% acidity value.

[0113] Example 7: Effect of buffer type on the stability of pembrolizumab-containing pharmaceutical formulations In this embodiment, the effect of buffer type on the stability of pembrolizumab-containing pharmaceutical formulations was investigated.

[0114] First, each of the pharmaceutical preparations listed in Table 13 was manufactured using the same method as described above.

[0115] [Table 13]

[0116] Each of the formulations in Table 13 was subjected to temperature stress using the same method as in Example 1, and then SEC analysis was performed to measure the purity of the samples.

[0117] The results are shown in Table 14. Table 14 shows the purity of formulations with different types of buffers, expressed as HMW content.

[0118] [Table 14]

[0119] As shown in Table 14, formulations containing histidine, namely 8C and 8D, had higher Δ%HMW values ​​compared to formulations containing other buffers. Therefore, it was confirmed that formulations containing histidine as a buffer are less stable than formulations containing other types of buffers.

[0120] Example 8: Stability of a pharmaceutical formulation containing high-concentration pembrolizumab In this example, the stability of a pharmaceutical formulation containing a high concentration of pembrolizumab was confirmed.

[0121] First, each of the pharmaceutical preparations listed in Table 15 was manufactured using the same method as described above.

[0122] [Table 15]

[0123] Of the formulations in Table 15, 9A through 9D and 9K were subjected to temperature stress using the same method as in Example 1, and then SEC analysis was performed to measure the purity of the samples. The results are shown in Table 16.

[0124] [Table 16]

[0125] As shown in Table 16, formulations 9A to 9D, which contain a high concentration of 150 mg / ml pembrolizumab but do not contain a buffer, showed superior stability compared to formulation 9K, which contains a buffer.

[0126] Furthermore, after applying temperature stress to each of the formulations in Table 15, the % acidity value was measured using icIEF as described above. The results are shown in Table 17.

[0127] [Table 17]

[0128] As shown in Table 17, formulations 9A to 9J, which contain a high concentration of 150 mg / ml pembrolizumab but do not contain a buffer, showed superior stability compared to formulation 9K, which contains a buffer.

[0129] Furthermore, each of the pharmaceutical preparations listed in Table 18 was manufactured using the same method as described above.

[0130] [Table 18]

[0131] After applying temperature stress to each formulation in Table 18, the % acidity value was measured using icIEF as described above. The results are shown in Table 19.

[0132] [Table 19]

[0133] As shown in Table 19, formulations 10A to 10I, which contain high concentrations of pembrolizumab at 200 mg / ml or 250 mg / ml but do not contain a buffer, showed superior stability compared to formulation 9K, which contains a buffer.

Claims

1. (a) an anti-PD-1 antibody or its antigen-binding fragment, wherein the anti-PD-1 antibody is pembrolizumab. (b) A stabilizer, wherein the stabilizer is a polyol, an amino acid or a pharmaceutically acceptable salt thereof, or a mixture thereof. The polyol is sorbitol, trehalose, or a mixture thereof. The amino acid is lysine, arginine, a pharmaceutically acceptable salt thereof, or a mixture thereof. A stable anti-PD-1 antibody pharmaceutical formulation that does not contain buffer and has a pH of 4.5 to 6.

5.

2. The pharmaceutical preparation according to claim 1, wherein the preparation has a pH of 5.0 to 5.

5.

3. The pharmaceutical formulation according to claim 1, wherein the anti-PD-1 antibody comprises a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3, and a light chain CDR1 having the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:

6.

4. The pharmaceutical preparation according to claim 1, wherein the concentration of the anti-PD-1 antibody or its antigen-binding fragment is 5 to 250 mg / ml.

5. The pharmaceutical preparation according to claim 1, which is free of surfactants or further contains surfactants.

6. The pharmaceutical formulation according to claim 5, wherein the surfactant is a polysorbate, a poloxamer, a sorbitan ester of another fatty acid, or a mixture thereof.

7. The pharmaceutical preparation according to claim 1, further comprising an antioxidant.

8. The pharmaceutical preparation according to claim 7, wherein the antioxidant is methionine.

9. The pharmaceutical preparation according to claim 1, wherein the pharmaceutical preparation is for subcutaneous injection or intravenous injection.

10. The concentration of the anti-PD-1 antibody or its antigen-binding fragment is 5 to 200 mg / mL. The pharmaceutically acceptable formulation according to claim 1, wherein the stabilizer is 1.0 to 15.0% (w / v) trehalose, its hydrate, or a mixture thereof; 1.0 to 20.0% (w / v) sorbitol, its hydrate, or a mixture thereof; 0.1 to 300.0 mM arginine, lysine, a pharmaceutically acceptable salt thereof, or a mixture thereof; or a mixture of two or more thereof.

11. A pharmaceutical preparation according to claim 1 for the treatment of cancer.

12. The step of adding a stabilizer to a solvent to prepare a mixed solution; and The step of adding pembrolizumab or its antigen-binding fragment to the aforementioned mixed solution; or The step of adding pembrolizumab or its antigen-binding fragment to a solvent to prepare a solution of pembrolizumab or its antigen-binding fragment; and A method for producing a stable pharmaceutical formulation according to claim 1, comprising the step of adding a stabilizer to the solution, wherein the production step is carried out without adding a buffer, and the stabilizer is a polyol, an amino acid or a pharmaceutically acceptable salt thereof, or a mixture thereof. The polyol is sorbitol, trehalose, or a mixture thereof. The amino acid is lysine, arginine, a pharmaceutically acceptable salt thereof, or a mixture thereof. method.

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