Stable anti-PD-1 antibody pharmaceutical formulations

Abstract

The present invention provides a stable anti-PD-1 antibody pharmaceutical formulation, a method for treating cancer, and a method for manufacturing the same. [Solution] The present invention provides a stable anti-PD-1 antibody pharmaceutical formulation comprising an anti-PD-1 antibody or its antigen-binding fragment, a stabilizer, and a buffer, 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 a great need to develop stable anti-PD-1 antibody pharmaceutical formulations to enhance drug stability. It is being widely discussed. Pharmacological formulations of anti-PD-1 antibodies require buffing for drug stability. a. Contains stabilizers and surfactants.

[0003] However, some buffers are prone to autodegradation under stress conditions, and antibodies This can affect the stability of pharmaceuticals. Also, in the case of phosphoric acid, pH in a freezing environment... This condition cannot be maintained, which affects the stability of the antibody.

[0004] Therefore, it contains a buffer that may affect the stability of anti-PD-1 antibody pharmaceutical preparations. Therefore, there is still a need for the development of formulations that can stabilize antibody proteins. [Overview of the project] [Problems that the invention aims to solve]

[0005] One embodiment comprises (a) an anti-PD-1 antibody or its antigen-binding fragment, and (b) a stabilizer. Furthermore, it does not contain buffer and has a stable anti-PD-1 inhibitor with a pH of approximately 4.5 to 6.5. This provides pharmaceutical preparations for the body.

[0006] Another embodiment includes the step of administering the pharmaceutical formulation to an individual, thereby treating cancer in the individual. This provides a method for doing so.

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

[0008] All technical terms used herein are defined in relation to the present invention unless otherwise specified. It is used in a sense that is generally understood by those skilled in the art within the field. Furthermore, this specification includes: The preferred methods and samples are described, but similar or equivalent methods are also included. This is also included within the scope of the present invention. Furthermore, the numerical values ​​described herein are not explicitly stated. Even if it is included in the meaning of "approximately," all of the references listed in this specification are considered to include the meaning of "approximately." The contents of these publications are, as a whole, integrated into this specification by reference.

[0009] One embodiment comprises (a) an anti-PD-1 antibody or its antigen-binding fragment, and (b) a stabilizer. Furthermore, it does not contain buffer and has a stable anti-PD-1 inhibitor with a pH of approximately 4.5 to 6.5. We provide pharmaceutical preparations for the body.

[0010] Unlike commonly known antibody-containing pharmaceutical preparations, the aforementioned pharmaceutical preparation uses a buffer. Although it does not contain [specific substances], a pH of 4.5 to 6.5 is a desirable environment for storing anti-PD-1 antibodies. Furthermore, an even more desirable environment is one where a pH of approximately 5.0 to 5.5 can be maintained. The pH of the aforementioned pharmaceutical preparation is, for example, pH 4.5 to 6.3, or 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. pH 8 to 6.0, pH 5.0 to 6.0, pH 5.2 to 6.0, pH 4.5 or 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 pharmaceutical formulation is also about pH 4.5 to about pH 5.5. In another specific example , the pH of the pharmaceutical formulation is also about pH 5.0 to about pH 5.5.

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

[0012] The term "antibody" refers to any form of antibody having the activity of specifically binding 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" is a fragment that can bind to the PD-1 antigen in an anti-PD-1 antibody, and includes, for example, Fab fragments, F(ab’)2 fragments, Fc fragments, or scFv fragments , but is not limited thereto. "Pembrolizumab antibody" is a humanized antibody used in cancer immunotherapy and is sold under the name KEYDTRUDA(R) as one of various trade names. The pembrolizumab is used for melanoma, non-small cell lung cancer (NSCLC), N ​​Lung cancer, including SCLC), head and neck squamous cell carcinoma (HNSCC) Head and neck cancers, including cancer, and classic Hodgkin lymphoma (cHL) Hodgkin lymphoma (including ma), urothelial carcinoma, renal cell carcinoma, gastric cancer, high-frequency microsatellite cancer Microsatellite instability-high cancer (MSI-H), mismatch repair Deficiency (dMMR: mismatch repair deficient): solid tumors, cervical cancer, liver cancer, Merkel It is also used in the treatment of cell carcinoma (MCC: Merkel cell carcinoma), etc. (Pembroli) Zumab is also the active pembrolizumab present in commercially available Keytruda. This also includes biosimilars or biobetters. The pembrolizumab is selected from one or more of the group consisting of SEQ ID NOs: 1 to 3. A heavy chain variable region containing an amino acid sequence, and selected from the group consisting of SEQ ID NOs: 4 to 6. It also contains a light chain variable region that includes one or more amino acid sequences. This includes heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1 (NYYMY), and SEQ ID NO: 2 (GINPSNGGT Heavy chain CDR2 having the amino acid sequence NFNEKFK, and the amino acid sequence of SEQ ID NO: 3 (RDYRFDMGFDY) The amino acid sequence of heavy chain CDR3 having an acid no acid sequence, and SEQ ID NO 4 (RASKGVSTSGYSYLH) Light chain CDR1 having the amino acid sequence of SEQ ID NO: 5 (LASYLES), Light chain CDR2 having the amino acid sequence of SEQ ID NO: 5 (LASYLES), It also contains a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6 (QHSRDLPLT). The pembrolizumab is sequence number 7 (VQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGL EWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKG PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNV DHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPSQEEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL A heavy chain variable region having the amino acid sequence GK, and Sequence ID No. 8 (IVLTQSPATLSLSPGERATLSCRA SKGVSTSSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGG TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL Light chain variable region containing the amino acid sequence SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC But so.

[0014] The term "biosimilar" is also called an equivalent biopharmaceutical. Because the product is not a synthesized chemical product, but is produced through cells, The process of replicating an original drug with a generic version is called the process of creating a generic version of the same product. This is not possible. Therefore, a duplicated drug related to biopharmaceuticals is not the same as the original drug. However, they are called biosimilars because they are similar.

[0015] The term "biobetter" refers to a biopharmaceutical that is based on an original biopharmaceutical and has an effect. It is a drug that has been improved in terms of effectiveness, safety, and convenience. It is superior to existing biopharmaceuticals. It is called a biobetter in the sense of (er). This biobetter is a genetically modified technology It can be manufactured using biotechnology techniques such as cell culture and other bioengineering technologies.

[0016] Pembrolizumab is a programmed cell death protein 1 (PD-1:prog It targets the rammed cell death protein 1) receptor. Pembrolizumab targets cancer cells IgG4 isotactic blocks protective mechanisms, allowing the immune system to destroy cancer cells. It is a p-antibody. Pembrolizumab was approved for medical use in the United States in 2014. Furthermore, in 2017, pembrolizumab was found to be used for specific genetic abnormalities (anomalies), such as If the excision is incomplete due to mismatch repair deficiency (dMMR) or microsatellite instability, It is permitted for solid tumors that are unresectable or metastatic. Furthermore, pembrolizumab can be used alone or in combination with other chemotherapy agents. The pembrolizumab may also be administered by intravenous or subcutaneous injection.

[0017] Pembrolizumab can also be produced by common methods known in the industry. For example, U S9,834,605 ​​and WO2008 / 156712A1 are used by persons skilled in the art in the industry. This describes a method that can be used to manufacture brolizumab. For example, the pem Brolizumab targets immunoglobulin light chain genes and immunoglobulin heavy chain genes in host cells. It can also be produced through recombinant expression.

[0018] The aforementioned anti-PD-1 antibody, for example, pembrolizumab or its antigen-binding fragment, is in an aqueous solution. In this context, it has a buffering function for pH. The anti-PD-1 antibody, for example, pembroliz The concentration of mab or its antigen-binding fragment corresponds to a buffering function of pH 4.5 to pH 6.5. It is also a suitable amount to provide the anti-PD-1 antibody, for example, pembroliz The concentration of mab or its antigen-binding fragment is also an effective amount for treating cancer. -1 The concentration of the antibody, for example, pembrolizumab or its antigen-binding fragment, is determined by the aforementioned pharmaceutical manufacturing process. The choice of agent is also made considering factors such as stability and viscosity. The anti-PD-1 antibody, for example, pembrose The concentration of lizumab or its antigen-binding fragment is, for example, 5 to 300 mg / mL, or less than 5. 250 mg / mL, 5 to 200 mg / mL, 10 to 200 mg / mL, 10 Ishi 165 mg / mL, 15 to 160 mg / mL, 5 to 45 mg / mL, 10 40 mg / mL, 15 to 35 mg / mL, 20 to 30 mg / mL, 130 mg / mL Ishi 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, It is also approximately 150 mg / mL, approximately 200 mg / mL, or approximately 250 mg / mL. A PD-1 antibody, such as pembrolizumab or its antigen-binding fragment, is administered at the aforementioned concentration. This contributes to the stability of pharmaceutical formulations, the viscosity of pharmaceutical formulations suitable for administration, and the maintenance of pH. It is possible.

[0019] The term "stabilizer" refers to a substance that maintains its state when left unattended or stored. This refers to a substance added to prevent changes or chemical reactions. The aforementioned stabilizer is a polyol. , or one or more selected from the group consisting of amino acids and metal salts.

[0020] In this specification, the term "polyol" refers to a polyol containing multiple hydroxyl groups. The excipient has multiple hydroxyl groups. The polyol is a sugar, sugar alcohol and It also contains sugar acid. The aforementioned sugar represents a soluble carbohydrate. The aforementioned sugar is They are monosaccharides, disaccharides, oligosaccharides, or polysaccharides. The sugar alcohols are derived from sugars. It is an organic compound in which each carbon atom has one hydroxyl group. The sugar acid is a chain The polyol is a sugar having a carboxyl group in one or both of the components. Fructose, mannose, galactose, sucrose, lactose, maltose, A pouch made of rehalose, mannitol, sorbitol, and polyethylene glycol. The selected force is one or more. The polyol is sorbitol, sucrose, tre It may also be haloth, mannose, maltose, mannitol, or a mixture thereof. The polyols mentioned include the anhydrides of polyols. For example, trehalose is trehalose. Furthermore, it also contains trehalose dihydrate. The concentration of the polyol is as described above. In pharmaceutical formulations, anti-PD-1 antibodies, such as pembrolizumab or its antigen binding, are used. Free to maintain the stability of the fragments and the viscosity of the liquid pharmaceutical formulation, within a range desirable to do so. It is adjusted to each specific polyol, sugar alcohol or sugar acid individually They can be different.

[0021] The concentration of the aforementioned sugar is 1.0 to 15.0% (w / v), 3.0 to 15.0% (w / v) v), 5.0 to 15.0% (w / v), 7.0 to 15.0% (w / v), 1.0 or 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, approximately 4.7% (w / v)) ), 6.5 to 8.5% (w / v) (for example, about 6.8% (w / v), about 7.0% (w / v), approximately 7.2% (w / v), or 7.8 to 8.2% (w / v) (for example, approximately 7.8%(w / v), approx. 7.9%(w / v), approx. 8.0%(w / v), approx. 8.1%(w / v), or approximately 8.2% (w / v). The sugars mentioned above are, for example, sucrose, gluco syl Yes. In one specific example, the sugar is 1.0 to 15.0% (w / v), 4.0 to 10.0% (w / v), 6.0 to 8.0% (w / v), or approximately 7.0% (w / v) It is also sucrose. In other specific examples, the sugar is 1.0 to 15.0% (w / v), 4.0 to 10.0% (w / v), 7.0 to 8.0% (w / v), or approximately It also contains 7.6% (w / v) trehalose.

[0022] The concentration of the sugar alcohol is 1.0 to 20.0% (w / v), for example, less than 1.0 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 0.0% (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 0.0% (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 It is also 5.0% (w / v). The aforementioned sugar alcohols are, for example, sorbitol, mannitol. Tor, its hydrate, or a mixture thereof. In one specific example, the sugar alcohol It also contains approximately 4.0% (w / v) sorbitol.

[0023] The aforementioned amino acids are glycine, alanine, valine, leucine, isoleucine, proline, Phenylalanine, tyrosine, tryptophan, serine, threonine, cysteine, meth Onion, asparagine, glutamine, lysine, arginine, histidine, aspartic acid , glutamic acid, a pharmaceutically acceptable salt thereof, or a mixture thereof. Mino acids include, for example, glycine, proline, phenylalanine, tyrosine, and tryptophan. This may also include lysine, arginine, their pharmaceutically acceptable salts, or mixtures thereof. In one specific example, the amino acid is arginine, a pharmaceutically acceptable salt thereof, or It is also a mixture of those. In another specific example, the amino acid is proline, its drug It is also a scientifically acceptable salt, or a mixture thereof. Furthermore, in another specific example, The amino acids are lysine, its pharmaceutically acceptable salts, or mixtures thereof. In yet another specific example, the amino acid is arginine, lysine, and its pharmaceutically acceptable It is also a tolerable salt, or a mixture thereof.

[0024] As the aforementioned stabilizer, the concentration of the amino acid 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 or 300.0 mM, 25.0 or 300.0 mM, 30.0 or 300.0 mM 50.0 to 300.0 mM, 80.0 to 300.0 mM, 100.0 to 3 0.0 mM, 120.0 to 300.0 mM, 0.1 to 250.0 mM, 0.5 or 250.0 mM, 1.0 or 250.0 mM, 5.0 or 250.0 mM, 1 0.0 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 mM Stone 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 M, 10.0 to 200.0 mM, 25.0 to 200.0 mM, 30.0 to 2 0.0 mM, 50.0 to 200.0 mM, 80.0 to 200.0 mM, 100 0.0 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 0.0 mM, 10.0 to 160.0 mM, 25.0 to 160.0 mM, 30.0 mM Stone 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 Stone 100.0 mM, 5.0 to 100.0 mM, 10.0 to 100.0 mM, 2 5.0 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 0.0 mM, 1.0 to 50.0 mM, 5.0 to 50.0 mM, 10.0 to 50 0.0 mM, 25.0 to 50.0 mM, or 30.0 to 50.0 mM, 0.1 Stone 40.0 mM, 0.5 to 40.0 mM, 1.0 to 40.0 mM, 5.0 mM or less 40.0 mM, 10.0 to 40.0 mM, 25.0 to 40.0 mM, or 3 0.0 to 40.0 mM, 0.1 to 30.0 mM, 0.5 to 30.0 mM, 1 0.0 to 30.0 mM, 5.0 to 30.0 mM, 10.0 to 30.0 mM, 2 5.0 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 0.1 to 10.0 mM, 0.5 to 10.0 mM, 1.0 to 10.0 mM, and It is also 5.0 to 10.0 mM. In one specific example, the amino acid is less than 1.0 mM. 10.0 mM, 1.0 to 5.0 mM, 2.0 to 10.0 mM, 2.0 to 5 Arginine in concentrations of 0.0 mM, 2.0 to 4.0 mM, or approximately 3.3 mM, and its pharmacologically It is also an acceptable salt, or a mixture thereof. In another specific example, the amino acid is 2 4 to 29 mM glycine, a pharmaceutically acceptable salt thereof, or a mixture thereof. The concentration of the aforementioned amino acid does not affect the target pH of the pharmaceutical preparation, and is anti-PD. -1 antibodies, for example, pembrolizumab or its antigen-binding fragments, do not affect the stability of the antibody. Within a certain range, it can be freely adjusted, and each specific amino acid produces different results. It may be possible.

[0025] The aforementioned metal salts are NaCl, KCl, NaF, KBr, NaBr, Na2SO4, and NaS It can also be CN, CaCl2, MgCl2, or K2SO4. The metal salt is, for example, Na It is also Cl or Na2SO4. The concentration of the metal salt is 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 mM Stone 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, 50.0 to 125 0.0 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 10 0.0 mM, 5 to 80.0 mM, 10.0 to 80.0 mM, 30.0 to 80 It can be 0.0 mM, or 50.0 to 80.0 mM. In one specific example, the metal salt This ranges from 5.0 to 150.0 mM, 20.0 to 140.0 mM, or approximately 100.0 It is also sodium chloride in mM. The concentration of the metal salt in the pharmaceutical formulation of the present invention is anti Maintaining the stability of PD-1 antibodies, such as pembrolizumab or its antigen-binding fragment, It can be freely adjusted within the range that does not cause precipitation, and varies individually depending on each specific metal salt. It can be. The stabilizer is also a mixture of a polyol and an amino acid. The polyol and When using a mixture with the amino acid, the amino acid is one of the stabilizers and viscosity reducers. It can perform one or more roles.

[0026] The statement that a pharmaceutical preparation provided herein "does not contain component A" means the same as above. This means that the pharmaceutical preparation does not contain or substantially does not contain component A. "Substantially does not contain component A" means either it does not contain component A at all, or component A is present. Even if component A is present in trace amounts that do not substantially affect the properties of the pharmaceutical preparation, This can also be interpreted to include cases where it is present, or where it exists in an undetectable amount.

[0027] The aforementioned pharmaceutical preparation is an anti-PD-1 antibody, such as pembrolizumab or its antigen-binding agent. It does not include any separate buffers other than the fragment. In this specification, the term "buffer" is used. This indicates a composition in which a pharmaceutical formulation is added to resist pH changes. The buffer The buffer can maintain the pH of the formulation within an acceptable range. Generally, the acid-base conjugate component causes the aforementioned formulation to change its pH. To resist oxidation. Where the concentration of a buffer is mentioned herein, the concentration mentioned This indicates the molar concentration of the buffer in its free acid or free base form. The phrase "does not contain buffer" indicates a pharmaceutical preparation that is resistant to pH changes. This indicates that it does not contain the buffer. It indicates that the intended buffer is not present in the pharmaceutical formulation. It can also be interpreted that it contains an amount that prevents it from functioning as a buffer. The buffer is Histidine, phosphoric acid (sodium phosphate or potassium phosphate), malic acid, tartaric acid, co Succinate, citrate, acetate, carbonic acid, and the drug It is also a pharmaceutically acceptable salt, or a mixture thereof. In one specific example, the buffer is histidine, its pharmaceutically acceptable salts, or mixtures thereof. Other specific examples include In this case, the buffer may be phosphoric acid, a pharmaceutically acceptable salt thereof, or a mixture thereof. be.

[0028] The aforementioned pharmaceutical preparations are those that do not contain surfactants, or those that further contain surfactants. It is also. The aforementioned surfactant is also a nonionic surfactant. The aforementioned nonionic surfactant The agents are polysorbates, poloxamers, sorbitan esters of other fatty acids, or those. It is also a mixture of the following: The polysorbate is polysorbate 20, polysorbate 40, It is also polysorbate 60, polysorbate 80, or a mixture thereof. This is also poloxamer 188. In the aforementioned pharmaceutical preparation, if a surfactant is included, The concentration of the surfactant is based on the total volume of the pharmaceutical formulation or the reconstituted formulation. to 0.001 to 1% (w / v), 0.001 to 0.5% (w / v), 0.00 1 to 0.1% (w / v), 0.001 to 0.05% (w / v), none of the above. 0.02% (w / v), 0.005 to 1% (w / v), 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. 0.008 to 0.5% (w / v), 0.008 to 0.1% (w / v), 0.008 It is 0.05% (w / v), or 0.008 to 0.02% (w / v). In a specific example, the pharmaceutical preparation is 0.02 to 0.04% (w / v) polysol Contains 20% polysorbate or 80% polysorbate.

[0029] The aforementioned pharmaceutical preparations also further contain antioxidants. "Ant)" means a substance that prevents oxidation. The aforementioned antioxidants include amino acids, vitamins, and Enzymes, glutathione, methylsulfonyl sorbate, or mixtures thereof. Yes. The aforementioned antioxidants include amino acids such as methionine, L-cysteine, and L-carnitine. or a mixture thereof. The antioxidants include vitamins such as vitamin A and vitamin B. It may also be C, vitamin E, or a mixture thereof. The coenzyme is coenzyme Q It is also 10. In one specific example, the antioxidant is also methionine.

[0030] The concentration of the antioxidant in the pharmaceutical formulation is such that the anti-PD-1 antibody, for example, pemb, Within a range desirable to maintain the stability of lorizumab or its antigen-binding fragment, The pharmaceutically acceptable formulation may vary depending on the specific type of antioxidant. In the case of a drug containing an antioxidant, the concentration of the antioxidant is 1 to 70 mM, 1 to 50 mM. mM, 1 to 30 mM, 5 to 50 mM, 5 to 30 mM, 1 to 20 mM, 1 or 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, It can be approximately 5 mM, approximately 20 mM, approximately 30 mM, approximately 40 mM, or even approximately 50 mM. (Example) In this case, the pharmaceutical preparation contains approximately 5 mM methionine. In another specific example, The aforementioned pharmaceutical preparation contains approximately 30 mM methionine. In yet another specific example, The pharmaceutical preparation described contains approximately 50 mM methionine.

[0031] The aforementioned pharmaceutical preparation is also a liquid. The aforementioned pharmaceutical preparation is for subcutaneous or intravenous injection. It is also for use. The aforementioned pharmaceutical preparation further comprises a suitable aqueous carrier to be suitable for injection. Furthermore, the aqueous carrier is pharmaceutically acceptable and non-toxic when administered to humans. These are substances that can be used, such as water, saline solution, intravenous solution, dextrose, or mixtures thereof. It's also a mixed crop.

[0032] The aforementioned pharmaceutical preparation has an appropriate osmotic pressure range when administered by subcutaneous or intravenous injection. It is also the case that the osmotic pressure is, for example, 200 to 400 mOsm / kg, 200 to 35 0 mOsm / kg, 250 to 300 mOsm / kg, 250 to 290 mOsm / kg, 270 to 328 mOsm / kg, 250 to 269 mOsm / kg, or The osmotic pressure is 328 to 350 mOsm / kg. This osmotic pressure minimizes the pain that may occur during administration. It can be appropriately adjusted to achieve this.

[0033] The aforementioned pharmaceutical formulation may have a viscosity within an appropriate range when administered by subcutaneous or intravenous injection. The viscosity, measured at room temperature of 25°C ± 3°C, is, for example, 0.5 to 100 cp, or 0. 5 to 90 cp, 0.5 to 80 cp, 0.5 to 70 cp, 0.5 to 60 c p, 0.5 to 50 cp, 0.5 to 40 cp, 0.5 to 30 cp, none of 0.5 The viscosity is 20 cp, 0.5 to 15 cp, or 0.5 to 10 cp. The dosage can be appropriately adjusted to minimize any pain that may occur during administration.

[0034] In one specific example, in the pharmaceutical preparation, the anti-PD-1 antibody, for example, pemb The concentration of lorizumab or its antigen-binding fragment is 5 to 300 mg / mL, or 5 to 25 0 mg / mL, 5 to 200 mg / mL, 15 to 30 mg / mL, or 150 mg / mL The concentration is 250 mg / mL, and the stabilizers are sucrose, glucose, and galactose. Maltose, protose, trehalose, sorbitol, mannitol, arginine Lysine, proline, glycine, phenylalanine, tyrosine, tryptophan, and its water A compound, its pharmaceutically acceptable salt, a mixture thereof, or a metal salt, and the pharmaceutically acceptable The pH of the target formulation is between pH 4.5 and pH 6.5, or between pH 5.0 and pH 5.5.

[0035] In the above specific example, the stabilizer is sucrose, glucose, galactose, mal Tose, protose, trehalose, their hydrates, or mixtures thereof, and the stable The concentration of the phosphate agent is 1.0 to 15.0% (w / v) or 3.0 to 15.0% (w / v). 5.0 to 15.0% (w / v), 7.0 to 15.0% (w / v), 1.0% (none) 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 0.0 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), 02 to 5.0%(w / v), 3 0.0 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, approximately 7.8% (w / v), approximately 7.9% (w / v), approximately 8% (w / v), approximately 8. It is also 1% (w / v), or about 8.2% (w / v). The stabilizer is sorbitol. The stabilizer is 1. 0 to 20.0% (w / v), for example, 1.0 to 15.0% (w / v), 1.0 Stone 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 0.0% (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 0.0% (w / v), 3.5 to 6.0% (w / v), 4.0 to 6.0% (w / v) It is also 4.0 to 5.5% (w / v), or 4.0 to 5.0% (w / v). The aforementioned stabilizers are arginine, lysine, proline, glycine, phenylalanine, and tyrosine. N, tryptophan, its hydrate, its pharmaceutically acceptable salts, or mixtures thereof The concentration of the stabilizer is 0.1 to 300.0 mM, or 0.5 to 300.0 mM. mM, 1.0 to 300.0 mM, 5.0 to 300.0 mM, 10.0 to 30 0.0 mM, 25.0 to 300.0 mM, 30.0 to 300.0 mM, 50.0 or 300.0 mM, 80.0 or 300.0 mM, 100.0 or 300.0 mM M, 120.0 to 300.0 mM, 0.1 to 250.0 mM, 0.5 to 25 0.0 mM, 1.0 to 250.0 mM, 5.0 to 250.0 mM, 10.0 mM or less 250.0 mM, 25.0 to 250.0 mM, 30.0 to 250.0 mM, 5 0.0 to 250.0 mM, 80.0 to 250.0 mM, 100.0 to 250 0.0 mM, 120.0 to 250.0 mM, 0.1 to 200.0 mM, 0.5 mM or less 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 M, 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 0.0 mM, 50.0 to 160.0 mM, 80.0 to 160.0 mM, 100.0 or 160.0 mM, 120.0 or 160.0 mM, 130.0 or 150.0 mM mM, 0.1 to 100.0 mM, 0.5 to 100.0 mM, 1.0 to 100 0.0 mM, 5.0 to 100.0 mM, 10.0 to 100.0 mM, 25.0 mM (not applicable) 100.0 mM, 30.0 to 100.0 mM, 50.0 to 100.0 mM, 8 0.0 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 mM, 10.0 to 40.0 mM, 25.0 to 40.0 mM, or 30.0 mM. 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 mM or less 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 mM It is also 10.0 mM. The aforementioned stabilizer is approximately 1.0 to approximately 15.0% (w / v) Sucrose, trehalose, its hydrate, or mixtures thereof, and about 0.1 to about 3 0.0 mM Arginine, Lysine, Proline, Glycine, Phenylalanine, Tyrosine It is also a mixture of tryptophan, its pharmaceutically acceptable salts, or mixtures thereof. The stabilizer is approximately 1.0 to approximately 20.0% (w / v) of sorbitol, mannitol. Lu, its hydrate, or a mixture thereof, and about 0.1 to about 300.0 mM arginine, Lysine, proline, glycine, phenylalanine, tyrosine, tryptophan, and their drugs It is also a mixture with a scientifically acceptable salt, or a mixture thereof.

[0036] In the above specific example, the pharmaceutical preparation also further contains a surfactant. The surfactant is also a polysorbate or a poloxamer. The polysorbate is It is also polysorbate 20 or polysorbate 80. The surfactant concentration is 0.00 1 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), 0.005 to 0.05% (w / v), 0.0 0.5 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 It is also %(w / v).

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

[0038] In the pharmaceutical formulation of the present invention, the anti-PD-1 antibody, for example, pembrolizumab or The antigen-binding fragment can be stabilized. The term "stabilization" refers to the aforementioned anti- PD-1 antibodies, such as pembrolizumab or its antigen-binding fragments, are produced before and after administration. During the manufacturing process, storage, or preservation, its physical stability, chemical stability, and / or biological stability This means substantially retaining activity, the physical stability, the chemical stability and / or The 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 is as follows: It also satisfies condition 1 or higher.

[0040] Stability can be measured at a selected temperature for a selected period of time. For example, in one specific case, a stable formulation can be stored at 2°C to 8°C for 12 months. Or, it is a formulation in which no significant changes are observed for a longer period of time. In other specific examples, it is stable. The formulation showed significant changes at temperatures of 2°C to 8°C for 18 months or longer. It is a formulation that does not undergo odor control. In other specific examples, the stable formulation is odor-free at 23°C to 27°C. The formulation is one in which no significant changes are observed for three months or longer. In this case, the stable formulation can be stored at 23°C to 27°C for 6 months or longer. This is a formulation in which no significant changes are observed. In other specific examples, a stable formulation is found at 23°C. In formulations where no significant changes are observed at 27°C for 12 months or longer. Yes. In other specific examples, the stable formulation lasts for 18 months at 23°C to 27°C. A formulation in which no significant changes are observed for a period of time or longer. Stability groups related to antibody formulations. The criteria are as follows: When measured by SE-HPLC, the antibody monomer content should be 10% or less, for example. Then, 5% or less, or 2.5% or less, is degraded. Antibody potency is the control. The levels are within 60% to 140% or 80% to 120% of the group or standard antibody levels. For example, when measuring low molecular weight species (LMW) with SE-HPLC, less than 10% of the antibody, 5 These also include those with changes of less than % or less than 2.5%. For example, high fractions obtained by SE-HPLC. When the number of hemoglobin molecules (HMW) was measured, the antibody showed a variation of 10% or less, 5% or less, or 2.5% or less. It also has chemical properties. Furthermore, the concentration and pH of the formulation should be within ±10% and ±5% respectively. Alternatively, it may have a change of ±2.5% or less.

[0041] The aforementioned pharmaceutical preparation is stored in a stability incubator at a temperature of 40±2°C and a humidity of 75±5%. When left for 4 weeks under these conditions, HM was found to be within the range of 10% or less, 5% or less, or 2.5% or less. The wattage (W) or pH will change.

[0042] The aforementioned pharmaceutical preparation is divided into 0.3 to 1 mL portions and placed in 2 cc vials of Type I glass material. Place the vial (Schott) into the stirrer (Heidolph) and stir at room temperature at 400 rpm. When stirred at PM for 72 hours, the odor was within the range of 10% or less, 5% or less, or 2.5% or less. This causes the HMW or pH to change.

[0043] The pembrolizumab or its antigen-binding fragment still retains biological activity. If the chemically stable state is observed for a predetermined period of time, then the chemical state within the pharmaceutical preparation is considered to be stable. It possesses chemical stability. This chemical stability is due to the chemically modified form of pembrolizumab. It can also be evaluated by detection and quantification. Chemical deformation can be a change in size or charge. This includes the charge change, which is also a charge change resulting from, for example, deamidation. The size deformation was observed using size exclusion chromatography (SE-HPLC) and sodium dodecyl sulfate chromatography. Polyacrylamide gel electrophoresis (SDS-PAGE), capillary electrophoresis, dodecay Sodium dodecyl sulfate (CE-SDS: capillary electrophoresis) ,) analysis, and substrate-assisted laser desorption / ionization / time-of-flight mass spectrometry (MALDI / TOF) MS:matrix-assisted laser desorption / ionization / time-of-flight mass spectrome The charge change can also be evaluated using try, ). IEC: ion exchange chromatograph) and image capillary isoelectric focusing (ICI It is also evaluated by EF (imaged capillary isoelectric focusing). This activity is P This can be determined by D-1 ligand binding analysis. The PD-1 ligand binding analysis is By enzyme-linked immunosorbent assay (ELISA) This is also performed. The ELISA analysis determines the ratio of pembrolizumab binding to PD-1 ligand. This measures (%). Pembrolizumab sample and PD-1 on ELISA substrate. The degree of bonding y after reaction with Gand was determined by measuring the absorbance at 450 nm and the relative bonding ratio. You can obtain the relative binding rate (%).

[0044] The aforementioned anti-PD-1 antibody, for example, pembrolizumab or its antigen-binding fragment, is pharmaceutically The formulation possesses biological activity. This biological activity is, for example, found in pharmaceutical formulations. Within the context, the biological activity of the anti-PD-1 antibody or its antigen-binding fragment is determined by the pharmaceutical manufacturing process. Within approximately 30%, 20%, or 10% of the biological activity shown at the time of manufacture of the agent (or If it is within the analytical error range, it possesses biological activity. The said biological activity is, for example, It can also be determined by antigen-binding analysis.

[0045] Stabilization depends on temperature stress, for example, freezing and thawing at 40°C for one to four weeks. Freezing stress, for example, five repetitions of a cycle of freezing at -70°C and thawing at room temperature, or stirring stress For example, in a stirrer, apply a rotational force of 400 rpm for 72 hours to remove large particles. Using high-performance liquid chromatography (SE-HPLC: size-exclusion HPLC), This can be evaluated by measuring %HMW, %monomer, and / or %LMW. In one specific example, the pharmaceutical formulation of the present invention is equivalent to or equivalent to Keytruda(R). Alternatively, it may have even smaller Δ%HMW, Δ%LMW, or Δ% monomer values. Stable Chemical treatment also involves applying temperature stress, freeze-thaw stress, or stirring stress to the image. Using icIEF (ic-IEF), we measure the % acidity change. It can be evaluated by doing so. In one specific example, the pharmaceutical preparation of the present invention is Ke It may have a Δ% acidity value equivalent to or even lower than that of ytruda(R).

[0046] The stable pharmaceutical formulations provided herein have an antibody content of 25 mg / ml. (pH 5.5 or pH 5.0), 0.3 to 1 mL of the formulation, 2 Type I glass containers Stored in a cc vial (Schott) at 40°C for 4 weeks, and measured using a standard SEC. The change in %HMW, i.e., the %HMW value after 4 weeks minus the %HMW value at week 0, is 10.0%. The following are also 5.0% or less, or 2.5% or less.

[0047] The stable pharmaceutical formulations provided herein have an antibody content of 25 mg / ml. (pH 5.5 or pH 5.0), place the formulation in a polypropylene microtube. The % change in acidity was measured using icIEF after being stored at 40°C for 4 weeks, i.e., 4 The percentage acidity after one week minus the percentage acidity value at week 0 is 20.0% or less, 15.0% or less, or 10.0% or less. It's also below.

[0048] In one specific example, the pharmaceutical preparation is (i) an anti-PD-1 antibody or its antigen binding cleavage. (ii) a stabilizer and (iii) a buffer excluding histidine, in an amount of about 4.5 to It has a pH of approximately 6.5. The buffer, excluding the histidine, contains succinate and citrate. It can be acetate, phosphate, or a combination thereof.

[0049] Another embodiment provides a method for treating cancer with the aforementioned pharmaceutical preparations. The pharmaceutical preparations are as described above. The method of treating cancer is that the pharmaceutical preparations cure the cancer. It also includes a step of administering the drug to the individual in a manner that is effective for treatment. The individual is a Hi It is also a to

[0050] In the above method, the term "cancer" is typically defined as upward-regulated cell growth. It refers to or describes a pathological condition in mammals that is characterized by a specific feature. The aforementioned cancer is cancer This includes, but is not limited to, tumors, lymphomas, leukemias, blastomas, and sarcomas. i. The aforementioned cancers include, for example, squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, h. Dikin lymphoma, non-Hodgkin lymphoma, gastrointestinal cancer or gastrointestinal tract cancer, kidney cancer, ovarian cancer, liver cancer Lymphocytic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer Thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, pleoplastic glioma, cervical cancer Brain cancer, stomach cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon carcinoma, Merkel cell carcinoma, high frequency microsurgery Telyte-instability cancer, esophageal cancer, urothelial carcinoma, primary mediastinal giant B-cell lymphoma and head and neck It also includes cancer.

[0051] Another embodiment involves the steps of adding a stabilizer to a solvent to produce a mixed solution, and adding to the mixed solution Adding an anti-PD-1 antibody or its antigen-binding fragment, or adding an anti-PD-1 antibody to a solvent. Alternatively, the antigen-binding fragment thereof is added to produce a solution of the anti-PD-1 antibody or its antigen-binding fragment. A stable pharmaceutical formulation is manufactured, comprising the steps of: preparing the solution; and adding a stabilizer to the solution. The present invention provides a method in which the manufacturing step is carried out without adding a buffer.

[0052] In the above method, the solvent may be an aqueous solvent, such as water, or saline. The method involves adding the anti-PD-1 antibody or its antigen-binding fragment to a mixed solution. The method also further includes the step of adding a surfactant. or further comprising the step of adding an antioxidant to the mixed solution to which the antigen-binding fragment has been added. Also, the above method sets the pH of the formulation to approximately pH 4.5 to approximately pH 5.5, and approximately pH 5 It may also include a step to adjust the pH to 0.5, or approximately 5.0.

[0053] Manufacturing of a pharmaceutical preparation as claimed in the terms or elements referred to in the aforementioned pharmaceutical preparation. The parts mentioned in the explanation of the method are the pharmaceutical preparations requested in the aforementioned section. It is understood to be the point mentioned in the explanation related to that. [Effects of the Invention]

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

[0055] According to one method for treating cancer in an individual, it is possible to efficiently treat cancer in the individual. It is possible.

[0056] According to one embodiment of a method for producing a stable anti-PD-1 antibody pharmaceutical formulation, This allows for the efficient production of pharmaceutical formulations of anti-PD-1 antibodies. [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 the same as those described in this invention. These are merely illustrative examples of the invention, and do not limit the scope of the present invention. There isn't one.

[0059] Materials and methods 1. Size exclusion chromatography (SEC) The purity of the sample was examined by size exclusion chromatography. In Raffi, antibody monomers and high molecular weight species (HMW) And the percentage of low molecular weight species (LMW) is determined. In exclusion chromatography, the LMW elutes more slowly than the HMW. The presence of MW 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 subjected to each batch. The solution was diluted using a ferrite rod and loaded into the wells of a 96-well plate. DynoPro (R) Plate Reader TMThe sample was loaded into a Wyatt Technology II instrument. , while increasing the temperature at a rate of 0.15°C / min in the range of 25°C to 70°C, pharmaceutics The size of aggregates in the target formulation was measured via dynamic light scattering (DLS). Stability The evaluation involves measuring the temperature at which the size of the aggregate changes, T agg Large value This indicates that it is even more stable.

[0061] 3.HUNKY (ΔG, AggPath) measurement method Aggregates in the sample were examined using the HUNKY measurement method. (HUNKY instrument, Unchained Labs.) The device is capable of confirming protein stability through chemical denaturation, and the device is capable of confirming protein stability through chemical denaturation. Using a denaturing agent and a sample, C 1 / 2 ΔG, AggPath and ΔG trend The value It can be confirmed. C 1 / 2 This ranks the stability. ΔG is the stability Quantify. AggPath predicts aggregation. ΔG trend This provides details about aggregation. show.

[0062] 4. Imaging capillary isoelectric focusing (icIEF) (cfil) The % acidity value of each sample was measured using icIEF. Specifically, each sample was... , placed in a 1.5 mL microtube made of polypropylene, the tube The sample was exposed to a stable incubator under temperature stress conditions of 40°C for 4 weeks. It was implemented in the iCE3 system (Protein Simple, USA) and utilized iCECFR software. The charge variation values ​​of the proteins were measured. % acidity indicates the charge variation values ​​of the proteins. The measurement identifies charge variants of proteins under stress. If the protein undergoes a change, it will either aggregate or its charge will change. Therefore, charge mutations in proteins can also serve as one factor in confirming their 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. did.

[0064] First, in the composition of each pharmaceutical preparation listed in Table 1, pembrolizumab and surfactant A buffer solution was prepared by adding the buffer (without the actionant) and the stabilizer to sterile distilled water. Mubrolizumab administered via a dialysis cassette (Slide-A-Lyzer cassette, Thermo Fisher Scientific) After being placed in the container, the solution is then placed in a beaker containing the respective compositions listed in Table 1, and dialysis is performed. The existing pembrolizumab solution was replaced with the buffer solution. Finally, the pembrolizumab In the lorizumab solution, the surfactant PS80 from the aforementioned prepared buffer solution is added to a 1x concentration. It was added in this manner. Then, using each solution, pembrolizumab was added to a final concentration of 25 mg / mL. The concentration was adjusted.

[0065] [Table 1]

[0066] Example 1: Buffer's effect on the stability of pembrolizumab-containing pharmaceutical formulations. In this embodiment, the buffer affects the stability of the pembrolizumab-containing pharmaceutical formulation. The effects were confirmed. Specifically, temperature stress was applied to formulations 1A, 2I, 3A, 4A, and 5Ano. After adding the substance, the purity of the sample was measured.

[0067] The aforementioned temperature stress is applied by placing 0.3 mL of each of the formulations into a 2 cc bag of Type I glass material. Place the vial in a stable incubator and subject the vial to a temperature stress condition of 40°C for 4 It was exposed for a week. Specifically, the vial was placed in the stable constant temperature chamber at a temperature of 40±2°C. The samples were placed under conditions of °C and relative humidity of 75±5% for 4 weeks. For the formulations stored for 4 weeks, the above information was provided. In some cases, SEC analysis was performed.

[0068] The results are shown in Table 2 and Figure 1. Table 2 shows formulations with different buffers, and This table shows the purity of formulations subjected to 4 weeks of temperature stress, expressed as HMW content. Figure 1 shows the buff A diagram showing the Δ%HMW of formulations after applying temperature stress for 4 weeks to formulations with different properties. That is the case.

[0069] [Table 2]

[0070] As shown in Table 2 and Figure 1, the average Δ%HM of buffer-containing formulations over 4 weeks The W value and the Δ%HMW value of the formulation without buffer were 0.64% and 0.39%, respectively. The percentage is such that formulations without buffer are more stable than formulations with buffer. Yes, they are.

[0071] Example 2: Stabilization of pembrolizumab-containing pharmaceutical formulations by adjusting the content of buffer and surfactant. Effects on sex In this example, the content of the buffer and surfactant is pembrolizumab-containing pharmaceutical The effect on the stability of the formulation was confirmed. Specifically, 1A, 1B, 1C, 1D, 2A, 2 For formulations B, 2C, 2D, 2E, 2F, 2G, and 2H, the DLS measurement method was used to determine the T ag g The value was measured.

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

[0073]

Table 3

[0074] As shown in Table 3 and FIG. 2, the T values of the formulations that do not contain the surfactant PS80 or contain 0.02% thereof agg are higher than those of the formulations containing 0.10% or 0 .20% of the surfactant PS80. That is, the formulations containing a low content of agg PS80 less than 0.10%, namely 1A, 1B, 2A, 2B, 2E and 2F, show aggregation at a higher temperature compared to the formulations containing a high content of PS80 of 0.10% or more, namely 1C, 1D, 2C, 2D, 2G and 2 H. That is, the formulations containing a low content of PS80 less than 0.10% are more stable than the formulations containing a high content of PS80 of 0.10% or more.

[0075] On the other hand, for 2A, 2B, 2E, 2F, that is, when the PS80 concentration is less than 0.10% , the histidine content has no significant effect on the change in T agg .

[0076] Furthermore, among the preparations containing 20 mM histidine buffer, the preparations containing PS80, i.e. , 2B, 2C and 2D T agg Compared to the value, formulations that do not contain PS80, i.e., 2 A's T agg The values ​​were higher than those of the other formulations. Of these, formulations containing 0.02% PS80, i.e., 2F T agg The values ​​are 2E, 2G and T of 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 embodiment, buffer-free and 20 mM histidine buffer are used with Pembrol. The effect on the stability of zumab-containing pharmaceutical preparations was investigated. Specifically, preparations 1B and 2 For formulation B, the ΔG value was measured using the HUNKY measurement method.

[0078] Unlike the DLS measurement method, which measures protein aggregation due to temperature rise, the HUNKY measurement method does not use gua. Responding to chemical stress mediated by nidin-HCl (Gdn-HCl: guanidine-HCl) This measures stability. Specifically, the HUNKY measurement method measures stability between 0M and 5.5M. The ratio of absorbance intensities at 348 nm and 332 nm in a Gdn-HCl concentration gradient. The ratio, or the ratio (348 / 332), is measured. Due to protein denaturation, the aforementioned The ratio increases.

[0079] The results are shown in Table 4 and Figure 3. Table 4 shows the results for buffer-free and 20 mM hiss This table shows the composition of preparations containing thidine, and the measured ΔG values ​​for them. Figure 3 This refers to the denaturing agent concentration changes measured for buffer-free and 20 mM histidine-containing formulations. This is a diagram showing the ratio (348 / 332) due to the chemical process.

[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 affects the stability of pembrolizumab-containing pharmaceutical preparations. This indicates that the impact is not significant.

[0082] Example 4: Stabilization of pembrolizumab-containing pharmaceutical formulations by adjusting the content of buffer and surfactant. Effects on sex In this embodiment, the content of the buffer and surfactant is determined to be the same as that of pembrolizumab-containing pharmaceutics. The effect on the stability of the target formulation was confirmed. Specifically, 1A, 1B, 1C, 1D, 2A, After applying temperature stress to formulations 2B, 2C, 2D, 2E, 2F, 2G, and 2H, SEC The purity of the sample was measured via [a specific method / device].

[0083] The aforementioned temperature stress is applied by placing 0.3 mL of each of the formulations into a 2 cc bag of Type I glass material. Place the vial in a stable incubator and subject the vial to a temperature stress condition of 40°C for 4 It was exposed for a week. Specifically, the vial was placed in the stable constant temperature chamber at a temperature of 40± The samples were stored for 4 weeks under conditions of 2°C and 75±5% relative humidity. The preparations stored for 4 weeks were then examined as described above. Based on the above, an SEC analysis was performed.

[0084] The results are shown in Table 5 and Figure 4. Table 5 shows the results for different concentrations of buffer and PS80. The composition of the conditioned formulation and the purity of the formulation subjected to 4 weeks of temperature stress are shown in terms of HMW content. This is a table. Figure 4 shows formulations with different buffer and PS80 content, subjected to temperature testing for 4 weeks. It is a drawing showing the Δ%HMW of the formulation after addition of the lees.

[0085]

Table 5

[0086] As shown in Table 5 and Figure 4, at 4 weeks, the average Δ%HMW value of the formulation containing 20 mM histidine buffer and the Δ%HMW value of the formulation without buffer were 0 .51% and 0.42% respectively, and the formulation without buffer was more stable than the formulation containing 20 mM histidine ba ffer. On the other hand, the average Δ%HMW value of the formulation containing 40 mM histidine buffer was 0.80%, showing the lowest stability.

[0087] Also, in the formulation containing 20 mM histidine buffer, compared with the Δ%HMW values of the formulations containing PS80, namely 2B, 2C and 2D, the Δ%HMW value of the formulation without PS80, namely 2A, was the lowest. Also, in the formulation containing 40 mM histidine buffer compared with the Δ%HMW values of the formulations containing PS80, namely 3B, 3C and 3D, the Δ%HMW value of the formulation without P S80, namely 3A, was the lowest. This indicates that the formulation without buffer and PS80 is more stable than the formulations containing buffer and PS80.

[0088] Also, the pH of the formulation stored for 4 weeks under stress conditions was measured. Table 6 is a table showing the results of measuring the pH of the samples left for 4 weeks under the temperature stress condition of 40 °C

[0089]

Table 6

[0090] As shown in Table 6, compared with the initial values of the 20 mM histidine buffer group, the 40 mM histidine buffer group, and the buffer-free dosage form, under the stress condition of 40 °C / 4 weeks, the amount of pH change showed no significant difference. In the dosage form without buffer, it was confirmed that the pH buffering action proceeded in the same manner as in the dosage form containing buffer. 、pH change amount showed no significant difference. In the dosage form without buffer, it was confirmed that the pH buffering action proceeded in the same manner as in the dosage form containing buffer. ッファが含まれた剤形と同様に、pH緩衝作用が進められていることを確認した。

[0091] Example 5: How changes in buffer and pH affect the stability of pembrolizumab-containing pharmaceutical formulations. Boss's influence In this example, the influence of the presence or absence of buffer and the change in pH on the stability of the pembrolizumab-containing pharmaceutical preparation was confirmed. 学的製剤の安定性に及ぼす影響を確認した。

[0092] First, each pharmaceutical preparation described in Table 7 was manufactured by the same method as described above.

[0093]

Table 7

[0094] After applying temperature stress to each preparation in Table 7 by the same method as in Example 1, SEC analysis was performed to measure the purity of the sample.

[0095] The results are shown in Table 8. Table 8 is a table showing the purity of the preparations with different presence or absence of buffer and pH in terms of the HMW content. 剤の純度をHMW含量で示した表である。

[0096]

Table 8

[0097] As shown in Table 8, the buffer-free preparation had a higher %HM compared to the buffer-containing preparation. The W value was low. Also, the %HMW value of the formulation at pH 5.0 was lower than that of the formulation at pH 5.5. In particular, the formulation at pH 5.0 without buffer had the lowest %HMW value. Therefore, the buffer-free formulation is more stable than the buffer-containing formulation, and the formulation at pH 5.0 is more stable than the formulation at pH 5.5. It was confirmed that the stability of the formulation at pH 5.0 without buffer is the most excellent.

[0098] [[ID=INTENTIONALLY_LEFT_BLANK]] [[ID=INTENTIONALLY_LEFT_BLANK]] Also, after applying temperature stress to each formulation in Table 7, the % acidic value was measured using icIEF as described above.

[0099] The results are shown in Table 9. Table 9 is a table showing the % acidic values of formulations with different presence or absence of buffer and pH.

[0100]

Table 9

[0101] As shown in Table 9, the buffer-free formulation had a lower % acidic value than the buffer-containing formulation. Also, the formulation at pH 5.0 had a lower % acidic value than the formulation at pH 5.5. In particular, the formulation at pH 5.0 without buffer had the lowest % acidic value. Therefore, the buffer-free formulation is more stable than the buffer-containing formulation, and the formulation at pH 5.0 is more stable than the formulation at pH 5.5. It was confirmed that the stability of the formulation at pH 5.0 without buffer is the most excellent.

[0102] Example 6: Expansion of stabilizers

[0103] In this example, changing the type of stabilizer and adding an antioxidant to pembrolizumab The effect on the stability of the contained pharmaceutical preparations was 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 formulation in Table 10 was subjected to temperature stress in the same manner as in Example 1, followed by SEC. Analysis was performed to measure the purity of the sample.

[0106] The results are shown in Table 11. Table 11 shows the results for different types of stabilizers and antioxidants. This table shows the purity of formulations to which the additive has been added, expressed as HMW content.

[0107] [Table 11]

[0108] As shown in Table 11, without buffer, an antioxidant was added and the pH was set to 5.0. The formulations containing 7A, 7B, and 7C include a buffer and do not contain antioxidants. The formulation with a pH of 5.5, i.e., 6A, had a lower Δ%HMW value. A formulation containing an antioxidant and having a pH of 5.0 without a buffer is a formulation containing a buffer. Furthermore, it is even more stable than formulations with a pH of 5.5 that do not contain antioxidants. A formulation that does not contain sphagnum, has an antioxidant added, and has a pH of 5.0, is a type of stabilizer, As with claustrophobic acid, sorbitol, trehalose, arginine, or any combination thereof, It demonstrated excellent stability even after numerous modifications.

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

[0110] The results are shown in Table 12. Table 12 shows the results for different types of stabilizers and antioxidants. This table shows the % acidity values ​​of formulations to which the additive has been added.

[0111] [Table 12]

[0112] As shown in Table 12, without buffer, an antioxidant is added and the pH is 5.0. The formulations containing these substances, namely 7A, 7B, and 7C, include a buffer and do not contain antioxidants. A formulation having a pH of 5.5, i.e., one whose % acidity change is similar to or similar to that of 6A. The values ​​were low. In particular, 7A showed the best stability because 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 buffer type is used to ensure the stability of the pembrolizumab-containing pharmaceutical formulation. The impact was confirmed.

[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 formulation in Table 13 was subjected to temperature stress in the same manner as in Example 1, followed by SEC. Analysis was performed to measure the purity of the sample.

[0117] The results are shown in Table 14. Table 14 shows the purity of formulations with different buffer types. This table shows the degree in terms of HMW content.

[0118] [Table 14]

[0119] As shown in Table 14, the histidine-containing formulations, namely 8C and 8D, are other Compared to formulations containing the buffer, the Δ%HMW value was higher. Therefore, as a buffer, His We have confirmed that formulations containing thidine have lower stability compared to formulations containing other types of buffers. did.

[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. Ta.

[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 to 9D and 9K were subjected to temperature storage in the same manner as in Example 1. After adding the sulfate, SEC analysis was performed to measure the purity of the sample. The results are shown in Table 16. It is.

[0124] [Table 16]

[0125] As shown in Table 16, it contains a high concentration of pembrolizumab at 150 mg / ml. Formulations 9A to 9D, which do not contain the buffer, are superior to formulation 9K, which contains the buffer. It demonstrated stability.

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

[0127] [Table 17]

[0128] As shown in Table 17, it contains a high concentration of pembrolizumab at 150 mg / ml. Formulations 9A to 9J, which do not contain the buffer, are superior to formulation 9K, which contains the buffer. It demonstrated stability.

[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, icIEF was performed as described above. The % acidity value was measured using [a specific method / tool]. The results are shown in Table 19.

[0132] [Table 19]

[0133] As shown in Table 19, high-concentration PEMA of 200 mg / ml or 250 mg / ml Formulations 10A to 10I, which contain brolizumab but do not contain a buffer, It showed superior stability compared to formulation 9K containing α.

Claims

1. (a) an anti-PD-1 antibody or its antigen-binding fragment, (b) containing a stabilizer, A stable anti-PD-1 agent that does not contain buffer and has a pH of approximately 4.5 to 6.

5. Pharmaceutical preparations for the body.

2. The formulation has a pH of approximately 5.0 to approximately 5.5, according to the pharmaceutically acceptable method described in claim 1. Agent.

3. The anti-PD-1 antibody is a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1, and SEQ ID NO: 2 Heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 3, and heavy chain CD R3 and the light chain CDR1 having the amino acid sequence of SEQ ID NO: 4, and the amino acid sequence of SEQ ID NO: 5 A light chain CDR2 having the amino acid sequence of SEQ ID NO: 6 and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6, The pharmaceutical preparation according to claim 1.

4. The pharmaceutical preparation according to claim 1, wherein the anti-PD-1 antibody is pembrolizumab.

5. The concentration of the anti-PD-1 antibody or its antigen-binding fragment is approximately 5 to approximately 200 mg / ml. The pharmaceutical preparation according to claim 1.

6. The stabilizer is a polyol, an amino acid or a pharmaceutically acceptable salt thereof, or A pharmaceutical preparation according to claim 1, which is a mixture thereof.

7. The aforementioned polyols are sorbitol, sucrose, trehalose, mannose, and maltose. The pharmaceutical preparation according to claim 6, which is sucrose, mannitol, or a mixture thereof.

8. The aforementioned amino acids are glycine, proline, phenylalanine, tyrosine, and tryptophan. The claim is that lysine, arginine, a pharmaceutically acceptable salt thereof, or a mixture thereof. The pharmaceutical preparations described in 6.

9. The stabilizer contains approximately 1.0 to approximately 15.0% (w / v) sugar, and approximately 1.0 to approximately 20% 0% (w / v) sugar alcohol, approximately 0.1 to approximately 300.0 mM amino acids, or The pharmaceutical preparation according to claim 1, wherein the metal salt is approximately 1.0 to approximately 300.0 mM.

10. The stabilizer comprises about 1.0 to about 15.0% (w / v) of sugar, and about 0.1 to about The pharmaceutical preparation according to claim 1, wherein the amino acid is 300.0 mM.

11. The buffer consists of histidine, phosphoric acid, citric acid, malic acid, tartaric acid, succinic acid, and acetic acid. The claim 1 is a carbonic acid, a pharmaceutically acceptable salt thereof, or a mixture thereof. Pharmaceutical preparations.

12. Claim 1, which is free of surfactants or further contains surfactants. A listed pharmaceutical preparation.

13. The surfactants include polysorbates, poloxamers, and sorbitan esters of other fatty acids. The pharmaceutical preparation according to claim 12, which is a mixture thereof.

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

15. The pharmaceutical preparation according to claim 14, wherein the antioxidant is methionine.

16. The pharmaceutical preparation described in claim 1 is for subcutaneous injection or intravenous injection. formulation.

17. The concentration of the anti-PD-1 antibody or its antigen-binding fragment is approximately 5 to approximately 200 mg / mL. And, The stabilizer is approximately 1.0 to approximately 15.0% (w / v) of sucrose, trehalose. , its hydrate, or a mixture thereof; about 1.0 to about 20.0% (w / v) of sorbitol Calcium, mannitol, its hydrate, or mixture thereof; approximately 0.1 to approximately 300.0 mM Arginine, lysine, proline, glycine, phenylalanine, tyrosine, tryptophan n, a pharmaceutically acceptable salt thereof, or a mixture thereof; or a mixture of two or more thereof The pharmaceutical preparation according to claim 1.

18. A step of administering to an individual an amount of the pharmaceutical formulation according to claim 1 that is effective in treating cancer. A method for treating cancer in an individual, including the cancerous stage.

19. 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 Add pembrolizumab or its antigen-binding fragment to the solvent, and pembrolizumab or so The step of preparing an antigen-binding fragment solution; and A method for producing a stable pharmaceutical preparation, comprising the step of adding a stabilizer to the aforementioned solution; Furthermore, the manufacturing step is carried out without adding a buffer.

Images