Pharmaceutical composition of a stable anti-PD-1 antibody

Abstract

The present disclosure relates to a pharmaceutical composition comprising a stable anti-PD-1 antibody, and more specifically, to a pharmaceutical composition comprising pembrolizumab. The present composition effectively solves the problems of high-concentration antibody formulations in the manufacture, storage, transport, and delivery of pharmaceuticals by ensuring the stability of the composition while lowering the viscosity of the formulation.

Description

Technology Field

[0001] Cross-reference of related applications

[0002] The present application claims priority to Chinese Patent Application No. 202311848745.9 (Title of Invention: Pharmaceutical Composition of Stable Anti-PD-1 Antibody), filed with the Chinese Intellectual Property Office on December 28, 2023, and Chinese Patent Application No. 202411895668.7 (Title of Invention: Pharmaceutical Composition of Stable Anti-PD-1 Antibody), filed with the Chinese Intellectual Property Office on December 20, 2024, the entire contents of said applications are incorporated herein by reference.

[0003] Technology field

[0004] The present disclosure relates to pharmaceutical compositions of stable anti-PD-1 antibodies, and more specifically, to pharmaceutical compositions of pembrolizumab and therapeutic uses thereof. Background Technology

[0005] Pembrolizumab is an anti-PD-1 humanized monoclonal antibody that received FDA approval in 2014. It is administered via intravenous infusion in a formulation specification of 100 mg:4 ml. In recent years, subcutaneous administration routes have garnered attention in the field of biopolymers due to their advantages, such as convenience and excellent patient compliance. To improve patient convenience, the use of high-concentration protein solutions (i.e., >80 mg / mL) in pre-filled syringes has become a trend. At such concentrations, many protein solutions exhibit high viscosity, posing significant challenges for handling and delivery. When pre-filled syringes are used for subcutaneous injections, formulations with a viscosity of less than 15 centipoise (cP) are preferred; otherwise, injection force (also known as slip friction) may increase, which is a challenge for high-concentration solutions. Simultaneously, in ultrafiltration / microfiltration systems, high viscosity leads to high membrane back pressure and reduced flow rates. Increased viscosity in pre-filled syringes can lead to difficulties during administration; for instance, injecting high-viscosity solutions can cause increased leakage at the injection site and increase patient pain because it requires the use of larger-diameter needles. In the case of lyophilized high-concentration formulations, high viscosity can also result in prolonged redissolution times. Therefore, controlling solution viscosity is an urgent task when developing high-concentration protein formulations. Sucrose or trehalose are commonly used as stabilizers in biopharmaceutical formulations, but high concentrations of sucrose or trehalose typically result in high viscosity of the formulation. If the dosage of these stabilizers is reduced to control viscosity during the development of high-concentration formulations, the decrease in stabilizer dosage can potentially affect the stability of the formulation. Consequently, the high viscosity issue faced by high-concentration biopharmaceuticals poses significant challenges to the manufacturing, storage, transportation, and delivery of pharmaceuticals.

[0006] Currently, research has already reported that amino acid-based excipients can replace sucrose to achieve the effect of a stabilizer. In its published patent WO2019050780A1, MERCK SHARP & DOHME proposed a novel PEGylated amino acid capable of reducing the viscosity of high-concentration bio-prescriptions. The specification of another patent, US11633476B2, describes a high-concentration formulation using a single amino acid as a stabilizer; however, the results typically failed to simultaneously satisfy formulation stability, viscosity characteristics, and insoluble particulates. Consequently, only high-concentration formulations using sucrose as a stabilizer were ultimately registered under the claims of that patent. Therefore, there is a need to further optimize the formulation of high-concentration preparations; in particular, by optimizing the components of the stabilizer, it is necessary to control the viscosity of high-concentration formulations while ensuring their stability to meet the demands of industrial production.

[0007] In order to achieve a superior viscosity reduction effect and a more stable formulation composition, the present disclosure obtained a pembrolizumab antibody formulation composition containing an amino acid system through formulation screening. By using a combination of amino acids, or a combination of amino acids and sucrose, as a stabilizer and optimizing the mixing ratio to obtain an optimal composition ratio, the formulation containing the amino acid system of the present disclosure has superior SEC purity and IEC purity compared to existing formulations, and improves the stability of the formulation while effectively lowering the viscosity of the formulation to facilitate drug delivery.

[0008] The present disclosure provides a pharmaceutical composition of an anti-PD-1 antibody. The pharmaceutical composition is

[0009] a) Approximately 80–200 mg / ml of anti-PD-1 antibody;

[0010] b) a surfactant selected from polysorbate 80, polysorbate 20, and poloxamer 188;

[0011] c) A stabilizer that is a combination of arginine and proline, or a combination of arginine and sucrose;

[0012] d) an antioxidant that is methionine; and

[0013] e) Includes a hyaluronidase dispersant,

[0014] The pH is about 4.0 to 6.0, the content of arginine is about 0.1 to 50 mg / ml, the content of proline is about 0.1 to 50 mg / ml, and the content of sucrose is about 0.1 to 50 mg / ml.

[0015] In some embodiments, the anti-PD-1 antibody in the pharmaceutical composition of the present disclosure is in a high concentration state, and the concentration thereof is about 80 to 200 mg / ml, for example, the pharmaceutical composition is about 80 mg / ml, 85 mg / ml, 90 mg / ml, 95 mg / ml, 100 mg / ml, 105 mg / ml, 110 mg / ml, 115 mg / ml, 120 mg / ml, 125 mg / ml, 130 mg / ml, 135 mg / ml, 140 mg / ml, 145 mg / ml, 150 mg / ml, 155 mg / ml, 160 mg / ml, 165 mg / ml, 170 mg / ml, 175 mg / ml, 180 mg / ml, 185 mg / ml, 190 mg / ml, 195 mg / ml, 200 mg / ml anti-PD-1 antibody It contains PD-1 antibodies.

[0016] In some embodiments, the stabilizer in the pharmaceutical composition of the present disclosure is a combination of arginine and proline, wherein the content of arginine and proline is about 0.1 to 50 mg / ml each, for example, the content of arginine is about 50 mg / ml, 45 mg / ml, 40 mg / ml, 35 mg / ml, 30 mg / ml, 20 mg / ml, 15 mg / ml, 10 mg / ml, 5 mg / ml, 1 mg / ml, 0.1 mg / ml, preferably about 15 mg / ml, and the content of proline is about 50 mg / ml, 45 mg / ml, 40 mg / ml, 35 mg / ml, 30 mg / ml, 20 mg / ml, 15 mg / ml, 10 mg / ml, 5 mg / ml, 1 mg / ml, 0.1 mg / ml, preferably about 15 mg / ml. In some embodiments, the weight ratio of arginine to proline is about 1:2 to 2:1, preferably about 1:2, 1:1, 2:1.

[0017] In some embodiments, the stabilizer in the pharmaceutical composition of the present disclosure is a combination of arginine and sucrose, wherein the content of arginine and sucrose is about 0.1 to 50 mg / ml each, for example, the content of arginine is about 50 mg / ml, 45 mg / ml, 40 mg / ml, 35 mg / ml, 30 mg / ml, 20 mg / ml, 15 mg / ml, 10 mg / ml, 5 mg / ml, 1 mg / ml, 0.1 mg / ml, preferably about 20 mg / ml, and the content of sucrose is about 50 mg / ml, 45 mg / ml, 40 mg / ml, 35 mg / ml, 30 mg / ml, 20 mg / ml, 15 mg / ml, 10 mg / ml, 5 mg / ml, 1 mg / ml, 0.1 mg / ml, preferably about 20 mg / ml. In some embodiments, the weight ratio of the arginine and sucrose is about 1:2 to 2:1, preferably about 1:2, 1:1, 2:1.

[0018] In some embodiments, the concentration of methionine in the pharmaceutical composition of the present disclosure is about 0.1 to 50 mM, and may be, for example, about 0.1 mM, 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, preferably about 15 mM. Optionally, nitrogen gas is filled into the pharmaceutical composition.

[0019] In some embodiments, the surfactant in the pharmaceutical composition of the present disclosure is selected from polysorbate 80, polysorbate 20, and poloxamer 188, and the concentration is about 0.01 to 1 mg / ml, for example, about 0.01 mg / ml, 0.05 mg / ml, 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, and 1 mg / ml. Preferably, the surfactant is polysorbate 80, and the concentration thereof is preferably about 0.4 mg / ml.

[0020] In some embodiments, the concentration of hyaluronidase in the pharmaceutical composition of the present disclosure is about 1 to 10,000 IU / ml, preferably about 1 to 4,000 IU / ml, and more preferably about 1 to 2,000 IU / ml.

[0021] In some embodiments, the pharmaceutical composition of the present disclosure further comprises a buffer.

[0022] In some embodiments, the buffer of the pharmaceutical composition of the present disclosure is histidine-hydrochloric acid, and its concentration is about 0.1 to 30 mM, and may be, for example, about 0.1 mM, 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, and preferably about 10 mM.

[0023] In some embodiments, the anti-PD-1 antibody of the present invention is pembrolizumab, its heavy chain sequence is as shown in SEQ ID NO: 1, and its light chain sequence is as shown in SEQ ID NO: 2.

[0024] In some preferred embodiments, the present disclosure provides a pharmaceutical composition comprising about 80 to 200 mg / ml of pembrolizumab, about 0.1 to 30 mM of histidine-hydrochloride, about 0.01 to 1 mg / ml of polysorbate 80, about 0.1 to 50 mM of methionine, about 0.1 to 50 mg / ml of arginine, about 0.1 to 50 mg / ml of proline, and about 1 to 10000 IU / ml of hyaluronidase, having a pH of about 4.0 to 6.0 (e.g., may be about pH 4.0, pH 4.5, pH 5, pH 5.5, pH 6.0), and optionally filled with nitrogen gas as needed.

[0025] More preferably, the present disclosure provides a pharmaceutical composition comprising about 160 to 180 mg / ml of pembrolizumab, about 5 to 20 mM of histidine-hydrochloride, about 0.1 to 0.6 mg / ml of polysorbate 80, about 5 to 25 mM of methionine, about 5 to 20 mg / ml of arginine, about 5 to 20 mg / ml of proline, and about 1 to 4000 IU / ml of hyaluronidase, having a pH of about 5.0 to 6.0 (e.g., may be about pH 5.0, pH 5.2, pH 5.5, pH 5.7, pH 6.0), and optionally filled with nitrogen gas as needed.

[0026] In some preferred embodiments, the present disclosure provides a pharmaceutical composition comprising about 80 to 200 mg / ml of pembrolizumab, about 0.1 to 30 mM of histidine-hydrochloride, about 0.01 to 1 mg / ml of polysorbate 80, about 0.1 to 50 mM of methionine, about 0.1 to 50 mg / ml of arginine, about 0.1 to 50 mg / ml of sucrose, and about 1 to 10000 IU / ml of hyaluronidase, wherein the pH is about 4.0 to 6.0 (e.g., may be about pH 4.0, pH 4.5, pH 5, pH 5.5, pH 6.0), and optionally filled with nitrogen gas as needed.

[0027] More preferably, the present disclosure provides a pharmaceutical composition of an anti-PD-1 antibody comprising about 160 to 180 mg / ml of pembrolizumab, about 5 to 20 mM of histidine-hydrochloride, about 0.1 to 0.6 mg / ml of polysorbate 80, about 5 to 25 mM of methionine, about 5 to 25 mg / ml of arginine, about 5 to 25 mg / ml of sucrose, and about 1 to 4000 IU / ml of hyaluronidase, with a pH of about 5.0 to 6.0 (e.g., may be about pH 5.0, pH 5.2, pH 5.5, pH 5.7, pH 6.0), and optionally filled with nitrogen gas as needed.

[0028] In some preferred embodiments, the present disclosure provides a pharmaceutical composition of an anti-PD-1 antibody comprising about 160 to 180 mg / ml of pembrolizumab, about 0.1 to 0.6 mg / ml of polysorbate 80, about 5 to 25 mM of methionine, about 5 to 20 mg / ml of arginine, about 5 to 20 mg / ml of proline or about 5 to 20 mg / ml of sucrose, and about 1 to 4000 IU / ml of hyaluronidase, having a pH of about 5.0 to 6.0 (e.g., may be about pH 5.0, pH 5.2, pH 5.5, pH 5.7, pH 6.0), and optionally filled with nitrogen gas.

[0029] More preferably, the present disclosure provides a pharmaceutical composition of an anti-PD-1 antibody. The pharmaceutical composition

[0030] (1) containing about 155 mg / ml of pembrolizumab, about 1.5% of arginine, about 1.5% of proline, about 10 mM of histidine hydrochloride, about 0.4 mg / ml of polysorbate 80, about 15 mM of methionine, about 2000 IU / ml of hyaluronidase, with a pH of about 5.5, optionally filled with nitrogen gas;

[0031] (2) containing about 160 mg / ml of pembrolizumab, about 1.5% of arginine, about 1.5% of proline, about 10 mM of histidine hydrochloride, about 0.4 mg / ml of polysorbate 80, about 15 mM of methionine, about 2000 IU / ml of hyaluronidase, with a pH of about 5.5, optionally filled with nitrogen gas;

[0032] (3) containing about 165 mg / ml of pembrolizumab, about 1.5% of arginine, about 1.5% of proline, about 10 mM of histidine hydrochloride, about 0.4 mg / ml of polysorbate 80, about 15 mM of methionine, about 2000 IU / ml of hyaluronidase, with a pH of about 5.5, optionally filled with nitrogen gas;

[0033] (4) containing about 170 mg / ml of pembrolizumab, about 2% of arginine, about 1% of proline, about 10 mM of histidine hydrochloride, about 0.4 mg / ml of polysorbate 80, about 15 mM of methionine, about 2000 IU / ml of hyaluronidase, with a pH of about 5.5, optionally filled with nitrogen gas;

[0034] (5) containing about 170 mg / ml of pembrolizumab, about 1.5% of arginine, about 1.5% of proline, about 10 mM of histidine hydrochloride, about 0.4 mg / ml of polysorbate 80, about 15 mM of methionine, about 2000 IU / ml of hyaluronidase, with a pH of about 5.5, optionally filled with nitrogen gas;

[0035] (6) containing about 170 mg / ml of pembrolizumab, about 1% of arginine, about 2% of proline, about 10 mM of histidine hydrochloride, about 0.4 mg / ml of polysorbate 80, about 15 mM of methionine, about 2000 IU / ml of hyaluronidase, with a pH of about 5.5, optionally filled with nitrogen gas;

[0036] (7) containing about 175 mg / ml of pembrolizumab, about 1.5% of arginine, about 1.5% of proline, about 10 mM of histidine hydrochloride, about 0.4 mg / ml of polysorbate 80, about 15 mM of methionine, about 2000 IU / ml of hyaluronidase, with a pH of about 5.5, optionally filled with nitrogen gas;

[0037] (8) containing about 180 mg / ml of pembrolizumab, about 1.5% of arginine, about 1.5% of proline, about 10 mM of histidine hydrochloride, about 0.4 mg / ml of polysorbate 80, about 15 mM of methionine, about 2000 IU / ml of hyaluronidase, with a pH of about 5.5, optionally filled with nitrogen gas;

[0038] (9) containing about 170 mg / ml of pembrolizumab, about 1.5% of arginine, about 1.5% of proline, about 10 mM of histidine hydrochloride, about 0.5 mg / ml of poloxamer 188, about 15 mM of methionine, about 2000 IU / ml of hyaluronidase, with a pH of about 5.5, optionally filled with nitrogen gas;

[0039] (10) containing about 165 mg / ml of pembrolizumab, about 2% of arginine, about 2% of sucrose, about 10 mM of histidine hydrochloride, about 0.4 mg / ml of polysorbate 80, about 15 mM of methionine, about 2000 IU / ml of hyaluronidase, with a pH of about 5.0, optionally filled with nitrogen gas;

[0040] (11) containing about 170 mg / ml of pembrolizumab, about 2% of arginine, about 2% of sucrose, about 10 mM of histidine hydrochloride, about 0.4 mg / ml of polysorbate 80, about 15 mM of methionine, about 2000 IU / ml of hyaluronidase, with a pH of about 5.5, optionally filled with nitrogen gas;

[0041] (12) contains about 165 mg / ml of pembrolizumab, about 2% of arginine, about 2% of sucrose, about 10 mM of histidine hydrochloride, about 0.4 mg / ml of polysorbate 80, about 15 mM of methionine, about 2000 IU / ml of hyaluronidase, has a pH of about 5.5, and optionally contains nitrogen gas.

[0042] At the same time, the present disclosure further provides a lyophilized formulation obtained by freeze-drying the pharmaceutical composition of the present disclosure, or obtained as the pharmaceutical composition of the present disclosure after restoring said lyophilized formulation.

[0043] Additionally, the present disclosure further provides a method for treating a tumor, comprising the step of administering a pharmaceutical composition or a lyophilized preparation according to any one of the above claims to a subject. The tumor is nasopharyngeal cancer, small cell lung cancer, non-small cell lung cancer, gastric cancer, esophageal cancer, pancreatic cancer, colorectal cancer, melanoma, kidney cancer, breast cancer, liver cancer, ovarian cancer, cervical cancer, bladder cancer, or head and neck tumor.

[0044] The present disclosure further provides the use of the pharmaceutical composition or lyophilized preparation in the manufacture of a drug for the treatment of tumors. Herein, the tumor is nasopharyngeal cancer, small cell lung cancer, non-small cell lung cancer, gastric cancer, esophageal cancer, pancreatic cancer, colorectal cancer, melanoma, kidney cancer, breast cancer, liver cancer, ovarian cancer, cervical cancer, bladder cancer, head and neck tumor. Brief explanation of the drawing

[0045] Figure 1a is the SEC-HPLC purity for the sample of Example 1. Figure 1b is the nrCE-SDS purity for the sample of Example 1. Figure 2 is the SEC-HPLC purity for the sample of Example 2. Figure 3a is the SEC-HPLC purity for the sample of Example 3. Figure 3b is the IEC-HPLC purity for the sample of Example 3. Figure 4 is a viscosity comparison diagram for the sample of Example 3. Figure 5a is a distribution of insoluble particulates for the light-irradiated 10d sample of Example 3. Figure 5b is a distribution of insoluble particulates for the high temperature 14d sample of Example 3. Figure 6a is the SEC-HPLC purity for the sample of Example 4. Figure 6b is the nrCE-SDS purity for the sample of Example 4. Figure 6c is the rCE-SDS purity for the sample of Example 4. Specific details for implementing the invention

[0046] All publications, patents, and patent applications mentioned in this specification are incorporated by reference into this specification by means of citation, and said citation has the same effect as if each publication, patent, or patent application were specifically and individually incorporated by means of citation.

[0047] Before describing the present disclosure in detail below, it should be understood that the present disclosure is not limited to the specific methodologies, protocols, and reagents described herein and that they may be modified. Furthermore, it should be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0048] Some embodiments disclosed herein include numerical ranges, and certain aspects of the disclosure may be described in the manner of ranges. Unless otherwise stated, it should be understood that descriptions of numerical ranges or in the manner of ranges are for the sake of brevity and convenience only and should not be construed as strict limitations on the scope of the disclosure. Accordingly, descriptions in the manner of ranges should be deemed to specifically disclose all possible sub-ranges and all possible specific numerical points within said ranges, as specified in the disclosure. Regardless of the breadth or narrowness of said numbers, the principles set forth above apply equally. When described in the manner of ranges, said ranges include the endpoints of the ranges.

[0049] The term “approx.” includes and describes the value or parameter itself. For example, “approx.” x” includes and describes “x” itself. As used herein, when used in combination with a measurement or to modify a value, unit, constant, or series of values, the term “approx.” includes the value or parameter itself, as well as a variation of ±1% to 10%. In some embodiments, when used in combination with a measurement or to modify a value, unit, constant, or series of values, the term “approx.” means a variation of ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, or ±10%.

[0050] The term “Pembrolizumab” refers to a humanized antibody used in cancer immunotherapy, currently branded as Keytruda ® It is marketed as a commercial product of ). “Pembrolizumab” is the generic name for the above antibody and may refer only to the active ingredient itself, which includes two homologous heavy chains and two homologous light chains. In this case, the sequence of the heavy chain is indicated by SEQ ID NO:1, and the sequence of the light chain is indicated by SEQ ID NO:2.

[0051] The terms “preparation,” “drug preparation,” “prescription,” or “drug prescription” may be used interchangeably herein, and refer to a form that allows the active ingredient of a drug to be effectively present and does not contain other ingredients that are toxic to the subject to whom the preparation is to be administered.

[0052] The term “pharmaceutical composition” means a formulation or a combination of formulations containing one, two, or more active ingredients, wherein the active ingredients contained therein are allowed to exist in a form having biologically effective activity and do not contain additional ingredients having unacceptable toxicity to the subject receiving said formulation. In a specific narrow sense, for example, when containing only one drug active ingredient, “pharmaceutical composition” may be used interchangeably with “drug formulation” or “drug prescription.” Where the “pharmaceutical composition” exists in the form of a combination of individual formulations containing two or more different active ingredients, they may be administered simultaneously, sequentially, individually, or at a time interval for the purpose of jointly utilizing the biological activity of the various active ingredients to treat diseases.

[0053] The term "high concentration" refers to a preparation containing an active pharmaceutical ingredient (API) with a concentration of about 80 to 200 mg / ml.

[0054] The term "pharmaceuticalally acceptable excipient" refers to a substance or reagent that can be reasonably administered to a subject to provide a formulation containing an effective amount of an active pharmaceutical ingredient in a stable manner. Suitable pharmaceutically acceptable excipients are well known in the art and include, but are not limited to, buffers, stabilizers, surfactants, etc.

[0055] The term "stabilizer" refers to a pharmaceutically acceptable excipient used to protect the active medicinal ingredient and / or formulation from chemical and / or physical degradation during manufacturing, storage, and application.

[0056] The term "surfactant" refers to a pharmaceutically acceptable excipient used to protect protein preparations from mechanical stresses, such as stirring and shearing.

[0057] The term "antioxidant" refers to a compound that prevents the oxidation of active ingredients under storage conditions.

[0058] The term "dispersant" refers to a compound used to increase the penetration of an active substance into tissues (e.g., skin) and to facilitate the effective diffusion of the active substance. For example, there is hyaluronidase (also known as hyaluronic acid degrading enzyme), which hydrolyzes hyaluronic acid to lower its viscosity within the extracellular matrix, thereby allowing pharmaceutical formulations to disperse more easily within tissues.

[0059] The term "buffer" refers to a reagent that maintains the pH of a formulation solution within an acceptable range. The histidine-hydrochloride buffer system described herein may be prepared using histidine and dilute hydrochloric acid, or prepared using histidine and histidine hydrochloride. At the same time, the pharmaceutical composition described herein may be a self-buffering system that does not contain a buffer.

[0060] The term "self-buffering" means the exclusion of components traditionally used to introduce buffering capacity into a formulation, including, but not limited to, histidine buffers, phosphate buffers, citric acid buffers, acetate buffers, carbonate buffers, succinic acid buffers, tartaric acid buffers, maleic acid buffers, or mixtures thereof. In addition to any buffering capacity present in a formulation that does not contain buffering components, proteins are known to provide buffering capacity to the formulation. Therefore, this does not mean that the formulation cannot have any buffering capacity.

[0061] The term "freeze-dried formulation" refers to a composition obtained or obtainable through the freeze-drying process of a liquid formulation. Preferably, this is a solid composition with a moisture content of less than 5%, preferably less than 3%.

[0062] The term "insoluble particulates" includes, but is not limited to, particulates introduced during the formulation process, fatty acid particles generated by the decomposition of polysorbate, silicone oil, macromolecular particles resulting from protein aggregation, and other unknown particulate materials (e.g., possible packaging material fragments, excipient crystals, etc.), and the decomposition of polysorbate was not observed in this specification.

[0063] The term "treatment" refers to clinical interventions in the process of attempting to alter an individual or address cell-derived disease processes, which may include preventive measures or interventions in clinical pathological processes. Therapeutic effects include, but are not limited to, the prevention of disease onset or recurrence, symptom relief, reduction of direct or indirect pathological consequences of any disease, prevention of metastasis, slowing the progression of disease, improvement or alleviation of disease status, and alleviation or improvement of prognosis.

[0064] The terms “individual,” “object,” or “patient” mean any animal, e.g., mammal or marsupial. Individuals of the present disclosure include, but are not limited to, humans, non-human primates (e.g., crab-eating monkeys, rhesus monkeys, or other types of macaque monkeys), mice, pigs, horses, donkeys, cattle, sheep, rats, and any type of poultry.

[0065] The terms “disease,” “symptom,” or “disorder,” etc. refer to any change or abnormality in regulation that impairs or interferes with the normal function of a cell, tissue, or organ. For example, the said “disease” includes, but is not limited to, tumors, pathogen infections, autoimmune diseases, T-cell dysfunctional diseases, or defects in immune tolerance (e.g., transplant rejection).

[0066] The term "tumor" refers to a disease characterized by the pathological proliferation of cells or tissues, and the resulting metastasis or invasion into other tissues or organs. Tumor growth is typically uncontrolled and progressive, and does not induce or inhibit the proliferation of normal cells. Tumors include "cancer" and generally refer collectively to all malignant tumors.

[0067] Experimental Example 1 Size Exclusion Chromatography (SEC-HPLC)

[0068] Aggregates, monomers, and fragments were quantified using size exclusion chromatography. The method was performed on a Waters e2695-2489 HPLC system using an X-Bridge BEH 200 Å SEC (7.8 × 300 mm) column. The mobile phase consisted of 100 mM sodium phosphate and 150 mM sodium chloride (pH 6.8). The sample was diluted to 1 mg / ml with the mobile phase, and the injection volume was 20 μl. Isosolvent elution was performed for 30 minutes at a flow rate of 0.5 mL / min, and the detection wavelength was 215 nm. Integration was performed using Empower 3 software, and the percentage content of each component was calculated using the area percentage method.

[0069] Experimental Example 2 Capillary Electrophoresis (CE-SDS) Measurement

[0070] The non-reducing and reducing purity of the present composition were measured using the CE-SDS method. The measurements were performed using an uncoated silica capillary with an ID of 50 μM in a SCIEX PA800 plus capillary electrophoresis system. The effective separation length of the capillary was 20 cm and the total length was 30.2 cm, and detection was conducted at a PDA of 220 nm and a bandwidth of 10 nm. For the measurement of reducing purity, 5 kV-20S injection and 15 kV-30 min separation were set, and for the measurement of non-reducing purity, 10 kV-20S injection and 15 kV-40 min separation were set, and integration processing was performed using 32Karat software.

[0071] Experimental Example 3: Ion Exchange Chromatography (IEC-HPLC) Measurement

[0072] The charge heterogeneity of the present composition was measured using ion exchange chromatography. Various post-translational modifications of recombinant protein pharmaceuticals can alter the surface charge distribution characteristics of the protein, thereby inducing charge heterogeneity. This method was performed on a Waters e2695-2489 HPLC system using a weak cation exchange chromatography column, ProPac WCX-10 (4.0 mm × 250 mm, 10 μm, Thermo). Mobile phase A consisted of 24 mM MES and 4% acetonitrile (v / v) (pH 6.0), and mobile phase B consisted of 20 mM sodium phosphate, 95 mM NaCl, and 4% acetonitrile (v / v) (pH 8.0). The sample was diluted to 8 mg / ml with ultrapure water, and the injection volume was 10 μl. Elution was performed at a flow rate of 0.5 ml / min, and the detection wavelength was 280 nm. Integration was performed using Empower 3 software, and the percentage content of each component was calculated using the area percentage method.

[0073] Experimental Example 4 Insoluble Particulate Detection (FlowCam) Method

[0074] Insoluble particulates of the composition were measured using a flow image analyzer (FlowCam 8100), and the parameter settings were as follows: sample detection was performed with an injection volume of 200 μl and a flow rate of 0.05 ml / min, and Visual Spreadsheet was used as the analysis software.

[0075] Experimental Example 5: Measurement of Protein Content

[0076] Protein content was measured using a Lunatic microspectroscopy instrument. The blank was calibrated with purified water, and the UV absorption at 280 nm was corrected for light scattering at 330 nm to calculate the protein concentration according to Equation 1. In this case, the extinction coefficient ε is 1.42 (L / g·cm²). -1 )am.

[0077]

[0078] Equation 1

[0079] Experimental Example 6: Viscosity Measurement

[0080] The viscosity of the sample was measured using an m-VROC microfluidic rheometer, and the measurement temperature was 25℃.

[0081] The present disclosure is explained in more detail through the following examples. Even if the concentration of a specific substance is changed or another substance is added based on the present disclosure, any change that does not significantly affect the improvement of protein stability is still considered to be part of the present disclosure.

[0082] Example 1 Effect of Antioxidants on the Photostability of a Formula

[0083] To prepare the formulation solutions, the antibody concentration was adjusted to the required level using ultrafiltration concentration. After ultrafiltration concentration was completed, the excipients to be added were added to the antibody solution, and the protein concentration was finally adjusted to approximately 170 mg / ml. All formulation samples were sterile filtered using a 0.22 μm filter, filled into sterile 2 ml vials under controlled conditions, filled with nitrogen gas as needed, and sealed with coated rubber stoppers and aluminum-plastic combination caps. These formulations were subjected to high temperature (40°C ± 2°C) and light irradiation (illumination 5000 lx, UV 90 μw / cm²). 2 The stability of the samples was observed by storing them under conditions of , 25℃±2℃. Tables 1–2: Results of different formulations and stability:

[0084]

[0085]

[0086] The following can be confirmed through the data in Table 2, Figures 1a and 1b. After conducting a light irradiation test for 10 days, the SEC-HPLC main peak purity of Formula R5 was 2.0% and 1.5% higher, respectively, compared to Formulas R2 and R3, which contained only 15 mM methionine, and the nrCE-SDS main peak purity was 1.2% and 1.0% higher, respectively. The experimental results showed that adding nitrogen gas filling conditions simultaneously with the addition of methionine effectively prevents aggregate formation under light irradiation conditions.

[0087] Example 2 Stability of antibody formulations in amino acid systems

[0088] To prepare the formulation solutions, the antibodies were adjusted to the required concentration using ultrafiltration concentration. After ultrafiltration concentration was completed, the excipients to be added were added to the antibody solution, and the protein concentration was finally adjusted to approximately 170 mg / ml. All formulations were sterile filtered using a 0.22 μm filter, filled into sterile 2 ml vials under controlled conditions, and sealed with coated rubber stoppers and aluminum-plastic combination caps. These formulations were stored under high temperature conditions (40°C ± 2°C) to observe the stability of the samples. Tables 3–4: Results of different formulation formulations and stability

[0089]

[0090]

[0091] Based on the data in Table 4 and Figure 2, the following can be confirmed. Compared to formulation R6, the viscosity of formulation R8 decreased by approximately 27%, and the viscosity of formulation R9 decreased by approximately 50%. After storage under high temperature conditions (40°C, 30d), there was no significant difference in the SEC-HPLC main peak purity of formulation R8 compared to formulation R6, while the SEC-HPLC main peak purity of formulation R9 was slightly superior by 0.3%. These results demonstrate that the amino acid system within the formulation can effectively reduce the viscosity of high-concentration pembrolizumab formulations, while simultaneously indicating that the formulation possesses excellent thermal stability. Here, 3% arginine is preferred.

[0092] Example 3 Stability of Pembrolizumab Antibody Preparations According to Different Amino Acid Ratios

[0093] To prepare the formulation solutions, the antibodies were adjusted to the required concentration via ultrafiltration concentration. After ultrafiltration concentration was completed, the excipients to be added were added to the antibody solution, and the protein concentration was finally adjusted to the required level. All formulations were sterilely filtered using a 0.22 μm filter, filled into sterile 2 ml vials under controlled conditions, and sealed with coated rubber stoppers and aluminum-plastic combination caps. These formulations were subjected to high temperature (40°C ± 2°C) and light irradiation (illumination 5000 lx, UV 90 μw / cm²). 2 The stability of the samples was observed by storing them under conditions of , 25℃±2℃. Tables 5–8: Results of different formulations and stability:

[0094]

[0095]

[0096]

[0097]

[0098] The following can be confirmed through the data in Tables 6, 7, and 8, and Figures 3a, 3b, 4, 5a, and 5b. Compared to formulation R12 containing sucrose, there was no significant difference in SEC-HPLC main peak purity between samples containing amino acid systems and samples containing self-buffering systems after being left at 40°C for 14 days. After formulations R13, R14, R15, R16, R17, and R20 were left under light irradiation for 10 days, the SEC-HPLC main peaks decreased by approximately 2.0%, 2.2%, 2.5%, 2.3%, 2.3%, and 2.3%, respectively, and the magnitude of the decrease was smaller than that of formulation R12 (SEC-HPLC main peak decrease of approximately 3.0%). At the same time, viscosity test results showed that the sample viscosity of formulations R13, R14, R15, R16, R17, and R20 was noticeably lower compared to formulation R12. The above results explain that arginine and proline exert excellent effects on SEC-HPLC stability within pembrolizumab formulations, and that their effects are not only significantly superior to those of sucrose but can also greatly reduce the viscosity of the samples.

[0099] According to the insoluble particulate data results, when compared to the sucrose-based formulation (R12) after 10 days of light irradiation, the total number of insoluble particulates for the formulation containing 1% arginine and 2% proline (R14), the formulation containing 1.5% arginine and 1.5% proline (R15), the formulation containing 2% arginine and 1% proline (R16), and the formulation containing 2% arginine and 2% sucrose (R20) was found to be approximately 37.8% (20697 / 54704), 28.3% (15486 / 54704), 28.9% (15793 / 54704), and 48.7% (26627 / 54704), respectively, of the total number of insoluble particulates for formulation R12. After being stored at high temperature for 14 days, the total number of insoluble particulates in prescriptions R14, R15, R16, and R20 compared to prescription R12 was approximately 62.9% (22354 / 35536), 22.9% (8130 / 35536), 32.5% (11535 / 35536), and 25.7% (9133 / 35536), respectively, of R12. Notably, prescriptions R15 and R20 showed superior effects in terms of insoluble particulates compared to the formulation containing only arginine (R13).

[0100] Synthesizing the experimental results described above, formulations R15 and R20 were clearly superior to formulation R12 in all aspects, including purity, viscosity, and total number of insoluble particulates. At the same time, another formulation containing 1.5% arginine and 1.5% proline (R11) and a formulation containing 2% arginine and 2% sucrose (R10) also achieved excellent effects similar to formulations R15 and R20.

[0101] Example 4 Stability of Pembrolizumab Antibody Preparations According to Different Excipient Concentrations

[0102] To prepare the formulation solutions, the antibodies were adjusted to the required concentrations using ultrafiltration concentration. After ultrafiltration concentration was completed, the excipients to be added were added to the antibody solution, and the protein concentration was finally adjusted to the levels shown in Table 9. All formulations were sterile filtered using a 0.22 μm filter, filled into sterile 2 ml vials under controlled conditions, and sealed with coated rubber stoppers and aluminum-plastic combination caps. These formulations were subjected to high temperature (40°C ± 2°C) and light irradiation (illumination 5000 lx, UV 90 μw / cm²). 2 The stability of the samples was observed by storing them under conditions of , 25℃±2℃. Tables 9–11: Results of different formulations and stability:

[0103]

[0104]

[0105]

[0106] The following can be confirmed through the data in Tables 10 and 11 and Figures 6a, 6b, and 6c. After being left at 40°C for 14 days, there was no significant difference in the SEC-HPLC, nrCE-SDS, and rCE-SDS main peak purity of the self-buffered system formulation and other amino acid-containing formulations compared to formulation R23. After being left under light irradiation for 10 days, the SEC-HPLC main peaks of formulations R24, R26, R27, and R30 decreased by approximately 1.8%, 2.6%, 2.6%, and 2.3%, respectively, compared to formulation R23, and the magnitude of the decrease was smaller than that of formulation R23 (SEC-HPLC main peak decrease of approximately 3.5%). The trend of change in the SEC-HPLC main peaks of formulations R22, R25, R28, and R29 basically coincided with the trend of change in the main peaks of formulation R23. The magnitude of the main peak decline for nrCE-SDS and rCE-SDS in each other group essentially matched the magnitude of the main peak decline for prescription R21. The above results demonstrate that formulations containing a combination of arginine and proline or a combination of arginine and sucrose possess excellent stability.

[0107] The embodiments of the present disclosure described above are merely exemplary, and those skilled in the art can recognize or identify countless equivalents of specific compounds, materials, and processes without conducting ordinary experiments. All such equivalents fall within the scope of the present disclosure and are included in the claims.

Claims

Claim 1 A pharmaceutical composition of an anti-PD-1 antibody comprising: a) an anti-PD-1 antibody of about 80 to 200 mg / ml; b) a surfactant selected from polysorbate 80, polysorbate 20, and poloxamer 188; c) a stabilizer which is a combination of arginine and proline, or a combination of arginine and sucrose; d) an antioxidant which is methionine; and e) a dispersant which is hyaluronidase, wherein the pH is about 4.0 to 6.0, the content of the arginine is about 0.1 to 50 mg / ml, the content of the proline is about 0.1 to 50 mg / ml, and the content of the sucrose is about 0.1 to 50 mg / ml. Claim 2 A pharmaceutical composition according to claim 1, wherein the weight ratio of arginine and proline or the weight ratio of arginine and sucrose is about 1:2 to 2:1, preferably about 1:2, 1:1, or 2:

1. Claim 3 A pharmaceutical composition according to claim 1, comprising a buffer, wherein the content thereof is about 0.1 to 30 mM, and preferably, said buffer is histidine hydrochloride. Claim 4 A pharmaceutical composition according to any one of claims 1 to 3, wherein the content of the surfactant is about 0.01 to 1 mg / ml. Claim 5 A pharmaceutical composition according to any one of claims 1 to 3, wherein the content of the methionine is about 0.1 to 50 mM. Claim 6 A pharmaceutical composition according to any one of claims 1 to 3, wherein nitrogen gas is filled within the pharmaceutical composition. Claim 7 A pharmaceutical composition according to any one of claims 1 to 3, wherein the content of the hyaluronidase is about 1 to 10,000 IU / ml. Claim 8 A pharmaceutical composition in which, in any one of the aforementioned claims, the anti-PD-1 antibody is pembrolizumab. Claim 9 A pharmaceutical composition according to claim 8, wherein the heavy chain sequence of pembrolizumab is as shown in SEQ ID NO:1 and the light chain sequence is as shown in SEQ ID NO:

2. Claim 10 A pharmaceutical composition of an anti-PD-1 antibody comprising about 80 to 200 mg / ml of pembrolizumab, about 0.1 to 30 mM of histidine-hydrochloride, about 0.01 to 1 mg / ml of polysorbate 80, about 0.1 to 50 mM of methionine, about 0.1 to 50 mg / ml of arginine, about 0.1 to 50 mg / ml of proline, and about 1 to 10,000 IU / ml of hyaluronidase, wherein the pH is about 4.0 to 6.0 and optionally filled with nitrogen gas. Claim 11 A pharmaceutical composition of an anti-PD-1 antibody comprising about 160 to 180 mg / ml of pembrolizumab, about 5 to 20 mM of histidine-hydrochloride, about 0.1 to 0.6 mg / ml of polysorbate 80, about 5 to 25 mM of methionine, about 5 to 20 mg / ml of arginine, about 5 to 20 mg / ml of proline, and about 1 to 4000 IU / ml of hyaluronidase, wherein the pH is about 5.0 to 6.0 and optionally filled with nitrogen gas. Claim 12 In claim 10, the pharmaceutical composition comprises any one of the following: (1) comprising about 155 mg / ml of pembrolizumab, about 1.5% of arginine, about 1.5% of proline, about 10 mM of histidine-hydrochloride, about 0.4 mg / ml of polysorbate 80, about 15 mM of methionine, and about 2000 IU / ml of hyaluronidase, with a pH of about 5.5 and optionally filled with nitrogen gas; (2) comprising about 160 mg / ml of pembrolizumab, about 1.5% of arginine, about 1.5% of proline, about 10 mM of histidine-hydrochloride, about 0.4 mg / ml of polysorbate 80, about 15 mM of methionine, and about 2000 IU / ml of hyaluronidase, (3) containing about 165 mg / ml of pembrolizumab, about 1.5% of arginine, about 1.5% of proline, about 10 mM of histidine-hydrochloride, about 0.4 mg / ml of polysorbate 80, about 15 mM of methionine, about 2000 IU / ml of hyaluronidase, with a pH of about 5.5 and optionally filled with nitrogen gas; (4) containing about 170 mg / ml of pembrolizumab, about 2% of arginine, about 1% of proline, about 10 mM of histidine-hydrochloride, about 0.4 mg / ml of polysorbate 80, about 15 mM of methionine, about 2000 IU / ml of hyaluronidase, with a pH of about 5.5 and optionally filled with nitrogen gas; 5.5, optionally filled with nitrogen gas; (5) containing about 170 mg / ml of pembrolizumab, about 1.5% of arginine, about 1.5% of proline, about 10 mM of histidine hydrochloride, about 0.4 mg / ml of polysorbate 80, about 15 mM of methionine, about 2000 IU / ml of hyaluronidase, with a pH of about 5.5, optionally filled with nitrogen gas; (6) about 170 mg / ml of pembrolizumab, about 1% of arginine, about 2% of proline, about 10 mM of histidine hydrochloride, about 0.(7) containing 4 mg / ml of polysorbate 80, about 15 mM of methionine, about 2000 IU / ml of hyaluronidase, with a pH of about 5.5 and optionally filled with nitrogen gas; (7) containing about 175 mg / ml of pembrolizumab, about 1.5% of arginine, about 1.5% of proline, about 10 mM of histidine-hydrochloride, about 0.4 mg / ml of polysorbate 80, about 15 mM of methionine, about 2000 IU / ml of hyaluronidase, with a pH of about 5.5 and optionally filled with nitrogen gas; (8) containing about 180 mg / ml of pembrolizumab, about 1.5% of arginine, about 1.5% of proline, about 10 mM of histidine-hydrochloride, about (9) comprising 0.4 mg / ml of polysorbate 80, about 15 mM of methionine, about 2000 IU / ml of hyaluronidase, with a pH of about 5.5 and optionally filled with nitrogen gas; (10) comprising about 170 mg / ml of pembrolizumab, about 1.5% of arginine, about 1.5% of proline, about 10 mM of histidine-hydrochloride, about 0.5 mg / ml of poloxamer 188, about 15 mM of methionine, about 2000 IU / ml of hyaluronidase, with a pH of about 5.5 and optionally filled with nitrogen gas; (10) about 165 mg / ml of pembrolizumab, about 2% of arginine, about 2% of sucrose, about 10 mM of histidine-hydrochloride, about (11) containing 0.4 mg / ml of polysorbate 80, about 15 mM of methionine, about 2000 IU / ml of hyaluronidase, with a pH of about 5.0 and optionally filled with nitrogen gas; (12) containing about 170 mg / ml of pembrolizumab, about 2% of arginine, about 2% of sucrose, about 10 mM of histidine-hydrochloride, about 0.4 mg / ml of polysorbate 80, about 15 mM of methionine, about 2000 IU / ml of hyaluronidase, with a pH of about 5.5 and optionally filled with nitrogen gas; (12) about 165 mg / ml of pembrolizumab, about 2% of arginine, about 2% of sucrose, about 10 mM of histidine-hydrochloride, about 0.A pharmaceutical composition comprising 4 mg / ml of polysorbate 80, about 15 mM of methionine, about 2000 IU / ml of hyaluronidase, with a pH of about 5.5, and optionally filled with nitrogen gas. Claim 13 A lyophilized preparation obtained by freeze-drying a pharmaceutical composition according to any one of the aforementioned claims, or by obtaining a pharmaceutical composition according to any one of the aforementioned claims after restoring the lyophilized preparation. Claim 14 A method for treating a tumor, comprising the step of administering a pharmaceutical composition or lyophilized preparation according to any one of the above claims to a subject. Claim 15 In paragraph 14, the above tumor is a nasopharyngeal cancer, small cell lung cancer, non-small cell lung cancer, gastric cancer, esophageal cancer, pancreatic cancer, colorectal cancer, melanoma, kidney cancer, breast cancer, liver cancer, ovarian cancer, cervical cancer, bladder cancer, or head and neck tumor.

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