Stable pharmaceutical composition of Anti-PD-1 antibody

Abstract

Provided is a stable pharmaceutical composition comprising an anti-PD-1 antibody. More particularly, the present invention relates to a pharmaceutical composition of pembrolizumab. The stability of the composition is ensured while the viscosity of a formulation is reduced, effectively solving the problems of high-concentration antibody formulations in drug manufacturing, storage and transportation, and drug delivery.

Description

Pharmaceutical compositions of stable anti-PD-1 antibodies

[0001] This application claims priority to the Chinese patent application filed with the Chinese Patent Office on December 28, 2023, with application number 202311848745.9 and invention name “Stable anti-PD-1 antibody pharmaceutical composition”, and the Chinese patent application filed with the Chinese Patent Office on December 20, 2024, with application number 202411895668.7 and invention name “Stable anti-PD-1 antibody pharmaceutical composition”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present disclosure relates to a pharmaceutical composition of a stable anti-PD-1 antibody, and more specifically, to a pharmaceutical composition of pembrolizumab and therapeutic uses thereof. Background Art

[0003] Pembrolizumab, a humanized monoclonal antibody against PD-1, was approved by the FDA in 2014. Its dosage form is 100 mg / 4 ml, and it is administered by intravenous infusion. In recent years, subcutaneous administration has attracted attention in the biomacromolecule field due to its convenience and improved patient compliance. To improve patient convenience, the use of high-concentration protein solutions (i.e., >80 mg / mL) in prefilled syringes has become a trend. At these concentrations, many protein solutions become highly viscous, posing significant challenges for handling and delivery. For subcutaneous injections, particularly when using prefilled syringes, a formulation with a viscosity of less than 15 centipoise (cP) is preferred. Otherwise, injection forces (also known as glide forces) may increase, which is a challenge for highly concentrated solutions. Furthermore, in ultrafiltration / diafiltration systems, high viscosity can lead to high membrane backpressure and reduced flow rates. In prefilled syringes, increased viscosity can lead to injection difficulties. For example, injection of highly viscous solutions can increase leakage at the injection site and require the use of larger needle diameters, which can increase patient pain. For lyophilized high-concentration formulations, high viscosity can also lead to long reconstitution times. Therefore, controlling solution viscosity is a top priority when developing high-concentration protein formulations. Sucrose or trehalose are often used as stabilizers in biologic formulations, but higher sucrose or trehalose concentrations typically result in higher formulation viscosity. If their dosage is reduced during the development of high-concentration formulations to control product viscosity, the reduction in stabilizer dosage will potentially affect product stability. Therefore, the high viscosity problem faced by high-concentration biologics poses many challenges to drug manufacturing, storage, transportation, and drug delivery.

[0004] At present, studies have shown that amino acid excipients can replace sucrose to achieve the effect of a stabilizer. MERCKSHARP&DOHME proposed a new PEGylated amino acid in its published patent WO2019050780A1 that can reduce the viscosity of high-concentration biological preparations. The specification of another patent US11633476B2 records the prescription of a high-concentration preparation using a single amino acid as a stabilizer, but the result usually cannot take into account the stability, viscosity properties, insoluble particles, etc. of the preparation product. Therefore, in its claims, only the prescription of a high-concentration preparation using sucrose as a stabilizer was authorized. Therefore, there is an urgent need to further optimize the prescription of high-concentration preparations, especially to optimize the components of the stabilizer to achieve control of the viscosity of high-concentration preparations while ensuring the stability of the product to meet the needs of industrial production.

[0005] SUMMARY OF THE INVENTION

[0006] To achieve a superior viscosity-reducing effect and a more stable formulation, the present invention has obtained a formulation for a pembrolizumab antibody formulation containing an amino acid system through formulation screening. By using a combination of amino acids or a combination of amino acids and sucrose as stabilizers and optimizing the ratio to achieve the optimal composition ratio, the formulation containing the amino acid system disclosed herein has superior SEC purity and IEC purity compared to conventional formulations, effectively reducing the viscosity of the formulation while improving product stability and facilitating drug delivery.

[0007] The present disclosure provides a pharmaceutical composition of an anti-PD-1 antibody, comprising

[0008] a) about 80-200 mg / ml of an anti-PD-1 antibody;

[0009] b) a surfactant selected from the group consisting of polysorbate 80, polysorbate 20, and poloxamer 188;

[0010] c) a stabilizer, wherein the stabilizer is a combination of arginine and proline, or a combination of arginine and sucrose;

[0011] d) an antioxidant, wherein the antioxidant is methionine;

[0012] e) a diffusing agent, wherein the diffusing agent is hyaluronidase;

[0013] The pH of the pharmaceutical composition is about 4.0-6.0, the content of arginine is about 0.1-50 mg / ml, the content of proline is about 0.1-50 mg / ml, and the content of sucrose is about 0.1-50 mg / ml.

[0014] In some embodiments, the anti-PD-1 antibody in the pharmaceutical composition of the present disclosure is in a high concentration state, and its concentration is about 80-200 mg / ml. For example, the pharmaceutical composition contains 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, or 200 mg / ml of anti-PD-1 antibody.

[0015] 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 0.1-50 mg / ml, respectively, 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 1:2-2:1, preferably, about 1:2, 1:1, 2:1.

[0016] In some embodiments, the stabilizer in the pharmaceutical composition of the present invention is a combination of arginine and sucrose, wherein the content of the arginine and sucrose is about 0.1-50 mg / ml, respectively, 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 to sucrose is about 1:2-2:1, preferably, about 1:2, 1:1, 2:1.

[0017] In some embodiments, the concentration of methionine in the pharmaceutical composition of the present disclosure is about 0.1-50 mM, 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 added to the pharmaceutical composition.

[0018] In some embodiments, the surfactant in the pharmaceutical composition of the present disclosure is selected from polysorbate 80, polysorbate 20, and poloxamer 188, and its concentration is about 0.01-1 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, 1 mg / ml. The preferred surfactant is polysorbate 80, and its concentration is preferably about 0.4 mg / ml.

[0019] In some embodiments, the concentration of hyaluronidase in the pharmaceutical composition of the present disclosure is about 1-10,000 IU / ml, preferably, about 1-4,000 IU / ml, and more preferably, about 1-2,000 IU / ml.

[0020] In some embodiments, the pharmaceutical compositions of the present disclosure further comprise a buffer.

[0021] In some embodiments, the buffer of the pharmaceutical composition of the present disclosure is histidine-HCl, and its concentration is about 0.1-30 mM, for example, about 0.1 mM, 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, preferably about 10 mM.

[0022] In some embodiments, the anti-PD-1 antibody of the present disclosure is pembrolizumab, whose heavy chain sequence is shown in SEQ ID NO: 1, and whose light chain sequence is shown in SEQ ID NO: 2.

[0023] In some preferred embodiments, the present disclosure provides a pharmaceutical composition comprising about 80-200 mg / ml of pembrolizumab, about 0.1-30 mM histidine-HCl, about 0.01-1 mg / ml of polysorbate 80, about 0.1-50 mM methionine, about 0.1-50 mg / ml of arginine, about 0.1-50 mg / ml of proline, and about 1-10,000 IU / ml of hyaluronidase, with a pH of about 4.0-6.0, for example, about pH 4.0, pH 4.5, pH 5, pH 5.5, or pH 6.0, and optionally, nitrogen is added when necessary.

[0024] Further preferably, the present disclosure provides a pharmaceutical composition of an anti-PD-1 antibody, comprising about 160-180 mg / ml of pembrolizumab, about 5-20 mM histidine-hydrochloride, about 0.1-0.6 mg / ml of polysorbate 80, about 5-25 mM methionine, about 5-20 mg / ml of arginine, about 5-20 mg / ml of proline, and about 1-4000 IU / ml of hyaluronidase, wherein the pH is about 5.0-6.0, for example, about pH 5.0, pH 5.2, pH 5.5, pH 5.7, or pH 6.0, and optionally, nitrogen is added when necessary.

[0025] In some preferred embodiments, the present disclosure provides a pharmaceutical composition comprising about 80-200 mg / ml of pembrolizumab, about 0.1-30 mM histidine-HCl, about 0.01-1 mg / ml of polysorbate 80, about 0.1-50 mM methionine, about 0.1-50 mg / ml of arginine, about 0.1-50 mg / ml of sucrose, and about 1-10,000 IU / ml of hyaluronidase, with a pH of about 4.0-6.0, for example, about pH 4.0, pH 4.5, pH 5, pH 5.5, or pH 6.0, and optionally, nitrogen is added when necessary.

[0026] Further preferably, the present disclosure provides a pharmaceutical composition of an anti-PD-1 antibody, comprising about 160-180 mg / ml of pembrolizumab, about 5-20 mM histidine-hydrochloride, about 0.1-0.6 mg / ml of polysorbate 80, about 5-25 mM methionine, about 5-25 mg / ml of arginine, about 5-25 mg / ml of sucrose, and about 1-4000 IU / ml of hyaluronidase, and having a pH of about 5.0-6.0, for example, about pH 5.0, pH 5.2, pH 5.5, pH 5.7, or pH 6.0, and optionally, nitrogen is added when necessary.

[0027] In some preferred embodiments, the present disclosure provides a pharmaceutical composition of an anti-PD-1 antibody, comprising about 160-180 mg / ml of pembrolizumab, about 0.1-0.6 mg / ml of polysorbate 80, about 5-25 mM of methionine, about 5-20 mg / ml of arginine, about 5-20 mg / ml of proline or about 5-20 mg / ml of sucrose, and about 1-4000 IU / ml of hyaluronidase, and having a pH of about 5.0-6.0, for example, about pH 5.0, pH 5.2, pH 5.5, pH 5.7, or pH 6.0, and optionally, nitrogen is added when necessary.

[0028] More preferably, the present disclosure provides a pharmaceutical composition of an anti-PD-1 antibody, comprising:

[0029] (1) about 155 mg / ml of pembrolizumab, about 1.5% of arginine, about 1.5% of proline, about 10 mM of histidine-HCl, about 0.4 mg / ml of polysorbate 80, about 15 mM of methionine, about 2000 IU / ml of hyaluronidase, at about pH 5.5, optionally purged with nitrogen;

[0030] (2) about 160 mg / ml of pembrolizumab, about 1.5% of arginine, about 1.5% of proline, about 10 mM of histidine-HCl, about 0.4 mg / ml of polysorbate 80, about 15 mM of methionine, about 2000 IU / ml of hyaluronidase, at about pH 5.5, optionally purged with nitrogen;

[0031] (3) about 165 mg / ml of pembrolizumab, about 1.5% of arginine, about 1.5% of proline, about 10 mM of histidine-HCl, about 0.4 mg / ml of polysorbate 80, about 15 mM of methionine, about 2000 IU / ml of hyaluronidase, at about pH 5.5, optionally purged with nitrogen;

[0032] (4) about 170 mg / ml of pembrolizumab, about 2% of arginine, about 1% of proline, about 10 mM of histidine-HCl, about 0.4 mg / ml of polysorbate 80, about 15 mM of methionine, about 2000 IU / ml of hyaluronidase, at about pH 5.5, optionally purged with nitrogen;

[0033] (5) about 170 mg / ml of pembrolizumab, about 1.5% of arginine, about 1.5% of proline, about 10 mM of histidine-HCl, about 0.4 mg / ml of polysorbate 80, about 15 mM of methionine, about 2000 IU / ml of hyaluronidase, at about pH 5.5, optionally purged with nitrogen;

[0034] (6) about 170 mg / ml of pembrolizumab, about 1% of arginine, about 2% of proline, about 10 mM of histidine-HCl, about 0.4 mg / ml of polysorbate 80, about 15 mM of methionine, about 2000 IU / ml of hyaluronidase, at about pH 5.5, optionally purged with nitrogen;

[0035] (7) about 175 mg / ml of pembrolizumab, about 1.5% of arginine, about 1.5% of proline, about 10 mM of histidine-HCl, about 0.4 mg / ml of polysorbate 80, about 15 mM of methionine, about 2000 IU / ml of hyaluronidase, about pH 5.5, optionally purged with nitrogen;

[0036] (8) about 180 mg / ml of pembrolizumab, about 1.5% of arginine, about 1.5% of proline, about 10 mM of histidine-HCl, about 0.4 mg / ml of polysorbate 80, about 15 mM of methionine, about 2000 IU / ml of hyaluronidase, about pH 5.5, optionally purged with nitrogen;

[0037] (9) about 170 mg / ml pembrolizumab, about 1.5% arginine, about 1.5% proline, about 10 mM histidine-HCl, about 0.5 mg / ml poloxamer 188, about 15 mM methionine, about 2000 IU / ml hyaluronidase, about pH 5.5, optionally purged with nitrogen;

[0038] (10) about 165 mg / ml of pembrolizumab, about 2% arginine, about 2% sucrose, about 10 mM histidine-HCl, about 0.4 mg / ml of polysorbate 80, about 15 mM methionine, about 2000 IU / ml of hyaluronidase, about pH 5.0, optionally, nitrogen aerated;

[0039] (11) about 170 mg / ml of pembrolizumab, about 2% arginine, about 2% sucrose, about 10 mM histidine-HCl, about 0.4 mg / ml of polysorbate 80, about 15 mM methionine, about 2000 IU / ml of hyaluronidase, about pH 5.5, optionally purged with nitrogen;

[0040] (12) about 165 mg / ml of pembrolizumab, about 2% arginine, about 2% sucrose, about 10 mM histidine-HCl, about 0.4 mg / ml of polysorbate 80, about 15 mM methionine, about 2000 IU / ml of hyaluronidase, about pH 5.5, optionally, nitrogen aerated.

[0041] At the same time, the present disclosure also provides a lyophilized preparation, which is obtained by lyophilizing the pharmaceutical composition of the present disclosure, or the pharmaceutical composition of the present disclosure is obtained by re-dissolving the lyophilized preparation.

[0042] Furthermore, the present disclosure provides a method for treating tumors, comprising administering the pharmaceutical composition or lyophilized formulation of the present disclosure to a subject, wherein the tumor is nasopharyngeal carcinoma, 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 tumors.

[0043] The present disclosure also provides the use of the pharmaceutical composition or lyophilized preparation in preparing a drug for treating tumors, wherein the tumor is nasopharyngeal carcinoma, 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 tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] FIG1A SEC-HPLC purity of samples in Example 1.

[0045] Figure 1B shows the nrCE-SDS purity of the samples in Example 1.

[0046] Figure 2 SEC-HPLC purity of samples in Example 2.

[0047] FIG3A SEC-HPLC purity of samples in Example 3.

[0048] FIG3B shows the IEC-HPLC purity of the samples in Example 3.

[0049] FIG4 is a comparison chart of sample viscosities in Example 3.

[0050] FIG5A is a distribution diagram of insoluble particles in the sample after 10 days of illumination in Example 3. ...

[0051] FIG5B is a distribution diagram of insoluble particles in the sample at high temperature for 14 days in Example 3.

[0052] FIG6A SEC-HPLC purity of samples in Example 4.

[0053] Figure 6B shows the nrCE-SDS purity of samples in Example 4.

[0054] Figure 6C rCE-SDS purity of samples in Example 4. DETAILED DESCRIPTION

[0055] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0056] 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, as these may vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments 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 of ordinary skill in the art to which the present disclosure belongs.

[0057] Certain embodiments disclosed herein include numerical ranges, and certain aspects of the present disclosure may be described in terms of ranges. Unless otherwise stated, it should be understood that numerical ranges or descriptions in terms of ranges are intended for simplicity and convenience and should not be considered as strict limitations on the scope of the present disclosure. Therefore, descriptions in terms of ranges should be considered to specifically disclose all possible subranges and all possible specific numerical points within the range, as these subranges and numerical points have been clearly stated herein. Regardless of the width of the numerical value, the above principles apply equally. When describing in terms of ranges, the range includes the endpoints of the range.

[0058] The term "about" includes and describes the value or parameter itself. For example, "about x" includes and describes "x" itself. As used herein, when used in conjunction with a measurement or to modify a value, unit, constant, or a range of values, the term "about" includes, in addition to the value or parameter itself, a variation of ±1%-10%. In some embodiments, when used in conjunction with a measurement or to modify a value, unit, constant, or a range of values, the term "about" refers to a variation of ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, or ±10%.

[0059] The term "Pembrolizumab" is a humanized antibody used in cancer immunotherapy, currently marketed under the trade name "Pembrolizumab" is the common name for the antibody and may also refer to the active ingredient itself, which comprises two homologous heavy chains and two homologous light chains, wherein the sequence of the heavy chain is shown in SEQ ID NO: 1 and the sequence of the light chain is shown in SEQ ID NO: 2.

[0060] The terms "formulation" or "pharmaceutical formulation" or "formulation" or "pharmaceutical formulation" are used interchangeably herein to refer to a form that allows the pharmaceutically active ingredient to be effectively present and that contains no other components that are toxic to the subject to which the formulation is to be administered.

[0061] The term "pharmaceutical composition" refers to a preparation or combination of preparations containing one, two or more active ingredients, which allows the active ingredients contained therein to exist in a biologically effective form and does not contain additional ingredients that are unacceptably toxic to the subject to which the preparation is administered. In certain narrow cases, such as when only one active pharmaceutical ingredient is contained, "pharmaceutical composition" and "pharmaceutical preparation" or "pharmaceutical prescription" can also be used interchangeably. When a "pharmaceutical composition" is present in the form of a combination of separate preparations containing different, two or more active ingredients, it can be administered simultaneously, sequentially, separately or at intervals, with the purpose of exerting the biological activities of multiple active ingredients for the joint treatment of diseases.

[0062] The term "high concentration" refers to a formulation comprising an active pharmaceutical ingredient (API) at a concentration of about 80-200 mg / ml.

[0063] The term "pharmaceutically acceptable excipient" is a substance / agent that can be reasonably administered to a subject to provide an effective amount of an active pharmaceutical ingredient in a stable formulated form. Suitable pharmaceutically acceptable excipients are well known in the art and include, but are not limited to, buffers, stabilizers, surfactants, and the like.

[0064] The term "stabilizer" is a pharmaceutically acceptable excipient used to protect the active pharmaceutical ingredient and / or formulation from chemical and / or physical degradation during production, storage and use.

[0065] The term "surfactant" refers to a pharmaceutically acceptable excipient used to protect protein formulations from mechanical stresses such as agitation and shear.

[0066] The term "antioxidant" refers to a compound that prevents oxidation of the active ingredient under storage conditions.

[0067] The term "diffusing agent" refers to a compound used to increase the permeability of active substances into tissues (such as skin) and help the active substances diffuse effectively, such as hyaluronidase, also known as hyaluronidase, which can hydrolyze hyaluronic acid, thereby reducing the viscosity of hyaluronic acid in the extracellular matrix and making the drug preparation easier to disperse in the tissue.

[0068] The term "buffer" refers to an agent that maintains the pH of a formulation within an acceptable range. The histidine-hydrochloric acid buffer system herein can be formulated with histidine and dilute hydrochloric acid, or with histidine and histidine hydrochloride. Furthermore, the pharmaceutical compositions herein can be self-buffering systems, without a buffer.

[0069] The term "self-buffering" refers to the exclusion of components traditionally used to introduce buffering capacity into a formulation, such as buffers including, but not limited to, histidine buffers, phosphate buffers, citrate buffers, acetate buffers, carbonate buffers, succinate buffers, tartaric acid buffers, maleate buffers, or mixtures thereof. Proteins are known to provide buffering capacity to a formulation in addition to any buffering capacity present in a formulation without a buffer component. Therefore, this does not mean that a formulation cannot have any buffering capacity.

[0070] The term "lyophilized preparation" refers to a composition obtained or obtainable by a freeze-drying process of a liquid preparation. Preferably, it is a solid composition having a water content of less than 5%, preferably less than 3%.

[0071] The term "insoluble particles" includes but is not limited to particles introduced during the product formulation process, fatty acid particles produced by polysorbate degradation, silicone oil, macromolecular particles caused by protein aggregation, and other unknown particles, such as possible packaging material debris, excipient crystallization, etc. No degradation of polysorbate was observed in this article.

[0072] The term "treatment" refers to clinical intervention intended to alter the disease process in an individual or cell, and can be either preventative or interventional in the clinical pathological process. Therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the progression of the disease, improving or relieving the condition, and alleviating or improving the prognosis.

[0073] The term "individual" or "subject" or "patient" refers to any animal, such as a mammal or marsupial. Individuals of the present disclosure include, but are not limited to, humans, non-human primates (e.g., cynomolgus or rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and any type of poultry.

[0074] The terms "disease," "condition," or "disorder" refer to any change or disorder that damages or interferes with the normal function of a cell, tissue, or organ. For example, "disease" includes, but is not limited to, tumors, pathogen infection, autoimmune diseases, T-cell dysfunction, or immune tolerance deficiency (e.g., transplant rejection).

[0075] The term "tumor" refers to a disease characterized by the pathological proliferation of cells or tissues, and their subsequent migration or invasion into other tissues or organs. Tumor growth is typically uncontrolled and progressive, without inducing or inhibiting normal cell proliferation. Tumor includes "cancer" and refers broadly to all malignant tumors.

[0076] Experimental Example 1 Size Exclusion Chromatography (SEC-HPLC)

[0077] Size exclusion chromatography was used to quantify aggregates, monomers, and fragments. This method utilized the X Bridge BEH SEC was performed on a Waters E2695-2489 HPLC system using a 7.8 × 300 mm column. The mobile phase consisted of 100 mM sodium phosphate, 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. Isocratic elution was performed at a flow rate of 0.5 mL / min for 30 min, with detection at 215 nm. Integration was performed using Empower 3 software, and the percentage of each component was calculated by area normalization.

[0078] Experimental Example 2 Capillary electrophoresis (CE-SDS) determination

[0079] Capillary electrophoresis (CE-SDS) was used to determine the non-reduced and reduced purity of this product. This determination was performed on a SCIEX PA800 plus capillary electrophoresis system using a 50 μM ID uncoated quartz capillary with an effective separation length of 20 cm and a total length of 30.2 cm. A PDA 220 nm detection setting with a bandwidth of 10 nm was used. The reduced purity was determined at 5 kV for 20 seconds and the separation was 15 kV for 30 minutes, while the non-reduced purity was determined at 10 kV for 20 seconds and the separation was 15 kV for 40 minutes. Integration was performed using 32Karat software.

[0080] Experimental Example 3 Ion exchange chromatography (IEC-HPLC) determination

[0081] Ion exchange chromatography was used to determine the charge heterogeneity of this product. Various post-translational modifications of recombinant protein drugs can alter the surface charge distribution of proteins, leading to charge heterogeneity. This method utilized a weak cation exchange column, ProPac WCX-10 (4.0 mm × 250 mm, 10 μm, Thermo), and was run on a Waters e2695-2489 HPLC system. Mobile phase A consisted of 24 mM MES, 4% acetonitrile (v / v), pH 6.0; mobile phase B consisted of 20 mM sodium phosphate, 95 mM NaCl, 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 detection was performed at a wavelength of 280 nm. Integration was performed using Empower 3 software, and the percentage of each component was calculated by area normalization.

[0082] Experimental Example 4 Insoluble Particle Detection (FlowCam) Method

[0083] The insoluble particles of this product were determined using a flow cytometer FlowCam 8100 with the following parameters: sample volume 200 μl, flow rate 0.05 ml / min, and analysis software Visual Spreadsheet.

[0084] Experimental Example 5 Protein content determination

[0085] Protein content was determined using a Lunatic microspectrophotometer. Purified water was used for blank subtraction, and UV absorption at 280 nm was corrected for light scattering at 330 nm. Protein concentration was calculated according to Equation 1, where the extinction coefficient ε is 1.42 (L / g·cm-1).

[0086] Experimental Example 6 Viscosity Measurement

[0087] The viscosity of the samples was tested using an m-VROC microfluidic rheometer at a temperature of 25°C.

[0088] The present disclosure is further illustrated by the following examples. Changing the concentration of certain substances or adding other substances based on the present disclosure, but changes that do not significantly improve protein stability are still considered part of the present disclosure.

[0089] Example 1 Effect of Antioxidants on the Light Stability of Prescriptions

[0090] To prepare the formulation solution, the antibody is adjusted to the required concentration by ultrafiltration concentration. After ultrafiltration concentration is completed, the excipients to be added are added to the antibody solution, and finally the protein concentration is adjusted to about 170 mg / ml. All formulation samples are sterile filtered through a 0.22 μm filter and filled into sterile 2 ml tube bottles under controlled conditions. Nitrogen is filled when necessary and sealed with a film-coated rubber stopper and an aluminum-plastic combination cap. These preparations are placed in a high temperature (40 ° C ± 2 ° C), light (illuminance of 5000 lx, ultraviolet 90 μ w / cm 2 The stability of the samples was investigated under the conditions of 25℃±2℃. Table 1-2 Different formulations and stability results:

[0091] Table 1 Prescription composition

[0092] Table 2 Stability data Note: / indicates not detected; 0d data refer to the test results of prescription R2.

[0093] The data in Table 2 and Figures 1A and 1B show that after 10 days of illumination, the main peak purity of Formulation R5 was 2.0% and 1.5% higher than that of Formulations R2 and R3, which contained only 15 mM methionine, respectively, by SEC-HPLC, and 1.2% and 1.0% higher by nrCE-SDS, respectively. These results demonstrate that the addition of methionine and nitrogen saturation can effectively prevent aggregate formation under illumination.

[0094] Example 2 Stability of Antibody Preparations under Amino Acid Systems

[0095] To prepare the formulation solution, the antibody is adjusted to the desired concentration by ultrafiltration concentration. After ultrafiltration concentration is completed, the excipients to be added are added to the antibody solution, and finally the protein concentration is adjusted to about 170 mg / ml. All formulations are sterile filtered through a 0.22 μm filter and filled into sterile 2 ml vials under controlled conditions, sealed with a film-coated rubber stopper and an aluminum-plastic combination cap. These formulations are placed under high temperature (40°C ± 2°C) to investigate the stability of the samples. Tables 3 and 4 show different formulation formulations and stability results:

[0096] Table 3 Prescription composition

[0097] Table 4 Stability data Note: NA means not detected.

[0098] The data in Table 4 and Figure 2 indicate that compared to formulation R6, formulation R8 exhibited approximately 27% lower viscosity, while formulation R9 exhibited approximately 50% lower viscosity. After aging at high temperature (40°C for 30 days), the SEC-HPLC main peak purity of formulation R8 was comparable to that of formulation R6, while that of formulation R9 was slightly higher by 0.3%. These results demonstrate that the amino acid system within the formulation effectively reduces the viscosity of high-concentration pembrolizumab formulations and exhibits excellent thermal stability. 3% arginine is preferred.

[0099] Example 3 Stability of Pembrolizumab Antibody Formulations at Different Amino Acid Ratios

[0100] To prepare the formulation solution, the antibody is adjusted to the desired concentration by ultrafiltration concentration. After ultrafiltration concentration is completed, the excipients to be added are added to the antibody solution, and finally the protein concentration is adjusted to the desired concentration. All formulations are sterile filtered through a 0.22μm filter and filled into sterile 2ml tube bottles under controlled conditions, sealed with a film-coated rubber stopper and an aluminum-plastic combination cap. These preparations are placed at high temperature (40℃±2℃), light (illuminance of 5000lx, ultraviolet 90μw / cm 2The stability of the samples was investigated under the conditions of 25°C ± 2°C. Tables 5 to 8 show the different formulations and stability results:

[0101] Table 5 Prescription composition

[0102] Table 6 Stability results Note: NA means not detected.

[0103] Table 7 Results of insoluble particles after 10 days of illumination

[0104] Table 8 Results of insoluble particles at high temperature (40°C ± 2°C) for 14 days

[0105] The data in Tables 6, 7, and 8, as well as Figures 3A, 3B, 4, 5A, and 5B, show that compared to the sucrose-containing formulation R12, the samples containing the amino acid system and the self-buffered system showed no significant difference in SEC-HPLC main peak purity after 14 days at 40°C. The SEC-HPLC main peak of formulations R13, R14, R15, R16, R17, and R20 decreased by approximately 2.0%, 2.2%, 2.5%, 2.3%, 2.3%, and 2.3% after 10 days of exposure to light, respectively. This decrease was smaller than that of formulation R12 (the SEC-HPLC main peak decreased by approximately 3.0%). Furthermore, viscosity test results showed that the sample viscosities of formulations R13, R14, R15, R16, R17, and R20 were significantly lower than those of formulation R12. The above results show that arginine and proline play an excellent role in SEC-HPLC stability in pembrolizumab preparations and the effect is significantly better than sucrose and can significantly reduce sample viscosity.

[0106] The insoluble particle data results showed that after 10 days of illumination, compared with the sucrose system (R12), the total number of insoluble particles in 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) were approximately 37.8% (20697 / 54704), 28.3% (15486 / 54704), 28.9% (15793 / 54704), and 48.7% (26627 / 54704) of the total number of insoluble particles in formulation R12, respectively. After 14 days of high temperature, compared with prescription R12, the total number of insoluble particles of prescriptions R14, R15, R16, and R20 were approximately 62.9% (22354 / 35536), 22.9% (8130 / 35536), 32.5% (11535 / 35536), and 25.7% (9133 / 35536) of R12, respectively. It is worth noting that compared with the preparation prescription containing only arginine (R13), prescriptions R15 and R20 have better effects in terms of insoluble particles.

[0107] Based on the above experimental results, compared with prescription R12, prescriptions R15 and R20 have obvious advantages in purity, viscosity, and total number of insoluble particles. At the same time, another preparation prescription containing 1.5% arginine and 1.5% proline (R11) and a preparation prescription containing 2% arginine and 2% sucrose (R10) also achieved excellent results similar to prescriptions R15 and R20.

[0108] Example 4 Stability of Pembrolizumab Antibody Formulations at Different Excipient Concentrations

[0109] To prepare the formulation solution, the antibody is adjusted to the desired concentration by ultrafiltration concentration. After ultrafiltration concentration is completed, the excipients to be added are added to the antibody solution, and finally the protein concentration is adjusted to the protein concentration in Table 9. All formulations are sterile filtered through a 0.22 μm filter and filled into sterile 2 ml tube bottles under controlled conditions, sealed with a film-coated rubber stopper and an aluminum-plastic combination cap. These preparations are placed in a high temperature (40 ° C ± 2 ° C), light (illuminance of 5000 lx, ultraviolet 90 μ w / cm 2 The stability of the samples was investigated under the conditions of 25°C ± 2°C. Tables 9 to 11 show the different formulations and stability results:

[0110] Table 9 Prescription composition

[0111] Table 10 Stability results Note: / indicates not detected; *Indicates that R260h data is used as 0h control.

[0112] Table 11 Stability results Note: / indicates not detected. * Indicates that R260h data is used as 0h control.

[0113] From the data in Tables 10 and 11 and Figures 6A, 6B, and 6C, it can be found that after being placed at 40°C for 14 days, there was no significant difference in the purity of the main peaks of SEC-HPLC, nrCE-SDS, and rCE-SDS of the self-buffer system formulations and other amino acid-containing formulations compared with formulation R23; after being placed under light for 10 days, compared with formulation R23, 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, which was less than the decrease in formulation R23 (the SEC-HPLC main peak decreased by approximately 3.5%). The change trends of the SEC-HPLC main peaks of formulations R22, R25, R28, and R29 were basically consistent with that of the main peak of formulation R23; the decrease in the main peaks of nrCE-SDS and rCE-SDS in other groups was basically consistent with the decrease in the main peak of formulation R21. The above results indicate that the formulations containing a combination of arginine and proline or a combination of arginine and sucrose have excellent stability.

[0114] The embodiments of the present disclosure described above are merely exemplary, and any person skilled in the art will recognize or be able to determine the equivalents of numerous specific compounds, materials, and operations without requiring undue experimentation. All such equivalents are within the scope of the present disclosure and are encompassed by the claims.

Claims

1. A pharmaceutical composition of an anti-PD-1 antibody, which comprises a) an anti-PD-1 antibody at about 80 - 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; e) a spreading agent which is hyaluronidase; wherein the pH of the pharmaceutical composition is about 4.0 - 6.0, the content of arginine is about 0.1 - 50 mg / ml, the content of proline is about 0.1 - 50 mg / ml, and the content of sucrose is about 0.1 - 50 mg / ml.

2. The pharmaceutical composition according to claim 1, wherein the weight ratio of arginine to proline or the weight ratio of arginine to sucrose is about 1:2 - 2:1, preferably about 1:2, 1:1, 2:

1.

3. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition comprises a buffer in an amount of about 0.1 - 30 mM; preferably, the buffer is histidine - hydrochloride.

4. The pharmaceutical composition according to any one of claims 1 - 3, wherein the content of the surfactant is about 0.01 - 1 mg / ml.

5. The pharmaceutical composition according to any one of claims 1 - 3, wherein the content of methionine is about 0.1 - 50 mM.

6. The pharmaceutical composition according to any one of claims 1 - 3, wherein nitrogen is filled into the pharmaceutical composition.

7. The pharmaceutical composition according to any one of claims 1 - 3, wherein the content of hyaluronidase is about 1 - 10000 IU / ml.

8. The pharmaceutical composition according to any of the preceding claims, wherein the anti-PD-1 antibody is pembrolizumab.

9. The pharmaceutical composition according to claim 8, wherein the heavy chain sequence of the pembrolizumab is as shown in SEQ ID NO:1, and the light chain sequence is as shown in SEQ ID NO:

2.

10. A pharmaceutical composition of an anti-PD-1 antibody, which comprises pembrolizumab at about 80 - 200 mg / ml, histidine - hydrochloride at about 0.1 - 30 mM, polysorbate 80 at about 0.01 - 1 mg / ml, methionine at about 0.1 - 50 mM, arginine at about 0.1 - 50 mg / ml, proline at about 0.1 - 50 mg / ml, hyaluronidase at about 1 - 10000 IU / ml, and its pH is about 4.0 - 6.0, and optionally, nitrogen is filled.

11. A pharmaceutical composition of an anti-PD-1 antibody, which comprises pembrolizumab at about 160 - 180 mg / ml, histidine hydrochloride at about 5 - 20 mM, polysorbate 80 at about 0.1 - 0.6 mg / ml, methionine at about 5 - 25 mM, arginine at about 5 - 20 mg / ml, proline at about 5 - 20 mg / ml, and hyaluronidase at about 1 - 4000 IU / ml, with a pH of about 5.0 - 6.0, and optionally filled with nitrogen.

12. The pharmaceutical composition according to claim 10, which comprises: (1) pembrolizumab at about 155 mg / ml, arginine at about 1.5%, proline at about 1.5%, histidine hydrochloride at about 10 mM, polysorbate 80 at about 0.4 mg / ml, methionine at about 15 mM, hyaluronidase at about 2000 IU / ml, with a pH of about 5.5, and optionally filled with nitrogen; (2) pembrolizumab at about 160 mg / ml, arginine at about 1.5%, proline at about 1.5%, histidine hydrochloride at about 10 mM, polysorbate 80 at about 0.4 mg / ml, methionine at about 15 mM, hyaluronidase at about 2000 IU / ml, with a pH of about 5.5, and optionally filled with nitrogen; (3) pembrolizumab at about 165 mg / ml, arginine at about 1.5%, proline at about 1.5%, histidine hydrochloride at about 10 mM, polysorbate 80 at about 0.4 mg / ml, methionine at about 15 mM, hyaluronidase at about 2000 IU / ml, with a pH of about 5.5, and optionally filled with nitrogen; (4) pembrolizumab at about 170 mg / ml, arginine at about 2%, proline at about 1%, histidine hydrochloride at about 10 mM, polysorbate 80 at about 0.4 mg / ml, methionine at about 15 mM, hyaluronidase at about 2000 IU / ml, with a pH of about 5.5, and optionally filled with nitrogen; (5) pembrolizumab at about 170 mg / ml, arginine at about 1.5%, proline at about 1.5%, histidine hydrochloride at about 10 mM, polysorbate 80 at about 0.4 mg / ml, methionine at about 15 mM, hyaluronidase at about 2000 IU / ml, with a pH of about 5.5, and optionally filled with nitrogen; (6) pembrolizumab at about 170 mg / ml, arginine at about 1%, proline at about 2%, histidine hydrochloride at about 10 mM, polysorbate 80 at about 0.4 mg / ml, methionine at about 15 mM, hyaluronidase at about 2000 IU / ml, with a pH of about 5.5, and optionally filled with nitrogen; (7) pembrolizumab at about 175 mg / ml, arginine at about 1.5%, proline at about 1.5%, histidine hydrochloride at about 10 mM, polysorbate 80 at about 0.4 mg / ml, methionine at about 15 mM, hyaluronidase at about 2000 IU / ml, with a pH of about 5.5, and optionally filled with nitrogen; (8) Pembrolizumab at approximately 180 mg / ml, approximately 1.5% arginine, approximately 1.5% proline, approximately 10 mM histidine hydrochloride, approximately 0.4 mg / ml polysorbate 80, approximately 15 mM methionine, approximately 2000 IU / ml hyaluronidase, approximately pH 5.5, optionally filled with nitrogen; (9) Pembrolizumab at approximately 170 mg / ml, approximately 1.5% arginine, approximately 1.5% proline, approximately 10 mM histidine hydrochloride, approximately 0.5 mg / ml poloxamer 188, approximately 15 mM methionine, approximately 2000 IU / ml hyaluronidase, approximately pH 5.5, optionally filled with nitrogen; (10) Pembrolizumab at approximately 165 mg / ml, approximately 2% arginine, approximately 2% sucrose, approximately 10 mM histidine hydrochloride, approximately 0.4 mg / ml polysorbate 80, approximately 15 mM methionine, approximately 2000 IU / ml hyaluronidase, approximately pH 5.0, optionally filled with nitrogen; (11) Pembrolizumab at approximately 170 mg / ml, approximately 2% arginine, approximately 2% sucrose, approximately 10 mM histidine hydrochloride, approximately 0.4 mg / ml polysorbate 80, approximately 15 mM methionine, approximately 2000 IU / ml hyaluronidase, approximately pH 5.5, optionally filled with nitrogen; (12) Pembrolizumab at approximately 165 mg / ml, approximately 2% arginine, approximately 2% sucrose, approximately 10 mM histidine hydrochloride, approximately 0.4 mg / ml polysorbate 80, approximately 15 mM methionine, approximately 2000 IU / ml hyaluronidase, approximately pH 5.5, optionally filled with nitrogen.

13. A lyophilized preparation obtained by lyophilizing the pharmaceutical composition according to any one of the foregoing, or the lyophilized preparation, after reconstitution, gives the pharmaceutical composition according to any one of the foregoing.

14. A method for treating a tumor, the method comprising administering to a subject the pharmaceutical composition or lyophilized preparation according to any one of the foregoing.

15. The method according to claim 14, wherein the tumor is nasopharyngeal carcinoma, small cell lung cancer, non-small cell lung cancer, gastric cancer, esophageal cancer, pancreatic cancer, colorectal cancer, melanoma, renal cancer, breast cancer, liver cancer, ovarian cancer, cervical cancer, bladder cancer, head and neck tumors.

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