Pharmaceutical preparations containing anti-TIGIT antibodies

Abstract

The pharmaceutical formulation comprises an anti-TIGIT antibody, a stabilizer, a buffer solution, and a surfactant.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority from Chinese patent application CN202211713441.7, filed on December 27, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] The present invention relates to pharmaceutical formulations comprising anti-TIGIT antibodies and uses of such formulations. [Background technology]

[0003] T cell immunoreceptor (TIGIT) with Ig and ITIM domains is an immune checkpoint receptor expressed by immune cells (e.g., activated T cells and natural killer cells (NK cells)) and mediates immunosuppression. TIGIT's ligand (PVR, CD155) has been identified in dendritic cells (DCs), macrophages, and many human cancer cells, and studies have shown that the ligand can downregulate T cell activation and cytokine secretion upon binding to TIGIT. The inhibitory effect of TIGIT / PVR interaction can mediate the effective antitumor activity of immune cells. Given the important role of TIGIT in regulating immune checkpoints, therapeutic molecules and methods have been developed in the field to modulate TIGIT-mediated immune cell suppression for immunotherapy and cancer treatment, providing more drug options for patients.

[0004] Protein (e.g., monoclonal antibody) pharmaceutical formulations are suitable for parenteral administration, including intravenous, intramuscular, intraperitoneal, or subcutaneous injection. While their liquid formulations are easy to use, the proteins in liquid formulations are at risk of aggregation and degradation, potentially resulting in reduced stability. Because proteins, particularly monoclonal antibodies, contained in conventional pharmaceutical formulations have difficulty maintaining good physical, chemical, and biological stability during storage, especially under non-refrigerated conditions, protein pharmaceutical formulations have relatively strict storage requirements and generally must be stored at low temperatures. Protein (antibody) pharmaceutical formulations also incorporate protective agents to improve protein stability. Reported protective agents include surfactants, sugars and polyols, salts, amino acids / amino acid salts, and several polymeric compounds. However, antibody drugs have different structures and properties, and there is currently a lack of versatile protein formulations that meet the needs of the pharmaceutical industry. Therefore, there is a strong need in this field for pharmaceutical formulations that stabilize antibody drugs, particularly pharmaceutical formulations containing anti-TIGIT antibodies to maintain their stability. After extensive experimentation, the present applicant has discovered an excellent pharmaceutical preparation containing an anti-TIGIT antibody, thereby meeting this need. Summary of the Invention

[0005] The present invention provides a pharmaceutical formulation comprising an anti-TIGIT antibody that can maintain the physical and chemical stability of the anti-TIGIT antibody under long-term storage conditions and various environmental conditions.

[0006] One aspect of the present invention provides a pharmaceutical formulation comprising an anti-TIGIT antibody, the formulation including an anti-TIGIT antibody, a buffer, a stabilizer, a surfactant, and the like.

[0007] In some specific embodiments of the present invention, the pharmaceutical formulation comprising an anti-TIGIT antibody provided by the present invention comprises a single-domain antibody whose heavy chain variable region (VHH) comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1. In some specific embodiments of the present invention, the anti-TIGIT antibody in the pharmaceutical formulation has a concentration ranging from 15 to 25 mg / mL, with a preferred concentration being 20 mg / mL. In some specific embodiments of the present invention, the heavy chain of the anti-TIGIT antibody in the pharmaceutical formulation comprises the amino acid sequence set forth in SEQ ID NO: 2.

[0008] In some specific embodiments of the present invention, the pharmaceutical formulation comprising an anti-TIGIT antibody provided by the present invention comprises an acetate buffer or a histidine buffer, a stabilizer is sucrose, sorbitol, or proline, and a surfactant is polysorbate 20 or polysorbate 80.

[0009] More specifically, the present invention provides a composition comprising the following components: (i) an anti-TIGIT antibody that is a single domain antibody whose heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1; (ii) an acetate buffer or a histidine buffer; (iii) sucrose, sorbitol or proline; (iv) polysorbate 20 or polysorbate 80, The pH value of the pharmaceutical preparation is 5.1 to 5.7.

[0010] In some specific embodiments of the present invention, the pH value of the pharmaceutical preparation is 5.1, 5.3, 5.4 or 5.7, preferably 5.3 or 5.4, and most preferably 5.4.

[0011] In some specific embodiments of the present invention, the concentration of the buffer solution is 5 to 15 mM, preferably 10 mM or 15 mM, and most preferably 10 mM.

[0012] In some specific embodiments of the present invention, the component (iii) is sucrose.

[0013] In some specific embodiments of the present invention, the concentration of the component (iii) is 2% (w / v) to 6% (w / v), preferably 2% (w / v), 4% (w / v), or 6% (w / v), and more preferably 2% (w / v).

[0014] In some specific embodiments of the present invention, the component (iv) is polysorbate 80.

[0015] In some specific embodiments of the present invention, the concentration of the component (iv) is 0.01% (w / v) to 0.05% (w / v), preferably 0.03% (w / v) or 0.05% (w / v), and more preferably 0.03% (w / v).

[0016] In some specific embodiments of the present invention, the concentration of the anti-TIGIT antibody is 15 to 25 mg / mL, preferably 20 mg / mL.

[0017] In some specific embodiments of the present invention, the full-length heavy chain of the anti-TIGIT antibody comprises or consists of the amino acid sequence set forth in SEQ ID NO:2.

[0018] In some preferred embodiments of the present invention, the present invention comprises: (i) an anti-TIGIT antibody having a concentration of 15 to 25 mg / mL; (ii) a histidine buffer solution having a concentration of 5 to 15 mM; (iii) sucrose at a concentration of 2 to 6% (w / v); (iv) providing a pharmaceutical formulation containing an anti-TIGIT antibody, the pharmaceutical formulation comprising polysorbate 80 at a concentration of 0.01 to 0.05% (w / v); The pH value of the pharmaceutical preparation is 5.1 to 5.7.

[0019] The present invention provides (i) an anti-TIGIT antibody at a concentration of 20 mg / mL; (ii) a histidine buffer solution having a concentration of 10 mM; (iii) sucrose at a concentration of 2% (w / v); (iv) polysorbate 80 at a concentration of 0.03% (w / v), The pH value of the pharmaceutical preparation is 5.4.

[0020] Another aspect of the present invention relates to a method for reducing tumor burden in a patient or for treating a tumor in a patient, comprising administering to the patient an effective dose of the above-mentioned pharmaceutical preparation, wherein the above-mentioned pharmaceutical preparation is administered in combination with other drugs, preferably the above-mentioned pharmaceutical preparation is administered in combination with an anti-PD-1 antibody and / or an anti-PD-L1 antibody.

[0021] The present invention further relates to the use of the above pharmaceutical formulations for the preparation of medicaments for treating cancer, wherein the above pharmaceutical formulations are administered in combination with other drugs, preferably, the above pharmaceutical formulations are administered in combination with anti-PD-1 antibodies and / or anti-PD-L1 antibodies. Among them, the cancer or tumor is a solid tumor or a blood tumor. Among them, the cancer or tumor is selected from mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, synovial sarcoma, thymic cancer, cervical cancer, endometrial cancer, gastric tumor, melanoma, bile duct cancer, head and neck cancer, lymphoma, blood cancer, and combinations thereof.

[0022] The present invention also relates to a drug delivery device comprising the pharmaceutical formulation.

[0023] The pharmaceutical formulation provided by the present invention was subjected to high-temperature accelerated testing, repeated freezing and thawing, and long-term frozen storage stability studies, and the results showed that the anti-TIGIT antibody protein in the pharmaceutical formulation maintained good stability.

[0024] Surprisingly, the pharmaceutical formulations comprising the anti-TIGIT antibodies unexpectedly exhibit the advantages of least aggregation, least particle count, lowest turbidity, least acidic variants, lowest pH drift, best thermal stability, best high temperature accelerated stability, best freeze-thaw stability, and best long-term frozen storage stability. [Brief explanation of the drawings]

[0025] [Figure 1a] 1 shows the results of the change in SEC polymer % over time in a high-temperature accelerated test of the pharmaceutical formulation containing the anti-TIGIT antibody of Example 1. [Figure 1b] This shows the results of the change in CEX acidic peak % over time in a high-temperature accelerated test of a pharmaceutical formulation containing an anti-TIGIT antibody according to Example 1. Among these, CP45 represents a pharmaceutical formulation with a pH of 4.5, CP50 represents a pharmaceutical formulation with a pH of 5.0, CP55 represents a pharmaceutical formulation with a pH of 5.5, CP60 represents a pharmaceutical formulation with a pH of 5.0, and CP65 represents a pharmaceutical formulation with a pH of 6.5. [Figure 2a] 1 shows the results of the change in SEC polymer % over time in a high-temperature accelerated test of a pharmaceutical formulation containing an anti-TIGIT antibody according to Example 2. [Figure 2b] This shows the results of the change in CEX acidic peak % over time in a high-temperature accelerated test of a pharmaceutical formulation containing an anti-TIGIT antibody according to Example 2. CP55 represents a pharmaceutical formulation containing a citric acid-sodium dihydrogen phosphate buffer, C55 represents a pharmaceutical formulation containing a citrate buffer, A55 represents a pharmaceutical formulation containing an acetate buffer, and H55 represents a pharmaceutical formulation containing a histidine buffer. [Figure 3a] 1 shows the results of the change in SEC polymer % over time in a high-temperature accelerated test of a pharmaceutical formulation containing an anti-TIGIT antibody according to Example 3. [Figure 3b] This shows the results of the change in CEX acidic peak % over time in a high-temperature accelerated test of a pharmaceutical formulation containing an anti-TIGIT antibody according to Example 3. Among these, 5 mM represents a pharmaceutical formulation with a buffer concentration of 5 mM, 15 mM represents a pharmaceutical formulation with a buffer concentration of 15 mM, and 30 mM represents a pharmaceutical formulation with a buffer concentration of 30 mM. [Figure 4a]1 shows the results of the change over time in the average particle size of proteins in a high-temperature accelerated test of a pharmaceutical formulation containing an anti-TIGIT antibody according to Example 4. [Figure 4b] 1 shows the results of changes in Pk1% over time in a high-temperature accelerated test of a pharmaceutical formulation containing an anti-TIGIT antibody according to Example 4. PS20 represents a pharmaceutical formulation containing polysorbate 20, and PS80 represents a pharmaceutical formulation containing polysorbate 80. [Figure 5a] 1 shows the results of the change in SEC polymer % over time in a high-temperature accelerated test of the pharmaceutical formulation containing the anti-TIGIT antibody according to Example 5. [Figure 5b] 1 shows the results of the change over time in CEX acidic peak % in a high-temperature accelerated test of a pharmaceutical formulation containing an anti-TIGIT antibody according to Example 5. [Figure 6] FIG. 10 shows a JMP software profiler model diagram for screening the pH value, stabilizer, and surfactant concentrations of a pharmaceutical formulation containing an anti-TIGIT antibody according to Example 6. [Figure 7a] The pharmaceutical formulation containing the anti-TIGIT antibody according to Example 7 was subjected to repeated freezing and thawing, and the results of the change over time in the subvisible particle level (SVP) in the formulation are shown. [Figure 7b] The pharmaceutical formulation containing the anti-TIGIT antibody according to Example 7 was repeatedly frozen and thawed, and the results of the changes in the SEC polymer in the formulation over time are shown. [Figure 7c] The pharmaceutical formulation containing the anti-TIGIT antibody of Example 7 was repeatedly frozen and thawed, and the results of changes in SEC fragments in the formulation over time are shown. [Figure 7d] The pharmaceutical preparation containing the anti-TIGIT antibody of Example 7 was repeatedly frozen and thawed, and the results of the change in IgG% in the preparation over time are shown. [Figure 7e] 1 shows the results of the time-dependent change in the CEX acidic peak in the pharmaceutical formulation containing the anti-TIGIT antibody of Example 7 after repeated freezing and thawing. [Figure 7f] 1 shows the results of the time course of subvisible particle levels in a pharmaceutical formulation containing another anti-TIGIT antibody according to Example 7 after repeated freezing and thawing. [Figure 7g]The pharmaceutical formulation containing another anti-TIGIT antibody according to Example 7 was subjected to repeated freezing and thawing, and the results show the changes in the SEC polymer in the formulation over time. [Figure 7h] 1 shows the results of time-dependent changes in SEC fragments in a pharmaceutical formulation containing another anti-TIGIT antibody according to Example 7 after repeated freezing and thawing. [Figure 7i] The results of Example 7 show the change in IgG% in a pharmaceutical formulation containing another anti-TIGIT antibody over time after repeated freezing and thawing. [Figure 7j] 1 shows the results of the time course of the CEX acidic peak in a pharmaceutical formulation containing another anti-TIGIT antibody according to Example 7 after repeated freezing and thawing. [Figure 8a] 10 shows the results of the time-dependent change in SEC fragments in a high-temperature accelerated test of a pharmaceutical formulation containing an anti-TIGIT antibody according to Example 8. [Figure 8b] 1 shows the results of the change in IgG% over time in a high-temperature accelerated test of a pharmaceutical preparation containing an anti-TIGIT antibody according to Example 8. [Figure 8c] 1 shows the results of changes in CEX acidic peak over time in a high-temperature accelerated test of a pharmaceutical formulation containing an anti-TIGIT antibody according to Example 8. [Figure 8d] 10 shows the results of a CEX acidic peak in a high-temperature accelerated test of a pharmaceutical formulation containing an anti-TIGIT antibody according to Example 8. [Figure 8e] 1 shows the results of the change over time of the SEC polymer in a high-temperature accelerated test of a pharmaceutical formulation containing an anti-TIGIT antibody according to Example 8. [Figure 8f] 1 shows the results of the change over time of the SEC polymer in a high-temperature accelerated test of a pharmaceutical formulation containing an anti-TIGIT antibody according to Example 8. [Figure 8g] 10 shows the results of time-dependent changes in SEC fragments in a high-temperature accelerated test of a pharmaceutical formulation containing an anti-TIGIT antibody according to Example 8. [Figure 8h] 1 shows the results of the change in IgG% over time in a high-temperature accelerated test of a pharmaceutical preparation containing an anti-TIGIT antibody according to Example 8. [Figure 8i]1 shows the results of the change over time in icIEF acidic peaks in a high-temperature accelerated test of a pharmaceutical formulation containing an anti-TIGIT antibody according to Example 8. [Figure 9a] 1 shows the results of the time course of SEC polymer in a high-temperature accelerated test of a pharmaceutical formulation containing a preferred anti-TIGIT antibody of the present invention. [Figure 9b] 1 shows the results of SEC fragment changes over time in a high-temperature accelerated test of a pharmaceutical formulation containing a preferred anti-TIGIT antibody of the present invention. [Figure 9c] 1 shows the results of the change in IgG% over time in a high-temperature accelerated test of a pharmaceutical formulation containing a preferred anti-TIGIT antibody of the present invention. [Figure 9d] 1 shows the results of the time course of icIEF acidic peaks in a high-temperature accelerated test of a pharmaceutical formulation containing a preferred anti-TIGIT antibody of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention will be described in more detail below with reference to embodiments, but these embodiments should not be construed as limiting the present invention.

[0027] definition As used herein, "pharmaceutical formulation" or "formulation" refers to a sterile composition containing a pharmaceutically active drug, suitable for parenteral administration (including, but not limited to, intravenous, intramuscular, subcutaneous, aerosol, pulmonary, etc.), e.g., containing a biologically active protein, and containing pharmaceutically acceptable excipients, diluents, and other additives. For example, pharmaceutical formulations may include liquids (e.g., directly administrable aqueous solutions) and lyophilized powders.

[0028] As used herein, "antibody" includes whole antibodies and any other antigen-binding fragments, such as monoclonal antibodies (including human or murine antibodies, humanized antibodies, or chimeric antibodies), polyclonal antibodies, and multispecific antibodies, and antibody-binding fragments include, but are not limited to, Fv, Fab, Fab', F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies composed of antibody fragments.

[0029] As used herein, "anti-TIGIT antibody" or "anti-TIGIT antibody protein" refers to an antibody capable of binding to TIGIT with sufficient affinity, thereby enabling the antibody to be used as a diagnostic and / or therapeutic agent targeting TIGIT. According to specific embodiments of the present invention, an antibody that specifically binds to TIGIT is a single-domain antibody whose heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1. In some specific embodiments of the present invention, the full-length heavy chain of the anti-TIGIT antibody comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2. As used herein, a single-domain antibody, also referred to as a nanobody, refers to a heavy chain single-domain antibody comprising only a VHH region or fragment, wherein the VHH may comprise the heavy chain variable region of a camelid heavy chain antibody. The anti-TIGIT antibody of the present invention is described in patent application WO2021139777A1, the entire text of which is incorporated herein by reference.

[0030] As used herein, a "stabilizer" is a reagent that can prevent or reduce the chemical and / or physical instability of a protein. Stabilizers include sugars, alcohols, acids, salts, etc. In an exemplary embodiment of the present invention, the stabilizer is selected from sucrose, sorbitol, glycine, proline, arginine hydrochloride, etc., for purposes of studying the stabilizer class.

[0031] As used herein, a "surfactant" is an organic substance having a hydrophilic and hydrophobic amphoteric structure and can be divided into anionic, cationic, and nonionic surfactants. Surfactants include polysorbates, polyethylene glycols, polypropylene glycols, etc. In an exemplary embodiment of the present invention, the surfactant is selected from polysorbate 20 or polysorbate 80.

[0032] As used herein, a "buffer system" refers to one or more buffering agents or buffer solutions and / or their acid / base conjugates, particularly one buffering agent or buffer solution and / or its acid / base conjugate. Also, as used herein, a "buffer" refers to a compound solution known to be safe for use in pharmaceutical preparations and capable of maintaining or controlling the pH value of the pharmaceutical preparation within a desired range. For example, buffer solutions include phosphate buffer, acetate buffer, citrate buffer, and certain amino acid buffers (e.g., arginine or histidine). In exemplary embodiments of the present invention, citrate-disodium hydrogen phosphate buffer, citrate buffer, acetate buffer, and histidine buffer are selected for buffer study. Among these, the histidine buffer is a buffer containing the histidine amino acid. Examples of histidine buffer solutions include histidine hydrochloride, histidine acetate, histidine phosphate, and histidine sulfate. However, in some specific embodiments of the present invention, the histidine buffer solution used is a histidine-histidine hydrochloride buffer.

[0033] As used herein, "stability" refers to the resistance of antibodies and other active ingredients to chemical degradation and physical change under given manufacturing, preparation, transportation, and storage conditions. Specifically, stability of a pharmaceutical formulation means that at least 90%, 95%, 98%, 99%, or 99.5% of the initial or reference amount of antibody or other active ingredient is retained under given manufacturing, preparation, transportation, and / or storage conditions. The amount of antibody or other active ingredient can be detected or determined by methods known in the art, such as ultraviolet spectrophotometry, chromatography, or SDS-PAGE.

[0034] As used herein, "clarity" refers to the degree of cloudiness of an aqueous solution, i.e., turbidity. When particles are present in an aqueous solution, light passing through the solution causes light scattering and light absorption, which in turn determines the degree of turbidity of the solution. For example, in this technical field, a transparency detector is used to visually detect the clarity and visible impurities of an aqueous solution sample, and the degree of opalescence is classified as clear, slightly opalescent, heavily opalescent, or opalescent. Furthermore, the turbidity of an aqueous solution is measured using a 350 nm absorbance detection method, and the higher the absorbance value, the higher the turbidity of the sample.

[0035] As used herein, "mean protein particle size" or "mean hydrated protein particle size" refers to the (hydrated) particle diameter of a protein in an aqueous solution. In the art, protein particle diameter is typically detected by dynamic light scattering (DLS). In this specification, "polydispersity coefficient" refers to the distribution of peaks in all particle sizes, and "Pk1%" refers to the percentage of the size range of a single particle in the size range of the entire particle range, to describe the degree of dispersion of the particle size distribution.

[0036] As used herein, "pH drift" refers to a change in the pH value of a pharmaceutical formulation relative to the pH value of the initial buffer system after the pharmaceutical formulation undergoes an exchange of ultrafiltration concentrate. For example, the initial pH value of the buffer system of a pharmaceutical formulation is 5.0, but after the pharmaceutical formulation undergoes an exchange of ultrafiltration concentrate, the pH value of the buffer system of the pharmaceutical formulation changes from the initial value of 5.0 to 5.2.

[0037] As used herein, "high-temperature accelerated testing" refers to placing a pharmaceutical formulation sample in an environment higher than room temperature to detect changes in formulation quality over a certain period of time, where the temperature typically includes 30°C, 40°C, 50°C, etc.

[0038] As used herein, "coagulation temperature T" agg (also called thermal aggregation temperature) refers to the temperature at which protein molecules begin to aggregate during the temperature increase process.

[0039] As used herein, "freeze-thaw" refers to placing a pharmaceutical preparation sample in an environment below 0°C until the pharmaceutical preparation is completely frozen, and then removing the pharmaceutical preparation sample and placing it at room temperature to thaw it. "Freeze-thaw cycle" refers to the number of times the freeze-thaw procedure is repeated, including temperatures such as -20°C, -40°C, -60°C, and -80°C.

[0040] As used herein, "long-term frozen storage test" refers to the process of storing a pharmaceutical formulation sample in an environment with a temperature lower than 0°C to detect changes in formulation quality over a certain period of time, including temperatures such as -20°C, -40°C, -60°C, and -80°C.

[0041] As used herein, "charge isomer" refers to a heterogeneous protein resulting from differences in the net charge and charge distribution on the protein surface. It is generally divided into acid isomers and base isomers, and these isomers exhibit different chromatographic and electrophoretic behavior from the main protein. When detecting pharmaceutical formulations by cation exchange chromatography (CEX), the main component is usually represented by a relatively abundant main peak, with acid isomers represented by an acidic peak appearing relatively early in the main peak, and base isomers represented by a basic peak appearing relatively later in the main peak. In addition, in the art, charge isomers are also detected by capillary isoelectric focusing (icIEF).

[0042] As used herein, "treatment" is a method for obtaining beneficial or desired results (including clinical results). Achieving beneficial or desired results includes, but is not limited to, one or more of the following: alleviating one or more symptoms of the disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or slowing the progression of the disease), delaying or slowing the progression of the disease, improving the disease state, increasing or improving quality of life, weight gain, and / or extending survival. "Treatment" also covers reducing the pathological consequences of cancer.

[0043] As used herein, "effective dose or amount" refers to a dose that can produce a therapeutic effect in an individual upon administration. This dose will vary depending on various factors, such as the purpose of treatment, frequency of treatment, the individual's weight and tolerance, the severity of symptoms, the risk of side effects, and the route of administration.

[0044] As used herein, "about" or "approximately" refers to a variation within an acceptable error range of a particular value that is determinable by one of ordinary skill in the art and that is dependent in part on how the particular value is measured and determined, i.e., limited by the measurement system or tool. For example, in the examples herein, "about" can refer to a range of 10%, 5%, or 1% of a given value.

[0045] Pharmaceutical preparations The present invention relates to a pharmaceutical formulation, and more particularly to a pharmaceutical formulation of an anti-TIGIT antibody comprising an anti-TIGIT antibody, a buffer, a stabilizer, a surfactant, and the like.

[0046] In some specific embodiments of the present invention, the pharmaceutical formulation contains an anti-TIGIT antibody at a concentration of 15 to 25 mg / mL. In some specific embodiments of the present invention, the concentration of the anti-TIGIT antibody is 16 to 24 mg / mL, 17 to 23 mg / mL, 18 to 22 mg / mL, or 19 to 21 mg / mL. In an exemplary embodiment of the present invention, the concentration of the anti-TIGIT antibody is 20 mg / mL.

[0047] In some specific embodiments of the present invention, the buffer is an acetate buffer or a histidine buffer. In certain embodiments of the present invention, the buffer is an acetate buffer, while in other certain embodiments of the present invention, the buffer is a histidine buffer. In some specific embodiments of the present invention, the buffer concentration ranges from 5 to 30 mM. In certain specific embodiments of the present invention, the buffer concentration ranges from 10 to 25 mM. In preferred embodiments of the present invention, the buffer concentration ranges from 5 to 15 mM. In certain specific embodiments of the present invention, the buffer concentration ranges from 5 mM, 10 mM, or 15 mM. In exemplary embodiments of the present invention, the buffer is an acetate buffer having a concentration of 5 to 15 mM. In certain embodiments, the buffer is an acetate buffer having a concentration of 5 mM, 10 mM, or 15 mM, respectively. In other exemplary embodiments of the present invention, the buffer is a histidine buffer having a concentration of 5 to 15 mM. In certain embodiments, the buffer is a histidine buffer having a concentration of 5 mM, 10 mM, or 15 mM, respectively.

[0048] In some specific embodiments of the present invention, the stabilizer is sucrose, sorbitol, or proline; in one exemplary embodiment of the present invention, the stabilizer is sorbitol; in another exemplary embodiment of the present invention, the stabilizer is proline; and in one exemplary embodiment of the present invention, the stabilizer is sucrose. Regarding the type of stabilizer, pharmaceutical formulations according to some specific embodiments of the present invention have a stabilizer concentration of 2% to 6% (mass / volume ratio); in certain specific embodiments of the present invention, the stabilizer concentrations are 2%, 3%, 4%, 5%, and 6%, respectively. In exemplary embodiments of the present invention, the stabilizer is sucrose, and its concentration is 2% to 6%, while in certain embodiments, the sucrose concentrations are 2%, 4%, and 6%, respectively.

[0049] In some specific embodiments of the present invention, the surfactant is polysorbate 20 or polysorbate 80. In an exemplary embodiment of the present invention, the surfactant is polysorbate 20, while in another exemplary embodiment of the present invention, the surfactant is polysorbate 80. In certain embodiments of the present invention, the concentration of the surfactant is 0.01% to 0.05% (weight to volume), and in some exemplary embodiments of the present invention, the concentration of the surfactant is 0.02%, 0.03%, or 0.05%. In some exemplary embodiments of the present invention, the surfactant is polysorbate 20, and the concentration is 0.02%, 0.03%, or 0.05%. In some preferred exemplary embodiments of the present invention, the surfactant is polysorbate 80, and the concentration is 0.02%, 0.03%, or 0.05%, and more specifically, in preferred exemplary embodiments of the present invention, the concentration is 0.03% or 0.05%.

[0050] In some specific embodiments of the present invention, the pharmaceutical formulation comprises an anti-TIGIT antibody, a histidine buffer or acetate buffer, a stabilizer, and a surfactant, and has a pH range of 5.1 to 5.7, for example, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, or 5.7. In some exemplary embodiments of the present invention, the pH is 5.1, 5.3, 5.4, or 5.7.

[0051] Certain embodiments of the pharmaceutical formulation according to the present invention are preferred, and the preferred forms thereof are listed below: 1) A pharmaceutical preparation, which comprises the following ingredients: (i) an anti-TIGIT antibody that is a single domain antibody whose heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1; (ii) an acetate buffer or a histidine buffer; (iii) sucrose, sorbitol or proline; (iv) polysorbate 20 or polysorbate 80, The pH value of the pharmaceutical preparation is 5.1 to 5.7. 2) The pH value of the pharmaceutical preparation is 5.1, 5.3, 5.4 or 5.7, preferably 5.3 or 5.4, and most preferably 5.4. 3) The concentration of the buffer solution is 5 to 15 mM, preferably 10 mM or 15 mM, and most preferably 10 mM. 4) Component (iii) is sucrose. 5) The concentration of component (iii) is 2% to 6%, preferably 2%, 4%, or 6%, and more preferably 2%. 6) Component (iv) is polysorbate 80. 7) The concentration of component (iv) is 0.01% to 0.05%, preferably 0.03% or 0.05%, and more preferably 0.03%. 8) The concentration of the anti-TIGIT antibody is 15 to 25 mg / mL, preferably 20 mg / mL. 9) The full-length heavy chain of the anti-TIGIT antibody comprises or consists of the amino acid sequence shown in SEQ ID NO:2. 10) A pharmaceutical preparation, which comprises the following ingredients: an anti-TIGIT antibody at a concentration of 15 to 25 mg / mL; a histidine buffer solution having a concentration of 5 to 15 mM; sucrose at a concentration of 2-6%; Polysorbate 80 having a concentration of 0.01 to 0.05%, The pH value of the pharmaceutical preparation is 5.1-5.7. 11) A pharmaceutical preparation, which comprises the following ingredients: an anti-TIGIT antibody at a concentration of 20 mg / mL; a histidine buffer solution having a concentration of 10 mM; sucrose at a concentration of 2%; Polysorbate 80 at a concentration of 0.03%, The pH value of the pharmaceutical preparation is 5.4.

[0052] Use and Method The present invention relates to a method for reducing tumor burden in a patient or a method for treating a tumor in a patient, comprising administering to the patient an effective dose of the pharmaceutical preparation. Further, the present invention relates to the use of the pharmaceutical preparation for use in preparing a medicament for treating cancer. More specifically, the method for reducing tumor burden in a patient or the method and use for treating a tumor in a patient according to the present invention comprises administering to the patient an effective dose of the pharmaceutical preparation alone or simultaneously in combination with other drugs.

[0053] In some embodiments of the present invention, the pharmaceutical formulations are administered in combination with other drugs to reduce tumor burden or treat tumors in a patient, and the other drugs may be growth inhibitors, cytotoxic agents, radiotherapeutic agents, protein drugs, immunomodulators, or medicinal nutritional supplements, including, for example, cisplatin, carboplatin, taxol, docetaxel, gefitinib, irinotecan, afatinib, gemcitabine, anti-VEGF antibodies (e.g., bevacizumab), anti-PD-L1 antibodies, anti-HER2 antibodies, anti-c-Met antibodies, anti-BCMA antibodies, anti-PD1 antibodies, anti-CTLA4 antibodies, anti-LAG-3 antibodies, and anti-2B4 antibodies. In a specific embodiment of the present invention, the anti-TIGIT antibody pharmaceutical formulations are administered in combination with anti-PD-L1 antibody drugs. In a specific embodiment of the present invention, the anti-TIGIT antibody pharmaceutical formulations are administered in combination with anti-PD-1 antibody drugs.

[0054] In some embodiments of the present invention, the tumor or cancer is a solid tumor, while in other certain embodiments of the present invention, the tumor or cancer is a hematological tumor (e.g., leukemia). In some embodiments, the tumor or cancer is selected from mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, synovial sarcoma, thymic cancer, cervical cancer, endometrial cancer, melanoma, gastric tumor, bile duct cancer, head and neck cancer, lymphoma, hematological cancer, and combinations thereof. More specifically, in some embodiments of the present invention, the tumor or cancer is lung cancer, in some embodiments of the present invention, the tumor or cancer is gastric cancer, in some embodiments of the present invention, the tumor or cancer is melanoma, in some embodiments of the present invention, the tumor or cancer is lymphoma, and in some embodiments of the present invention, the tumor or cancer is cervical cancer.

[0055] The present invention provides a drug delivery device (e.g., a syringe or pre-filled syringe) containing the above-mentioned pharmaceutical formulation, and by administering the pharmaceutical formulation containing the above-mentioned anti-TIGIT antibody to a patient using this drug delivery device, the symptoms of the cancer or tumor are treated or improved.

[0056] The stability of pharmaceutical formulations can be evaluated by methods commonly used in the art, including light scattering detection, size exclusion chromatography detection, etc. Other methods for detecting the stability of pharmaceutical formulations used in the present invention are also known in the art. Sample detection methods used in specific embodiments of the present invention are described below.

[0057] Furthermore, the present invention also relates to the following embodiments: 1. A pharmaceutical preparation, which is (i) an anti-TIGIT antibody that is a single domain antibody whose heavy chain variable region comprises or consists of the amino acid sequence of SEQ ID NO: 1; (ii) 5 to 15 mM histidine buffer; (iii) 2% (w / v) to 6% (w / v) sucrose; (iv) 0.01% (w / v) to 0.05% (w / v) of polysorbate 80, wherein the pH value of the pharmaceutical preparation is 5.1 to 5.7; Pharmaceutical preparations. 2. The pharmaceutical formulation according to embodiment 1, wherein the concentration of the anti-TIGIT antibody is 15 to 25 mg / mL. 3. The pharmaceutical formulation of embodiment 1 or 2, wherein the concentration of the anti-TIGIT antibody is 20 mg / mL. 4. The pharmaceutical formulation according to any one of embodiments 1 to 3, wherein the full length heavy chain of the anti-TIGIT antibody comprises or consists of the amino acid sequence of SEQ ID NO:2. 5, 20 mg / mL anti-TIGIT antibody and 10 mM histidine buffer; 2% (w / v) sucrose, 0.03% (w / v) polysorbate 80, wherein the pH value of the pharmaceutical preparation is 5.1 to 5.7; A pharmaceutical formulation according to any one of embodiments 1 to 4. 6. The pharmaceutical formulation according to any one of embodiments 1 to 5, wherein the pH value of the pharmaceutical formulation is preferably 5.4. 7. The pharmaceutical formulation of any one of embodiments 1 to 6, for use in treating cancer in a subject. 8. The pharmaceutical preparation used in embodiment 7, wherein the cancer is a solid tumor or a blood tumor. 9. The pharmaceutical formulation for use in embodiment 7 or 8, wherein the cancer is selected from mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, synovial sarcoma, thymic cancer, cervical cancer, endometrial cancer, gastric tumor, melanoma, bile duct cancer, head and neck cancer, lymphoma, blood cancer, and combinations thereof. 10. The pharmaceutical formulation according to any one of embodiments 7 to 9, wherein the pharmaceutical formulation is administered in combination with other drugs. 11. The pharmaceutical formulation used in embodiment 10, wherein the other drug is an anti-PD-1 antibody and / or an anti-PD-L1 antibody. 12. The pharmaceutical formulation used in embodiment 10 or 11, wherein the other drug is an anti-PD-1 antibody. 13. Use of the pharmaceutical formulation of any one of embodiments 1 to 6 for the preparation of a medicament for treating cancer in a subject. 14. The use according to embodiment 13, wherein the cancer is a solid tumor or a blood tumor. 15. The use of embodiment 13 or 14, wherein the cancer is selected from mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, synovial sarcoma, thymic cancer, cervical cancer, endometrial cancer, gastric tumor, melanoma, bile duct cancer, head and neck cancer, lymphoma, blood cancer, and combinations thereof. 16. The use according to any one of embodiments 13 to 15, wherein the pharmaceutical preparation is administered in combination with other drugs. 17. The use of embodiment 16, wherein the other drug is an anti-PD-1 antibody and / or an anti-PD-L1 antibody. 18. The use of embodiment 16 or 17, wherein the other drug is an anti-PD-1 antibody. 19. A method for reducing tumor burden in a patient and / or treating a tumor in a patient, comprising administering to the patient an effective dose of a pharmaceutical formulation described in any one of embodiments 1 to 6. 20. The method of embodiment 19, wherein the tumor is a solid tumor or a hematological tumor. 21. The method of embodiment 19 or 20, wherein the tumor is selected from mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, synovial sarcoma, thymic cancer, cervical cancer, endometrial cancer, gastric tumor, bile duct cancer, head and neck cancer, lymphoma, blood cancer, and combinations thereof. 22. The method of any one of embodiments 19 to 21, wherein the pharmaceutical preparation is administered in combination with other drugs. 23. The method of embodiment 22, wherein the other agent is an anti-PD-1 antibody and / or an anti-PD-L1 antibody. 24. The method of embodiment 22 or 23, wherein the other agent is an anti-PD-1 antibody. 25. A drug delivery device comprising the pharmaceutical formulation of any one of embodiments 1 to 6.

[0058] Example Sample detection method 1. Appearance and detection of visible foreign matter The appearance of the sample includes color, clarity, and visible foreign matter. The specific procedure is as follows: After wiping the sample bottle clean, a sample bottle filled with water was used as a negative control to observe the appearance of the liquid in the bottle. First, the light illuminance of the transparency detector was adjusted to 1000-3750 Lux in a dark room, and the sample bottle was placed on the edge (25 cm) of the transparency detector's light shield. The sample clarity and visible foreign matter were inspected visually against a black background. Next, the color of the sample was observed visually against a white background under fluorescent lighting.

[0059] 2. Dynamic Light Scattering (DLS) The Wyatt DynaPro Plate Reader-III high-throughput dynamic static light scattering analyzer was used to measure the average protein particle size and polydispersity coefficient of the sample, and the PK1% was calculated. The specific procedure was as follows: 25 μL of sample was taken from the ultra-clean bench and placed into the microwells of a 384-well plate. After coating, the 384-well microplate was centrifuged at 2000 rpm for 2 minutes, and the average protein particle size of the sample in the 384-well plate was then detected using the Wyatt DynaPro Plate Reader-III high-throughput dynamic static light scattering analyzer. The specific detection parameter settings are shown in Table 1.

[0060] [Table 1]

[0061] 3. Size Exclusion Chromatography (SEC) An Agilent 1260 high performance liquid chromatograph was used for size exclusion chromatography detection (SEC), with detection parameters as shown in Table 2, and the formulation samples should be diluted to 1.0 mg / mL with the mobile phase before testing.

[0062] [Table 2]

[0063] 4.Dynamic particle morphology analysis (FlowCam) Dynamic particle morphology analysis was performed using a FlowCam 8100 particle analysis detector to measure the morphology and quantity of subvisible particles in the sample. The specific detection parameters were as shown in Table 3. 250 μL of sample was manually injected and detected once per sample.

[0064] [Table 3]

[0065] 5.350nm absorbance detection (A 350 ) The absorbance value was measured using a SpectraMax® 190 light absorption microplate reader (hereinafter referred to as the microplate reader) to measure the turbidity of the sample. The specific procedure was as follows: 100 μL of sample was taken and added to a 96-well plate, and the absorbance value of the sample in the 96-well plate at 350 nm was detected using the microplate reader.

[0066] 6. Preparation of antibody pharmaceutical formulations As shown in the table below, the anti-TIGIT antibody of the present invention is a single-domain antibody, the amino acid sequence of its heavy chain variable region (VHH) being shown in SEQ ID NO: 1 and the amino acid sequence of the full-length heavy chain being shown in SEQ ID NO: 2. This anti-TIGIT antibody can be obtained by techniques well known in the art, such as production in mammalian host cells by genetic engineering techniques or protein synthesis techniques.

[0067] [Table 4]

[0068] The purified anti-TIGIT antibody protein was concentrated by ultrafiltration, desalted, and the protein concentration was adjusted. Stabilizers, surfactants, and buffer solutions were then added according to the experimental design to prepare antibody pharmaceutical formulations for testing and detection in the following examples.

[0069] In the following examples, studies were conducted on anti-TIGIT antibody pharmaceutical formulations, including pH values, buffers, surfactants, stabilizers, etc., to determine pharmaceutical formulations that can maintain the stability of anti-TIGIT antibodies.

[0070] All of the reagents used in the examples of the present invention are commercially available products.

[0071] Example 1. pH value studies pH is a major factor affecting protein stability, and protein stability is expressed differently depending on the pH. In this example, a high-temperature (50°C) accelerated test was performed to examine the effect of different pHs on the stability of the anti-TIGIT antibody protein and determine the pH range that contributes to the stabilization of the anti-TIGIT antibody. As shown in Table 4, a single-factor test design was used to design five candidate formulations, and the pH values ​​screened were 4.5, 5.0, 5.5, 6.0, and 6.5. Each formulation contained a citrate-disodium hydrogen phosphate buffer solution and 0.02% polysorbate 20 (also known as PS20 or Tween 20), with the buffer concentration at 15 mM and the protein concentration at 2.0 mg / mL.

[0072] [Table 5]

[0073] According to the research results, on the 0th day, it was revealed that there were no significant differences in appearance, protein concentration, initial protein average particle size, and charge isomers (CEX) among the formulations with different pH values. The initial aggregation temperatures of the proteins in each formulation were in the order of formulation CP55 (58.0 °C) ≈ CP50 (57.8 °C) > CP60 (56.2 °C) > CP45 (54.9 °C) > CP65 (51.5 °C), indicating that the thermal stability of the protein is relatively excellent when the pH is 5.0 and 5.5. As the pH increases, the content of the SEC polymer gradually increases, indicating that the higher the pH, the easier the protein is to aggregate.

[0074] After standing at 50 °C for 6 days, according to the appearance results, when the pH is 5.0 - 5.5, the sample exhibits slight opalescence and there are no visible particles; when the pH is 4.5, the opalescence of the sample increases; when the pH ≥ 6.0, there are visible particles in the sample. According to the results of the turbidity (A350) absorbance value, since relatively large-sized particles appeared in both the formulations with pH 4.5 and pH ≥ 6.0 during the 50 °C accelerated test, it was revealed that pH 4.5 and pH ≥ 6.0 can be excluded from the detection results of appearance and turbidity. According to the DLS results, after standing at 50 °C for 6 days, the protein average particle size (58.0 nm) of the formulation with pH 5.5 is larger than that (44.1 nm) of the formulation with pH 5.0, indicating that in the range of pH 5.0 - 5.5, the lower the pH, the smaller the protein average particle size, which can relieve protein aggregation and improve the stability of the protein colloid. As shown in Figure 1a, according to the SEC results, the amount of polymer increase in each formulation was in the order of CP45 (0.0%) < CP50 (0.3%) < CP55 (0.7%) < CP60 (1.2%) < CP65 (1.6%), indicating that the higher the pH, the more SEC polymer there is.

[0075] SDS-PAGE and CEX were used to examine the changes in protein degradation and charge isomers during the 50°C accelerated test. SDS-PAGE results indicated that formulations at pH ≥ 6.0 had relatively high levels of debris, suggesting that anti-TIGIT antibody was more readily degraded at pH ≥ 6.0. As shown in Figure 1b, CEX results indicated that the decrease in the content of the main peak and basic peak was primarily due to an increase in the content of the acidic peak. The increase in the acidic peak was as follows: CP65 (12.0%) > CP50 (11.2%) ≒ CP55 (10.9%) ≒ CP60 (10.3%) > CP45 (8.4%). The pH 6.5 formulation showed a relatively large increase in the acidic peak, so pH 6.5 was excluded. In summary, the pH range for anti-TIGIT antibody pharmaceutical formulations should be approximately 5.0 to 5.5.

[0076] Example 2. Buffer System Studies Buffer systems are commonly used to maintain the pH of a solution to maintain protein stability. The buffer systems used in this example included acetate buffer, histidine buffer (histidine-histidine hydrochloride buffer), citrate buffer (citric acid-sodium citrate buffer), and citrate-disodium hydrogen phosphate buffer. The buffer system screening study preliminarily examined the effect of various buffer systems on the stability of the anti-TIGIT antibody protein using a high-temperature (50°C) accelerated test. As shown in Table 5, four candidate formulations were designed with a single factor, with a buffer concentration of 15 mM, a pH of 5.5, and a surfactant concentration of 0.02% polysorbate 20.

[0077] [Table 6]

[0078] The study results revealed no significant differences in appearance, protein concentration, pH, or molecular isomer (SEC) between the different formulations on day 0. The initial protein aggregation temperatures for each formulation were as follows: A55 (61.5°C), H55 (61.2°C), CP55 (58.0°C), and C55 (57.1°C). The acetate and histidine buffer formulations had the highest initial protein aggregation temperatures and the best thermal stability. DLS results showed that the initial average protein particle size (8.2 nm) in acetate and histidine buffers was smaller than that in citrate-disodium hydrogen phosphate buffer and citrate buffer (9.1 nm and 9.0 nm, respectively), indicating that acetate and histidine buffers provided good protein colloidal stability.

[0079] After 6 days of incubation at 50°C, visual inspection revealed visible particles in the citrate buffer formulation, while the other formulations showed no visible particles and had a good appearance. Turbidity (A350) absorbance measurements showed that the citrate buffer formulation had the highest absorbance (0.089), indicating that the anti-TIGIT antibody produced relatively large particles in the citrate buffer formulation during the 50°C accelerated test. Therefore, citrate buffer was excluded. DLS results showed that after 6 days of incubation at 50°C, the average protein particle size was CP55 (58.0 nm) > C55 (20.2 nm) > A55 (9.1 nm) ≒ H55 (9.5 nm). Citrate-disodium hydrogen phosphate and citrate buffer formulations produced relatively large particles <1 μm during the 50°C accelerated test. Therefore, citrate-disodium hydrogen phosphate buffer was excluded. As shown in Figure 2a, the SEC polymer content of each formulation was C55 (0.9%) > CP55 (0.7%) > A55 (0.2%) > H55 (0.0%), indicating that acetate and histidine buffers showed the lowest SEC polymer content. Therefore, acetate and histidine buffers are suitable buffer options. SDS-PAGE and CEX results (shown in Figure 2b) revealed no significant differences in the degradation tendency or charge isomer changes of anti-TIGIT antibodies in the four buffers. In summary, acetate or histidine buffers should be used as the buffer for pharmaceutical formulations containing anti-TIGIT antibodies.

[0080] Example 3: Buffer System Concentration Studies The buffer concentration simultaneously affects the buffering capacity of the buffer system and protein stability. While increasing the buffer concentration generally improves the buffering capacity, it may also decrease protein stability, making it necessary to strike a balance between the two. In this example, a high-temperature (50°C) accelerated test was conducted to preliminarily examine the effect of buffer concentration on the stability of anti-TIGIT antibodies. In this example, histidine buffer and 0.02% polysorbate 20 (PS20) were selected for each formulation, with a pH of 5.5. Three single-factor candidate formulations were designed, with buffer concentrations ranging from 5 to 30 mM (mmol / L) (see Table 6) and a protein concentration of 2 mg / mL.

[0081] [Table 7]

[0082] The study results showed that on day 0, there were no significant differences in appearance, protein concentration, pH, or molecular isomer (SEC) between formulations with different buffer concentrations. The initial aggregation temperatures of the protein for each formulation were F-5mM (64.6°C) > F-15mM (61.2°C) > F-30mM (58.8°C), indicating that the lower the buffer concentration, the higher the initial aggregation temperature and the better the thermal stability. DLS results showed that the initial average protein particle size (7.2 nm) of the formulation with a 5mM buffer concentration was smaller than that of the formulations with 15mM and 30mM buffer concentrations (8.2 nm for both). This suggests that formulations with low buffer concentrations had a relatively smaller average protein particle size and better protein colloidal stability compared to formulations with high buffer concentrations.

[0083] After 6 days at 50°C, visual inspection showed that formulations with each buffer concentration had a clean appearance with no visible particles. The turbidity (A350) absorbance values ​​were F-30mM (0.031) > F-15mM (0.021) > F-5mM (0.013), indicating that formulations with higher buffer concentrations exhibited larger particle sizes during the 50°C accelerated test. DLS results showed that after 6 days at 50°C, the average protein particle size was F-30mM (24.9nm) > F-15mM (9.5nm) > F-5mM (7.6nm), indicating that formulations with 30mM buffer exhibited a relatively large number of particles <1µm during the 50°C accelerated test, and therefore the 30mM buffer concentration was excluded. As shown in Figure 3a, the SEC polymer gain for each formulation was as follows: F-30 mM (0.2%) > F-15 mM (0.0%) > F-5 mM (-0.3%). This indicates that the higher the buffer concentration in the formulation, the greater the polymer gain. Therefore, the polymer analysis study revealed that the optimal buffer concentration range for the formulation was 5–15 mM.

[0084] After standing at 50°C for 6 days, according to the results of SDS-PAGE, there was no significant difference in proteolysis among the formulations with different buffer concentrations. According to the results of SEC, the order of the increase in SEC fragments of each formulation was F-30 mM (0.3%) < F-15 mM (0.4%) < F-5 mM (0.5%). Although there was no significant difference among the formulations, it was revealed that as the buffer concentration increased, the amount of fragment increase tended to decrease. Referring to Figure 3b, according to the results of CEX, the decrease in the content of the main peak and the basic peak was mainly represented by the increase in the content of the acidic peak. The order of the increase in the acidic peak of each formulation was F-30 mM (12.0%) > F-15 mM (11.7%) > F-5 mM (10.8%). Thus, it was revealed that as the buffer concentration in the formulation increased, the amount of increase in the acidic peak tended to rise. Therefore, formulations with a buffer concentration of 5 - 15 mM can reduce proteolysis and charge heterogeneity. From the results of the above analysis search, the lower the buffer concentration, the smaller the initial protein average particle size of the anti-TIGIT antibody in the formulation, the tighter the protein structure, and the higher the thermal stability. Therefore, the buffer concentration range of the pharmaceutical formulation containing the anti-TIGIT antibody was selected as 5 - 15 mM.

[0085] Example 4. Study on Surfactants In this example, the effect of surfactant type on the stability of the anti-TIGIT antibody was preliminarily investigated by a high-temperature (50°C) accelerated test. Each formulation in this example selected a histidine buffer at pH 5.5 with a buffer concentration of 15 mM, and investigated the effect of polysorbate 20 (also called polysorbate 20, Tween 20 or PS20) and polysorbate 80 (also called polysorbate 80, Tween 80 or PS80) on the stability of the anti-TIGIT antibody. The information of each formulation is as shown in Table 7.

[0086]

Table 8

[0087] The study results showed that there were no significant differences in appearance, protein concentration, pH, initial average protein particle size, molecular isomer (SEC), and charge isomer (CEX) between the two formulations on day 0. The initial protein aggregation temperatures of the two formulations were 61.2°C and 62.1°C, respectively, with the F-PS80 formulation having a slightly higher initial protein aggregation temperature and better thermal stability.

[0088] After 6 days of incubation at 50°C, visual inspection revealed that both formulations had good appearance, with no visible particles. Turbidity (A350) and absorbance measurements revealed that the F-PS80 formulation had a slightly lower absorbance (0.019) than the F-PS20 formulation (0.021). DLS results, as shown in Figures 4a and 4b, showed that after 6 days of incubation at 50°C, the average protein particle size was F-PS20 (9.5 nm) > F-PS80 (8.6 nm). The protein in the F-PS20 formulation exhibited multiple peaks (Pk1 89.4%), while the protein in the F-PS80 formulation maintained a single peak (Pk1 100%). This suggests that oligomers appeared in the F-PS20 formulation during the 50°C accelerated test, resulting in an increased average protein particle size and a polydisperse particle size distribution.

[0089] After 6 days of incubation at 50°C, SDS-PAGE showed no significant difference in proteolysis between the two formulations, and SEC showed the same increase in SEC fragments (0.4%) for both formulations. CEX showed that the decrease in the main peak and basic peak content was mainly due to an increase in the acidic peak content, with the acidic peak increase for the F-PS20 and F-PS80 formulations being 11.7% and 11.4%, respectively.

[0090] In summary, polysorbate 20 and polysorbate 80 were selected as surfactants for pharmaceutical formulations of anti-TIGIT antibodies. The polysorbate 80 formulation showed a relatively slow increase in protein particle size and a monodisperse particle size distribution, making polysorbate 80 superior to polysorbate 20 in inhibiting anti-TIGIT antibody protein aggregation.

[0091] Example 5 Stabilizer Type Study In this example, a high-temperature (50°C) accelerated test was conducted to preliminarily examine the effect of stabilizers on the stability of the anti-TIGIT antibody. For each formulation in this example, a 15 mM histidine buffer solution at pH 5.5 and a 0.02% polysorbate 20 (PS20) solution were selected, and the effect of sucrose, sorbitol, glycine, proline, arginine hydrochloride, and sodium chloride on the stability of the anti-TIGIT antibody was examined (Table 8).

[0092] [Table 9]

[0093] The results of the study showed that on day 0, there were no significant differences in appearance, protein concentration, pH, initial protein average particle size, and molecular isomer (SEC) between formulations with different stabilizers. The initial protein aggregation temperature of each formulation was F グリシン (62.1℃)>F スクロース (61.2℃)≒F ソルビトール (61.0℃)≒F プロリン (60.8℃)>F アルギニン (56.7℃)>F NaCl (54.6℃) and F アルギニン and F NaCl The formulation has a relatively low initial aggregation temperature and relatively poor thermal stability. スクロース The formulation was found to have a slightly lower initial acidic peak level (22.9%), while the other formulations had initial acidic peak levels of 24.0% to 24.7%.

[0094] After standing at 50°C for 6 days, the appearance showed that F グリシン A small amount of visible particles appeared in the formulation, F アルギニン and F NaCl Although a large amount of visible particles appeared in the formulation, F スクロース , F ソルビトール and F プロリン The formulation had no visible particles and good appearance. The results of turbidity (A350) and absorbance values ​​showed that the absorbance values ​​were: F NaCl (0.197)>F アルギニン (0.039)>F グリシン (0.031)>F プロリン (0.022)≒F スクロース (0.021)≒F ソルビトール (0.018), F NaCl , F アルギニン and F グリシン The formulations all showed relatively large particle sizes during the 50°C accelerated test. Therefore, NaCl, arginine, and glycine can be excluded based on the appearance and turbidity results. After 6 days at 50°C, the average protein particle size was, in order, F スクロース (9.5 nm) ≒ F ソルビトール (9.2nm)>F プロリン (8.9 nm). Referring to FIG. 5a, the amount of SEC polymer increase for each formulation is NaCl (0.5%)>F アルギニン (0.4%)>F グリシン (0.1%) ≒ F プロリン (-0.1%) ≒ F スクロース (0.0%) ≒ F ソルビトール (0.0%), F NaCl and F アルギニン The protein polymer in the formulation increased relatively much, but F プロリン , F スクロース and F ソルビトール It was found that none of the polymers in the formulations increased significantly.

[0095] After standing at 50°C for 6 days, SDS-PAGE showed that F グリシン , F アルギニン and F NaClThe formulations showed relatively high levels of proteolysis. SEC results showed that each formulation showed an increase in SEC fragments of 0.3% to 0.5%. Referring to Figure 5b, the CEX results showed that the increase in the acidic peak of each formulation was in the order of F. グリシン (14.1%)>F スクロース (11.7%) ≒ F ソルビトール (11.0%) ≒ F プロリン (10.5%)>F NaCl (7.9%)≒F アルギニン (7.7%), F グリシン Glycine was excluded because the formulation showed the greatest increase in acidic peak components. Therefore, based on the results of the proteolysis and charge heterogeneity studies, sucrose, sorbitol, and proline were selected as suitable stabilizers.

[0096] In summary, sucrose, sorbitol, and proline can all significantly suppress the aggregation and degradation of anti-TIGIT antibody proteins and maintain protein stability, and therefore, sucrose, sorbitol, or proline is selected as the stabilizer for pharmaceutical formulations containing anti-TIGIT antibodies, with sucrose being preferred.

[0097] Example 6. Screening of pH values ​​and stabilizer concentrations In this example, acetate buffer was used, and a 23-point full factorial experiment (3 factors, 2 levels) including one center point was conducted. The pH, sucrose, and polysorbate 80 concentrations determined in the previous example were used as factors, as shown in Table 9. The pH values ​​were 4.8, 5.3, and 5.8, respectively; the sucrose concentrations were 1.70%, 5.85%, and 10.00%, respectively; and the polysorbate 80 concentrations were 0.01%, 0.03%, and 0.05%, respectively. As shown in Table 10, a total of nine candidate formulations were selected in this example. The basic physicochemical properties and protein stability data of each formulation were examined through accelerated testing at high temperature (40°C), and the preferred pH, polysorbate 80, and sucrose concentrations for maintaining the stability of the anti-TIGIT antibody in each formulation were further screened.

[0098] [Table 10]

[0099] [Table 11]

[0100] The anti-TIGIT antibody protein was concentrated by ultrafiltration, desalted, and adjusted for protein concentration. Stabilizers, surfactants, and buffer solutions were then added according to the experimental design to prepare nine target formulations. The formulation samples were filtered through a 0.22 μm disposable sterile filter in a biological safety cabinet, and 0.6 mL of each sample was dispensed into 3 mL sterile vials under sterile conditions. Each vial was fitted with a 13 mm rubber stopper and a 13 mm aluminum-plastic composite cap. Each formulation was sampled and analyzed according to the experimental design shown in Table 11.

[0101] [Table 12]

[0102] The study results showed that on day 0, there were no significant differences between the different formulations in appearance, protein concentration, charge isomer (CEX), SEC initial fragment content, or intact IgG content. There were significant differences between the formulations in protein aggregation temperature (Tagg), initial protein average particle size, and initial polymer content. As shown in Table 12, after 28 days of incubation at 40°C, there were significant differences between the different formulations in SEC polymer gain, DLS protein average particle size gain, DLS Pk1% drop (aggregation), SEC fragment gain, intact IgG drop (degradation), and acidic peak increase (charge heterogeneity).

[0103] [Table 13] [Table 14]

[0104] The above experimental data was imported into JMP (registered trademark) 15.0.0 software, and multiple linear regression and binomial equation fitting were performed for each factor using the standard least squares method. The analysis results are shown in Table 13. The model P values ​​for the initial protein mean particle size, measured pH, SEC polymer increase, DLS protein mean particle size increase, and DLS Pk1% decrease were all less than 0.1, indicating that the selected model was reliable and statistically significant, and its adjusted coefficient of determination (R 2 ) are all greater than 0.95, suggesting a good fit between the model and the experimental values, and the regression model can be used to analyze and predict the experimental results instead of the true test points.

[0105] [Table 15]

[0106] Referring to Table 14, the model analysis results show that the pH value and sucrose concentration have a significant positive effect on the initial average protein particle size, i.e., the higher the pH value and sucrose concentration, the larger the initial average protein particle size; the pH value has a significant positive effect on the increase in polymer, i.e., the higher the pH, the greater the increase in polymer; the polysorbate 80 concentration has a significant negative effect on the increase in DLS average protein particle size and the decrease in Pk1%, i.e., the higher the polysorbate 80 concentration, the smaller the increase in DLS particle size and the decrease in Pk1%; but the sucrose concentration has a relatively significant positive effect on the increase in DLS particle size and the decrease in Pk1%, i.e., the higher the sucrose concentration, the greater the increase in DLS particle size and the decrease in Pk1%.

[0107] [Table 16] [Table 17]

[0108] From the above experimental results, as shown in Table 15, each response factor was predicted and profiled using JMP (registered trademark) 15.0.0 software, and the importance of any response factor with a model P value of less than 0.1 was set to 0.2.

[0109] [Table 18]

[0110] As shown in Figure 6, the results of the intention profiler showed that when the pH was 4.8 to 5.1, the intention to prepare gradually increased, and when the pH was 5.1 (corresponding to the measured pH of 5.4), the intention was at its maximum. When the pH was greater than 5.1 (corresponding to the measured pH of 5.4), the intention to prepare gradually decreased. As the sucrose concentration decreased, the intention to prepare tended to increase. When the sucrose concentration was 2%, the intention to prepare approached its maximum value. As the polysorbate 80 concentration increased, the intention to prepare tended to increase, but when the polysorbate 80 concentration was in the range of 0.01 to 0.05%, the intention to prepare was relatively large. In summary, the pharmaceutical formulation containing anti-TIGIT antibody was selected to have a pH range of 4.8 to 5.4 (corresponding to an actual pH of 5.1 to 5.7), a sucrose concentration of 2% to 6%, and a polysorbate 80 concentration of 0.01 to 0.05%. However, the results of the intention profiler revealed that the preferred pH and concentrations of each component were a theoretical pH of 5.1, i.e., an actual pH of 5.4, a sucrose concentration of 2%, and a polysorbate 80 concentration of 0.03%.

[0111] Example 7. Study of component concentrations 7.1 Sucrose concentration studies The target formulation was prepared according to the formulation information shown in Table 16, and samples were sampled and detected according to the experimental design shown in Table 17. Shaking tests and freeze-thaw tests were conducted to investigate and verify that a sucrose concentration in the formulation ranging from 2% to 6% contributes to the stability of the anti-TIGIT antibody protein.

[0112] [Table 19]

[0113] [Table 20]

[0114] Without freeze-thawing, all formulations showed no significant differences in basic physicochemical properties (appearance, protein concentration, pH), molecular isomers (SEC, CE-SDS), charge isomers (CEX), or subvisible particle (SVP) levels. As shown in Table 18, the initial protein particle size was as follows: Formulation F1 (6.7 nm), Formulation F3 (6.9 nm), and Formulation F4 (7.3 nm). Figures 7a, 7b, 7c, 7d, and 7e show that after 10 freeze-thaw cycles at temperatures ranging from -20°C to room temperature, there were no significant changes in protein appearance, subvisible particle levels, or purity (SEC, CEX, and CE-SDS) among the formulations.

[0115] [Table 21] [Table 22] [Table 23]

[0116] In summary, after freezing and thawing 10 times at temperatures ranging from -20°C to room temperature, there was no significant change in the purity of the anti-TIGIT antibody protein or the number of particles of submicron or larger size in each formulation. Therefore, the anti-TIGIT antibody protein has good freeze-thaw stability in formulations with sucrose concentrations ranging from 2% to 6%. At the same time, taking into account the results of the initial protein average particle size and the results of the stabilizer concentration screening studies in Examples 5 and 6, a sucrose concentration of 2% is preferred.

[0117] 7.2 Polysorbate 80 concentration studies The target formulation was prepared according to the formulation information in Table 19, and samples were sampled and detected according to the experimental design in Table 20. Shaking tests and repeated freeze-thaw tests were conducted to verify that a polysorbate 80 concentration in the formulation ranging from 0.03% to 0.05% contributes to the stability of the anti-TIGIT antibody protein.

[0118] [Table 24]

[0119] [Table 25]

[0120] As shown in Table 21, on day 0, there were no significant differences between the two formulations in basic physicochemical properties (appearance, protein concentration, pH, osmolality), initial protein particle size, molecular isomers (SEC, CE-SDS), charge isomers (CEX), and subvisible particle (SVP) levels. After 28 days of shaking at 25°C and 200 rpm, both formulations had no visible particles and good appearance. The 0.05% polysorbate 80 formulation had slightly fewer subvisible particles (SVPs) than the 0.03% polysorbate 80 formulation, but the difference was not significant. Furthermore, there were no significant changes in the average protein particle size or purity (SEC, CEX, and CE-SDS) between the two formulations. Therefore, the results of the aggregation, degradation, and charge heterogeneity analyses indicated that both 0.03% and 0.05% polysorbate 80 concentrations could maintain the stability of the TIGIT antibody protein.

[0121] [Table 26] [Table 27]

[0122] As shown in Table 18 and Figures 7f-7j above, after 10 cycles of freezing and thawing at temperatures ranging from -20°C to room temperature, there were no significant changes in the basic physicochemical properties of the protein (appearance, protein concentration, pH, osmotic pressure), subvisible particle (SVP) level, average protein particle size, and purity (SEC, CEX, and CE-SDS) of each formulation. The results of the freeze-thaw study indicated that the anti-TIGIT antibody protein in each formulation had good freeze-thaw stability. Studies of protein polymers, degradation, and charge heterogeneity revealed that both 0.03% and 0.05% polysorbate 80 concentrations could maintain the stability of the anti-TIGIT antibody protein, but the preferred concentration of polysorbate 80 was 0.03%.

[0123] Example 8. Optimization of buffer concentration and buffer system Example 3 of the present application preliminarily confirmed that the appropriate buffer concentration range is 5 to 15 mM, and that the lower the buffer concentration, the better the protein stability. However, during the study of sucrose and polysorbate concentrations in the above example, the applicant found that if a 5 mM acetate buffer system was selected, pH drift would occur after the formulation was concentrated and exchanged using a centrifuge tube. For example, the pH increased by 0.1 to 0.2 in Table 23. Because pH drift increases the difficulty of controlling the concentration and exchange process, this example investigated the type and concentration of buffer that would prevent pH drift.

[0124] 8.1 Optimization of buffer concentration In this example, as shown in Table 22, the formulations were selected to have an acetate buffer system at pH 5.1 with buffer concentrations of 10, 15, 20, and 25 mmol / L, a protein concentration of 20.0 mg / mL, 3.42% sucrose, and 0.02% polysorbate 80 (PS80), and four candidate formulations were designed for each single factor.

[0125] [Table 28]

[0126] As shown in Table 23, after the formulations containing acetate buffer (buffer concentrations are 10, 15, 20, and 25 mM) were concentrated and buffer-exchanged using centrifuge tubes, the pH increased by 0.1 - 0.2, and the lower the buffer concentration, the greater the increase in pH. It was revealed that increasing the buffer concentration in the acetate buffer does not reduce the phenomenon of pH drift after concentrating and buffer-exchanging the formulation. On day 0, there were no significant differences in the appearance, protein concentration, molecular isomers (SEC, CE-SDS), and charge isomers (CEX) of each formulation. The initial aggregation temperature Tagg was in the order of F1 - 10 mM acetic acid (62.9 °C) > F2 - 15 mM acetic acid (62.1 °C) > F3 - 20 mM acetic acid (61.4 °C) > F4 - 25 mM acetic acid (60.8 °C), and the initial average protein particle size was in the order of F1 - 10 mM acetic acid (5.2 nm) < F2 - 15 mM acetic acid (6.1 nm) < F3 - 20 mM acetic acid (6.7 nm) < F4 - 25 mM acetic acid (7.3 nm).

[0127] After the above formulations were left standing at 40 °C for 28 days, all formulations had no visible particles, had good appearance, and there were no significant differences in the subvisible particle (SVP) level. The average protein particle size was in the order of F1 - 10 mM acetic acid (5.7 nm) < F2 - 15 mM acetic acid (6.4 nm) < F3 - 20 mM acetic acid (7.0 nm) < F4 - 25 mM acetic acid (7.6 nm).

[0128] As shown in Figures 8a and 8b, according to the results of CE-SDS and SEC fragments, it was revealed that there were no significant differences in purity among the formulations. As shown in Figure 8c, according to the results of CEX, the decrease in the content of the main peak and basic peak was mainly represented by the increase in the acidic peak content, and it was revealed that there were no significant differences in the increase amount of the acidic peak of each formulation. At the same time, combined with the study of the buffer concentration in Example 3, the buffer concentration of the formulation containing the anti-TIGIT antibody may be 10 mM - 15 mM, preferably 10 mM or 15 mM, and from the results such as protein polymers, fragments, and average particle size, acetate buffer may be used as the buffer for the formulation.

[0129]

Table 29

[0130] 8.2 Optimization of buffer systems Considering that the phenomenon of pH drift occurs after acetate buffer formulation is concentrated and exchanged, and that pH drift increases the difficulty of controlling the concentration and exchange process, the applicant optimized the buffer system to obtain a buffer that does not have pH drift.

[0131] In this example, the formulation was selected to contain 10 mM acetate or histidine buffer at pH 5.1, a protein concentration of 20.0 mg / mL, 3.42% sucrose, and 0.02% polysorbate 80 (PS80), and two candidate formulations were designed for each single factor (see Table 24).

[0132] [Table 32]

[0133] As shown in Table 25, the pH of the acetate buffer (10 mM buffer concentration) formulation increased by 0.2 after concentration / solution exchange, while the pH of the histidine buffer (10 mM buffer concentration) formulation remained unchanged after concentration / solution exchange. In the high-temperature accelerated test, on day 0, there were no significant differences between the formulations in terms of appearance, protein concentration, initial average protein particle size, molecular isomers (SEC, CE-SDS), or charge isomers (CEX). The initial protein aggregation temperature (Tag) was F1-acetate buffer (62.9°C) > F2-histidine buffer (60.9°C). The Tagg temperature of the acetate buffer formulation was slightly higher than that of the histidine buffer formulation, but the difference was not significant. This suggests that 10 mM histidine buffer is beneficial for maintaining the pH stability of anti-TIGIT antibody formulations and, at the same time, maintaining the stability of the anti-TIGIT antibody protein.

[0134] As shown in Table 25, after 28 days of storage at 40°C, there were no significant differences in appearance, subvisible particle level, average protein particle size, or SEC polymer between the formulations. At the same time, as shown in Table 25 and Figure 8d, the results of CE-SDS and SEC fragment analysis revealed no significant differences between the different formulations. Therefore, based on the results of protein polymer and degradation analysis, the buffer solution can be a 10 mM acetate buffer or a histidine buffer.

[0135] As shown in Table 25 and Figure 8e, the CEX results showed that the decrease in the contents of the main peak and basic peak was mainly due to an increase in the content of the acidic peak, with the increase in the acidic peak for each formulation being F1-10 mM acetate buffer (12.9%) > F2-10 mM histidine buffer (10.9%), and the histidine buffer formulation produced fewer acidic peaks than the acetate buffer. Therefore, the charge heterogeneity analysis results showed that histidine buffer is superior to acetate buffer in reducing protein charge heterogeneity.

[0136] In summary, both acetate buffer and histidine buffer can maintain the stability of anti-TIGIT antibodies and suppress the production of acidic peaks. Therefore, an acetate buffer or histidine buffer with a concentration of 10 mM to 15 mM may be selected as the buffer for a formulation containing an anti-TIGIT antibody. However, since histidine buffer formulations do not cause pH drift, the buffer is preferably a histidine buffer.

[0137] [Table 33] [Table 34] [Table 35]

[0138] 8.3 Optimization of pH value Considering the effects of pH and pH drift factors determined in the above examples, further investigation of the stability of anti-TIGIT antibody protein formulations in the pH range of 5.1 to 5.7 is warranted. In this example, the preferred formulation from the above examples, i.e., 20 mg / mL anti-TIGIT antibody protein, 10 mM histidine buffer, 20 mg / mL (2%) sucrose, and 0.3 mg / mL (0.03%) polysorbate 80, was selected. Three candidate formulations were designed for each single factor (see Table 26). The stability of the anti-TIGIT antibody protein at different pH values ​​was evaluated using a high-temperature (40°C) accelerated test. Sampling was performed according to the experimental design in Table 27.

[0139] [Table 36]

[0140] [Table 37]

[0141] As shown in Table 28, on day 0, the formulations with different pH levels appeared as a colorless, slightly opalescent liquid with no visible particles, relatively small protein particle sizes, relatively low SEC polymer, debris, and acidic peak contents, and relatively high intact antibody and heavy chain content levels.

[0142] After 28 days at 40°C, the formulations with different pH levels remained colorless and slightly opalescent, with no visible particles. FlowCam results revealed no significant change in the subvisible particles. As shown in Table 28 and Figures 8f and 8g, compared to day 0, the average protein particle size increased only 0.1-0.2 nm, indicating a modest increase in particle size. Pk1 remained at 100%, and the SEC polymer increased by 0.3-0.5%, with only a slight increase in the polymer. Therefore, the analysis of average protein particle size and polymer indicated that the anti-TIGIT antibody protein in each formulation exhibited good stability at pHs of 5.1, 5.4, and 5.7, with the anti-TIGIT antibody protein exhibiting the best stability at pH 5.4.

[0143] As shown in Table 28 and Figures 8h and 8i, the results of CE-SDS and icIEF revealed that the formulations with pH 5.1, 5.4, and 5.7 showed relatively little degradation of the anti-TIGIT antibody protein and little increase in the acidic peak, and the formulation with pH 5.4 showed the least degradation of the anti-TIGIT antibody protein and little increase in the acidic peak. Therefore, the results of protein degradation and charge heterogeneity analysis showed that the anti-TIGIT antibody protein in each formulation had good stability in the formulations with pH 5.1, 5.4, and 5.7, with the pH of 5.4 showing the best stability of the anti-TIGIT antibody protein.

[0144] In summary, the anti-TIGIT antibody protein in the above formulation has relatively excellent stability within the pH range of 5.1 to 5.7, but the protein stability is best at pH 5.4.

[0145] [Table 38] [Table 39]

[0146] Discussion of Results: By combining the pH screening studies, stabilizer type and concentration studies, buffer type and concentration screening studies, and surfactant screening studies from the above examples, we obtained a formulation capable of maintaining the stability of the anti-TIGIT antibody. In the study of stabilizer types, sucrose, sorbitol, and proline were all found to be capable of maintaining the stability of the anti-TIGIT antibody. Considering the risk of sorbitol crystallizing during long-term frozen storage of the original solution and the relatively high cost of proline as an additive for pharmaceutical formulations, and taking into account the results from the above examples, sucrose was determined to be the preferred stabilizer for the formulation of the present invention, as it was able to maintain the stability of the anti-TIGIT antibody in the formulation relatively well. Furthermore, the anti-TIGIT antibody exhibited good freeze-thaw stability at sucrose concentrations ranging from 2% to 6%, with the initial average particle size of the anti-TIGIT antibody being best at a sucrose concentration of 2%. In buffer screening, both acetate buffer and histidine buffer (histidine-histidine hydrochloride buffer) were found to be able to maintain the stability of the anti-TIGIT antibody. However, taking into account the pH stability factor, histidine buffer was found to be the preferred buffer. In buffer concentration screening studies, the selectable buffer concentration range was 5 to 15 mM, with 10 mM being the preferred concentration. In combination with the buffer and its concentration, a pH value in the range of 5.1 to 5.7 was found to be able to maintain the stability of the anti-TIGIT antibody protein, and the anti-TIGIT antibody was most stable at a pH of 5.4. In surfactant screening studies, both polysorbate 20 and polysorbate 80 were found to be able to maintain the stability of the anti-TIGIT antibody protein in pharmaceutical formulations. However, compared with polysorbate 20, polysorbate 80 caused the anti-TIGIT antibody protein to increase in particle size more slowly and exhibited a more monodisperse particle size distribution, further contributing to the stability of the anti-TIGIT antibody. At the same time, the selectable concentrations of polysorbate 20 or 80 are 0.03% to 0.05%, with 0.03% and 0.05% being preferred, and 0.03% being preferred.To summarize the studies in the above examples, preferred forms of anti-TIGIT antibody formulations are shown in Table 29, and preferred forms of anti-TIGIT antibody formulations are shown in Table 30.

[0147] [Table 40]

[0148] [Table 41]

[0149] The preferred formulations of anti-TIGIT antibody protein shown in Table 30 were subjected to high temperature (40°C) accelerated testing to verify the stability of the anti-TIGIT antibody protein in these formulations, and the study conditions and detection methods are as shown in Table 31.

[0150] [Table 42]

[0151] On day 0, the formulation appeared as a colorless, slightly opalescent liquid with no visible particles, very low polymer and debris levels (both 0.1%), a relatively small average protein particle size (5.4 nm), a relatively low acidic peak content (17.6%), and a relatively high intact IgG level (98.2%).

[0152] After 28 days of incubation at 40°C, the formulation remained a colorless, slightly opalescent liquid with no visible particles, as shown in Table 32 and Figures 9a-9b. FlowCam results revealed no significant increase in subvisible particles. Compared to day 0, the average protein particle size increased by only 0.2 nm, a modest increase in particle size, and the particle size distribution remained single-peaked. The SEC polymer increased by 0.2%, but the increase in the amount of polymer was relatively small. Therefore, aggregation analysis indicated that the anti-TIGIT antibody had good stability in this formulation. As shown in Table 32 and Figures 9c-9d, after 28 days of incubation at 40°C, the SEC debris increased by 0.3%, the CE-SDS analysis showed a 1.4% decrease in intact IgG, and the icIEF analysis showed a 12.5% ​​increase in the acidic peak, with the main peak and basic peak decreasing by 9.2% and 3.4%, respectively.

[0153] Overall, the anti-TIGIT antibody protein exhibited relatively little degradation and little increase in the acidic peak in the above formulation, demonstrating good stability. In summary, the results of protein degradation and charge heterogeneity analysis demonstrated that the anti-TIGIT antibody protein exhibited good stability in the above formulation.

[0154] [Table 43] [Table 44]

[0155] The above anti-TIGIT antibody formulation was frozen and stored at -40°C for at least 24 hours, and then subjected to three repeated freeze-thaw cycles and long-term frozen storage under the repeated freeze-thaw conditions shown in Table 33 and the long-term frozen storage conditions shown in Table 34, respectively, and the appearance, protein concentration, pH, average protein particle size, Flowcam, SEC, icIEF, and CE-SDS were detected.

[0156] [Table 45]

[0157] [Table 46]

[0158] Referring to Table 35, after the anti-TIGIT antibody formulation was frozen and thawed three times, the appearance of the formulation remained a colorless, slightly opalescent liquid, with no visible foreign matter or particles. The protein concentration and pH value were stable, and there was no significant increase in the average particle size of the protein, no significant increase in SEC polymer, and no significant increase in SEC fragments. The icIEF results showed no significant changes in the acidic peak, main peak, and basic peak. The CE-SDS results showed no significant change in NR-MP%, and no significant trend in the Flowcam particle count was observed.

[0159] [Table 47]

[0160] Referring to Table 36, after 18 months of long-term frozen storage of the anti-TIGIT antibody formulation, the appearance of the formulation remained a colorless, slightly opalescent liquid, with no visible foreign matter or particles. The protein concentration and pH value were stable, there was no significant increase in the average particle size of the protein, no significant increase in SEC polymer, and no significant increase in SEC fragments. The icIEF results showed no significant changes in the acidic peak, main peak, and basic peak. The CE-SDS results showed no significant changes in NR-MP%, NGHC, and R-MP, and no significant trend in the Flowcam particle count was observed.

[0161] Therefore, the pharmaceutical preparations containing the anti-TIGIT antibody provided by the present invention can maintain the stability of the anti-TIGIT antibody during long-term frozen storage and freeze-thawing.

[0162] [Table 48] [Table 49]

[0163] After reading the above content of the present invention, those skilled in the art will be able to make various changes and modifications to the present invention, but it should be understood that these equivalents or equivalent forms are also included in the scope limited by the claims attached hereto.

Claims

1. (i) an anti-TIGIT antibody that is a single domain antibody whose heavy chain variable region comprises or consists of the amino acid sequence of SEQ ID NO: 1; (ii) 5-15 mM histidine buffer; (iii) 2% (w / v) to 6% (w / v) sucrose; and (iv) 0.01% (w / v) to 0.05% (w / v) of polysorbate 80, wherein the pH value of the pharmaceutical preparation is 5.1 to 5.7; Pharmaceutical preparations.

2. the concentration of the anti-TIGIT antibody is 15 to 25 mg / mL; The pharmaceutical formulation of claim 1.

3. The concentration of the anti-TIGIT antibody is 20 mg / mL. The pharmaceutical formulation according to claim 1 or 2.

4. The full-length heavy chain of the anti-TIGIT antibody comprises or consists of the amino acid sequence of SEQ ID NO:

2. The pharmaceutical formulation according to any one of claims 1 to 3.

5. 20 mg / mL of anti-TIGIT antibody; 10 mM histidine buffer; 2% (w / v) sucrose; 0.03% (w / v) polysorbate 80, wherein the pH value of the pharmaceutical preparation is 5.1 to 5.7; The pharmaceutical formulation according to any one of claims 1 to 4.

6. The pH value of the pharmaceutical preparation is preferably 5.4; The pharmaceutical formulation according to any one of claims 1 to 5.

7. for use in treating cancer in a subject; The pharmaceutical formulation according to any one of claims 1 to 6.

8. the cancer is a solid tumor or a hematological tumor; The pharmaceutical formulation of claim 7.

9. The cancer is selected from mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, synovial sarcoma, thymic cancer, cervical cancer, endometrial cancer, gastric tumor, melanoma, bile duct cancer, head and neck cancer, lymphoma, blood cancer, and combinations thereof; 9. The pharmaceutical formulation according to claim 7 or 8.

10. The pharmaceutical formulation is administered in combination with other drugs. The pharmaceutical formulation according to any one of claims 7 to 9.

11. the other drug is an anti-PD-1 antibody and / or an anti-PD-L1 antibody; The pharmaceutical formulation of claim 10.

12. the other drug is an anti-PD-1 antibody; 12. The pharmaceutical formulation according to claim 10 or 11.

13. Use of the pharmaceutical formulation of any one of claims 1 to 6 for the preparation of a medicament for treating cancer in a subject.

14. the cancer is a solid tumor or a hematological tumor; 14. The use according to claim 13.

15. The cancer is selected from mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, synovial sarcoma, thymic cancer, cervical cancer, endometrial cancer, gastric tumor, melanoma, bile duct cancer, head and neck cancer, lymphoma, blood cancer, and combinations thereof; 15. Use according to claim 13 or 14.

16. The pharmaceutical formulation is administered in combination with other drugs. Use according to any one of claims 13 to 15.

17. the other drug is an anti-PD-1 antibody and / or an anti-PD-L1 antibody; 17. The use according to claim 16.

18. the other drug is an anti-PD-1 antibody; 18. Use according to claim 16 or 17.

19. A method for reducing tumor burden in a patient and / or for treating a tumor in a patient, comprising administering to the patient an effective dose of a pharmaceutical formulation according to any one of claims 1 to 6. method.

20. The tumor is a solid tumor or a hematological tumor.

20. The method of claim 19.

21. The tumor is selected from mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, synovial sarcoma, thymic cancer, cervical cancer, endometrial cancer, gastric tumor, bile duct cancer, head and neck cancer, lymphoma, blood cancer, and combinations thereof; 21. The method of claim 19 or 20.

22. The pharmaceutical formulation is administered in combination with other drugs. The method according to any one of claims 19 to 21.

23. the other drug is an anti-PD-1 antibody and / or an anti-PD-L1 antibody; 23. The method of claim 22.

24. the other drug is an anti-PD-1 antibody; 24. The method of claim 22 or 23.

25. The pharmaceutical formulation according to any one of claims 1 to 6, Drug delivery devices.

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