Subcutaneous anti-HER2 antibody-containing pharmaceutical composition

Abstract

The present invention relates to stable liquid pharmaceutical formulations containing high concentrations of trastuzumab, pertuzumab, or a mixture thereof, for convenient subcutaneous administration. The formulations of the present invention can be administered for the treatment of cancers such as breast cancer and metastatic gastric or gastroesophageal junction adenocarcinoma.

Description

[Technical Field]

[0001] Incorporate elements from an ASCII text file by reference.

[0002] This application incorporates the following sequence listing by reference: File name: P4330-PCT_seq listing, 6 KB, Creation date: April 14, 2023.

[0003] Cross-reference of related applications This application claims the benefits of U.S. Provisional Applications No. 63 / 333,930 and No. 63 / 333,935, filed on 22 April 2022, which are incorporated in their entirety by reference. [Background technology]

[0004] The HER2 (human epidermal growth factor receptor 2) proto-oncogene encodes a transmembrane protein. The HER2 protein is overexpressed in many adenocarcinomas, including breast cancer (e.g., ductal carcinoma), gastric cancer (e.g., gastric adenocarcinoma), and gastroesophageal cancer (e.g., gastroesophageal junction adenocarcinoma). Overexpression of the HER2 protein can potentially be utilized by targeting HER2-specific monoclonal antibodies (mAbs), thereby enabling the treatment of diseases such as HER2-positive breast and gastric cancer. Trastuzumab is a recombinant humanized monoclonal antibody that targets HER2. After binding to HER2 on the surface of tumor cells, trastuzumab induces antibody-dependent cell-mediated cytotoxicity in tumor cells overexpressing the HER2 protein.

[0005] The use of monoclonal antibodies in medicine is increasing year by year. In many cases, such monoclonal antibodies have been administered via intravenous (IV) routes. Most therapeutic monoclonal antibodies (mAbs) have been administered by conventional intravenous (IV) administration. IV administration requires patients to travel long distances to a medical facility where trained healthcare professionals are available to administer the IV infusion and determine the appropriate dose and infusion rate. Clinical administration is expensive and inconvenient for patients.

[0006] Furthermore, administering the appropriate amount of low-concentration monoclonal antibody via IV infusion is typically time-consuming (approximately 90 minutes) and inconvenient for patients (requiring visits to well-staffed clinics).

[0007] An alternative route of administration is subcutaneous (SC) administration, which allows patients to conveniently and economically self-administer the medication at home. This is more convenient for patients and leads to improved compliance and cost reduction for healthcare providers.

[0008] However, the amount of monoclonal antibody that can be injected via the subcutaneous route is limited. Therefore, a liquid formulation with suitable solubility and stability is required, as the amount is less than 1 to 2 mL. Therefore, a stable liquid pharmaceutical preparation containing a high concentration of antibodies such as trastuzumab was used. target The need arose to address the administration of SC for therapeutic purposes. [Overview of the project]

[0009] One embodiment of the present invention is a pharmaceutical composition comprising an anti-human epidermal growth factor receptor-2 (HER2) protein, a buffer with a pH of 4.6 to 6.5, one or more stabilizers, and an isotonic agent.

[0010] One embodiment of the present invention is a pharmaceutical composition comprising an anti-HER2 antibody, sucrose, and methionine.

[0011] In yet another embodiment of the present invention, the present invention relates to a pharmaceutical composition comprising an active pharmaceutical ingredient including an anti-HER2 antibody, methionine, and glutamic acid.

[0012] According to one embodiment of the present invention, the anti-HER2 antibody comprises trastuzumab or pertuzumab.

[0013] According to one embodiment of the present invention, the anti-HER2 antibody is trastuzumab or pertuzumab, and the trastuzumab or pertuzumab in the pharmaceutical composition has a concentration of 80 to 150 mg / mL.

[0014] According to one embodiment of the present invention, the stabilizer includes methionine, glutamic acid, arginine, N-α-acetylarginine, proline, glycine, lysine, glutamine, or a combination thereof.

[0015] According to one embodiment of the present invention, the methionine in the pharmaceutical composition has a concentration of 5 to 15 mM, and the glutamic acid has a concentration of 5 to 15 mM.

[0016] According to one embodiment of the present invention, the pharmaceutical composition further comprises an isotonic agent selected from trehalose, sucrose, mannitol, or sorbitol.

[0017] According to one embodiment of the present invention, the isotonic agent in the above pharmaceutical composition is sucrose.

[0018] According to one embodiment of the present invention, the sucrose has a concentration of 150 to 300 mM.

[0019] According to one embodiment of the present invention, the pharmaceutical composition substantially does not contain hyaluronidase.

[0020] According to one embodiment of the present invention, the pharmaceutical composition is configured for subcutaneous administration.

[0021] According to one embodiment of the present invention, the pharmaceutical composition has FcγRIII bonds with a relative potency (%) of 93.9 to 107.9 after being stored at a temperature of 5°C ± 3°C for 3 days.

[0022] According to one embodiment of the present invention, the pharmaceutical composition comprises: (a) trastuzumab or pertuzumab at 5 to 150 mg / mL, (b) methionine at 0 to 15 mM, (c) glutamic acid at 0 to 15 mM, (d) polysorbate 80 at 0 to 0.05% (w / v), (e) sucrose at 0 to 300 mM with a pH of 4.6 to 6.5, and (f) an acetate buffer at 5 to 40 mM.

[0023] According to one embodiment of the present invention, the pharmaceutical composition comprises: (a) trastuzumab or pertuzumab at 80 to 150 mg / mL, (b) methionine at 5 to 15 mM, (c) glutamic acid at 5 to 15 mM, (d) polysorbate 80 at 0.01 to 0.03% (w / v), (e) sucrose at 200 to 220 mM, and (f) an acetate buffer at 5 to 40 mM with a pH of 4.6 to 6.5.

[0024] Another embodiment of the present invention is a method for treating cancer in a subject who needs it, which comprises subcutaneously administering a therapeutically effective amount of the pharmaceutical composition to the subject.

[0025] According to one embodiment of the present invention, the cancer is breast cancer.

[0026] According to one embodiment of the present invention, the cancer is metastatic gastric cancer or gastroesophageal junction adenocarcinoma.

[0027] According to one embodiment of the present invention, the pharmaceutical composition is administered once every three weeks.

[0028] Another embodiment of the present invention is a prefilled syringe containing a 3 mL to 7 mL solution. The solution comprises: (a) trastuzumab 、 pertuzumab, or mixtures thereof (b) methionine, and (c) glutamic acid.

[0029] Another embodiment of the present invention is a pre-filled syringe containing 3 mL to 7 mL of solution. The solution comprises (a) trastuzumab or pertuzumab, (b) methionine, (c) glutamic acid, (d) an organic cosolvent, (e) sucrose, and (f) acetate buffer.

[0030] According to one embodiment of the present invention, the trastuzumab or pertuzumab has a concentration of 80 to 150 mg / mL.

[0031] According to one embodiment of the present invention, the methionine has a concentration of 5 to 15 mM, and the glutamic acid has a concentration of 5 to 15 mM.

[0032] According to one embodiment of the present invention, the solution further comprises sucrose.

[0033] Another embodiment of the present invention is a method for treating cancer in a subject requiring such treatment, comprising subcutaneously administering a therapeutically effective amount of the solution from a pre-filled syringe to the subject.

[0034] Another embodiment of the present invention is a method for stabilizing trastuzumab, comprising binding trastuzumab with methionine and glutamic acid in solution. The above composition has an FcγRIII binding affinity of 93.9 to 107.9 relative potency (%) after storage at a temperature of 5°C ± 3°C for 3 days.

[0035] While embodiments are described herein in a manner that enables the writing of a clear and concise specification, it is intended and will be understood that embodiments may be combined or separated in various ways without departing from the invention. For example, it will be understood that all preferred features described herein are applicable to all aspects of the invention described herein. [Brief explanation of the drawing]

[0036] [Figure 1]Figure 1 shows the amino acid sequences of the trastuzumab light chain (SEQ ID NO: 1) and the trastuzumab heavy chain (SEQ ID NO: 2), along with their corresponding domains. [Figure 2] Figure 2. Colloidal stability of high-concentration trastuzumab in different buffers. Scattering constant versus trastuzumab concentration. Figure 2A contains histidine buffer at pH 6.5. Figure 2B contains histidine buffer at pH 6.0. Figure 2C contains citrate buffer at pH 6.0. Figure 2D contains citrate buffer at pH 5.5. Figure 2E contains acetate buffer at pH 5.5, and Figure 2F contains acetate buffer at pH 5.0. [Figure 3] Figure 3 shows the stability of high-concentration trastuzumab in acetate buffer as measured by size exclusion chromatography. Figure 3 shows the antibody profile of high-concentration trastuzumab over 28 days under accelerated thermal stress at 40°C. Figure 3A is the antibody profile under acetate at pH 5.4. Figure 3B is the antibody profile under acetate at pH 5.0. Figure 3C is the antibody profile under acetate at pH 4.6. [Figure 4] Figure 4 shows the stability of high-concentration trastuzumab in acetate buffer by cation exchange chromatography. Figure 4 shows the charge variant profile of high-concentration trastuzumab antibody over 28 days under accelerated thermal stress at 40°C. Figure 4A shows the charge variant profile under acetate at pH 5.4. Figure 4B shows the charge variant profile under acetate at pH 5.0. Figure 4C shows the charge variant profile under acetate at pH 4.6. [Figure 5] Figure 5 shows the long-term stability of trastuzumab formulations as measured by SEC-HPLC analysis. Figure 5 shows the integrity of trastuzumab antibodies up to 24 months under different temperature conditions. Figure 5A shows antibody purity at 5°C. Figure 5B shows antibody purity at 25°C. Figure 5C shows antibody purity at 40°C. Figure 6 shows the long-term stability of trastuzumab formulations as measured by CEX analysis. [Figure 6]Figure 6 shows the primary peak profiles of trastuzumab antibody up to 24 months under different temperature conditions. Figure 6A shows the percentage of primary peaks at 5°C. Figure 6B shows the percentage of primary peaks at 25°C. Figure 6C shows the percentage of primary peaks at 40°C. [Figure 7] Figure 7 shows the stability of trastuzumab formulations as measured by SEC-HPLC analysis. Figure 7 shows the integrity of trastuzumab antibodies up to 3 months at different temperatures. Figure 7A shows the purity of the antibody at 5°C. Figure 7B shows the purity of the antibody at 25°C. Figure 7C shows the purity of the antibody at 40°C. [Figure 8] Figure 8 shows the stability of the trastuzumab formulation as measured by CEX analysis. Figure 8 shows the major peak profiles of the trastuzumab antibody up to 3 months at different temperatures. Figure 8A shows the percentage of major peaks at 5°C. Figure 8B shows the percentage of major peaks at 25°C. Figure 8C shows the percentage of major peaks at 40°C. [Figure 9] Figure 9 shows the stability of the pertuzumab formulation as measured by SEC-HPLC analysis. Figure 9 shows the integrity of the pertuzumab antibody up to 3 months under different temperatures. Figure 9A shows the purity of the antibody at 5°C. Figure 9B shows the purity of the antibody at 25°C. Figure 9C shows the purity of the antibody at 40°C. [Figure 10] Figure 10 shows the stability of pertuzumab formulations as measured by CEX analysis. Figure 10 shows the charge heterogeneity profiles of pertuzumab antibodies up to 3 months under different temperatures. Figure 10A shows the charge heterogeneity profile of sample example 41. Figure 10B shows the charge heterogeneity profile of sample example 42. Figure 10C shows the charge heterogeneity profile of sample example 43. Figure 10D shows the charge heterogeneity profile of sample example 44. [Figure 11] Figure 11. Antitumor activity of trastuzumab under different buffering conditions. Mean + / - SE (mm3) of tumor volume was plotted on the y-axis, and days after tumor transplantation were plotted on the x-axis. PBS buffer control - filled square, IV trastuzumab - filled circle, and high-concentration trastuzumab - filled triangle. [Figure 12] Figure 12. Pharmacokinetic (PK) studies examining the plasma concentration (μg / mL) of trastuzumab versus time in CD-1 mice after subcutaneous or intravenous administration of various formulations of trastuzumab at 10 mg / kg. The high-concentration trastuzumab compound and IV trastuzumab contain the same active pharmaceutical ingredient (API) (trastuzumab). Concentrations are based on median values. G3: Group 3, high-concentration trastuzumab SC. G5: Group 5, IV trastuzumab IV. Figure 12A. Observation time points ranged from 0 to 96 hours. Figure 12B. Observation time points ranged from 0.25 to 1344 hours. [Modes for carrying out the invention]

[0037] The present invention relates to a stable liquid pharmaceutical formulation containing a high concentration of anti-HER2 antibody, including trastuzumab, pertuzumab, or a mixture thereof, for convenient subcutaneous administration of low-volume formulations.

[0038] Anti-HER2 antibody Human epidermal growth factor receptor 2 (HER2) is a tyrosine kinase that binds to the surface of the cell membrane. HER2 proteins are known to be involved in signaling pathways that trigger cell proliferation and differentiation, as well as in cell malignancy associated with overexpression or activity. Overexpression of HER2 proteins may be inhibited using targeted therapeutic agents, including anti-HER2 antibodies. Anti-HER2 antibodies include, but are not limited to, trastuzumab and pertuzumab. Applications of inhibiting HER2 overexpression include, but are not limited to, the use of a mixture of trastuzumab and pertuzumab.

[0039] Trastuzumab Trastuzumab is a recombinant humanized monoclonal antibody that directly targets HER2. The amino acid sequence of the trastuzumab light chain is (SEQ ID NO: 1).

[0040] The amino acid sequence of the trastuzumab heavy chain is (SEQ ID NO: 2). Figure 1 shows the amino acid sequences of the trastuzumab light chain (SEQ ID NO: 1) and the trastuzumab heavy chain (SEQ ID NO: 2), along with their corresponding domains.

[0041] Trastuzumab has the CAS number 180288-69-1.

[0042] Pertuzumab Pertuzumab is a recombinant humanized monoclonal antibody that directly targets HER2. Pertuzumab The amino acid sequence of the light chain is (SEQ ID NO: 3). Pertuzumab The amino acid sequence of the heavy chain is (SEQ ID NO: 4). Pertuzumab has the CAS number 380610-27-5.

[0043] The concentration of trastuzumab or pertuzumab may be 5 to 150 mg / mL, 10 to 150 mg / mL, 20 to 150 mg / mL, 30 to 150 mg / mL, 40 to 150 mg / mL, 50 to 150 mg / mL, 60 to 150 mg / mL, 70 to 150 mg / mL, 80 to 150 mg / mL, 90 to 150 mg / mL, 100 to 150 mg / mL, 110 to 150 mg / mL, 120 to 150 mg / mL, 130 to 150 mg / mL, or 140 to 150 mg / mL. Furthermore, the concentration of trastuzumab may be 95 to 145 mg / mL, 100 to 140 mg / mL, 105 to 135 mg / mL, 110 to 130 mg / mL, 115 to 125 mg / mL, or 120 mg / mL.

[0044] The concentration of trastuzumab or pertuzumab may be freely adjusted within a range that does not substantially adversely affect the stability of the stable liquid pharmaceutical formulation according to the present invention.

[0045] In this specification, “pharmaceutically acceptable salt” means a salt that, within the bounds of sound medical judgment, is suitable for use in contact with human and lower animal tissues without causing excessive toxicity, irritation, or allergic reactions, and that is commensurate with a reasonable benefit / risk ratio. pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19. pharmaceutically acceptable salts of the compounds of the present invention include those derived from appropriate inorganic and organic acids and bases. Examples of pharmaceutically acceptable, non-toxic acid addition salts include inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, glutamic acid, or malonic acid, or salts of amino groups formed using other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include acetic acid, glutamine, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, bicarbonate, boric acid, butyric acid, camphoric acid, camphorsulfonic acid (citric acid, cyclopentanepropionic acid, digluconic acid, dodecyl sulfate, ethanesulfonic acid, formic acid, fumaric acid, glucoheptonic acid, glycerophosphate, gluconic acid, hemisulfate, heptanoic acid, hexanoic acid, hydroiodic acid, 2-hydroxyethanesulfonic acid, lactobionic acid, lactic acid, lauric acid, lauryl Examples include sulfuric acid, malic acid, maleic acid, malonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pectinic acid, persulfate, 3-phenylpropionic acid, phosphoric acid, picric acid, pivalic acid, propionic acid, stearic acid, succinic acid, sulfuric acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, undecanoic acid, and valerates. Pharmaceutically acceptable salts derived from appropriate bases include alkali metals, alkaline earth metals, ammonium and N + (C 1-4Examples include alkyl)4 salts. Typical alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium. Furthermore, pharmaceutically acceptable salts, where appropriate, include non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylic acids, sulfuric acid, phosphoric acid, nitric acid, lower alkyl sulfonic acids, and aryl sulfonic acids.

[0046] Pharmaceutical composition The compounds provided in accordance with the present invention are typically administered in the form of liquid pharmaceutical compositions. The present invention therefore provides pharmaceutical compositions comprising one or more of the compounds described herein, or pharmaceutically acceptable salts or esters, and one or more pharmaceutically acceptable carriers. In this specification, “pharmaceutically acceptable carrier” means a non-toxic carrier, adjuvant, excipient, etc., that does not impair the pharmacological activity of the formulated compound. Pharmaceutically acceptable carriers that may be used with the compounds described herein include, but are not limited to, buffers, isotonic agents, stabilizers, organic cosolvents, penetration enhancers, solubilizers, fillers, diluents, and combinations thereof.

[0047] The pharmaceutical composition of the present invention may be substantially free of hyaluronidase, which is a penetration enhancer. Hyaluronidase includes all commonly known hyaluronidases or modified hyaluronidases such as enzyme proteins having the amino acid sequence of GenBank:AAC70915.1 in humans. The hyaluronidase enzyme may be a glycoprotein such as rHuPH20. The term "substantially free of hyaluronidase" means that, when measured by art-known techniques such as ELISA, hyaluronidase is not detected in the composition, the amount of hyaluronidase is less than 10 units / dose, the amount of hyaluronidase is less than 5 units / dose, or the amount of hyaluronidase is less than 1 unit / dose.

[0048] Pharmaceutical composition: buffering agent The pharmaceutical composition may contain one or more buffers or buffering agents. The buffers may be L-histidine / histidine hydrochloride, sodium citrate / citric acid, L-histidine / acetic acid, phosphate, sodium acetate / acetic acid, acetic acid, or a combination thereof, in concentrations of 2 to 120 mM, 5 to 100 mM, 5 to 90 mM, 5 to 80 mM, 5 to 70 mM, 5 to 60 mM, 5 to 50 mM, 5 to 40 mM, 5 to 30 mM, 5 to 20 mM, 5 to 10 mM, 5 to 35 mM, 10 to 30 mM, 15 to 25 mM, or 20 mM.

[0049] The concentration of one buffer may be 2 to 120 mM, 5 to 100 mM, 5 to 90 mM, 5 to 80 mM, 5 to 70 mM, 5 to 60 mM, 5 to 50 mM, 5 to 40 mM, 5 to 30 mM, 5 to 20 mM, 5 to 10 mM, 5 to 35 mM, 10 to 30 mM, 15 to 25 mM, or 20 mM.

[0050] The buffer can stabilize the pH of the pharmaceutical composition to 4.6 to 6.5, 4.7 to 6.3, 4.8 to 6.1, 4.9 to 5.9, 5.0 to 5.7, 5.0 to 5.4, 5.1 to 5.5, 5.1 to 5.3, 4.6 to 5.4, 4.6 to 5.0, 5.0 to 5.4, 5.2 to 5.6, or 5.2.

[0051] Pharmaceutical composition: Isotonic agent The pharmaceutical composition may contain one or more isotonic agents. The isotonic agents may be, but are not limited to, polyols or sugar derivatives. The isotonic agents may be sucrose, trehalose, mannitol, sorbitol, or a combination thereof.

[0052] The concentration of one isotonic agent may be 20 to 300 mM, 50 to 290 mM, 80 to 280 mM, 110 to 270 mM, 140 to 260 mM, 170 to 250 mM, 180 to 240 mM, 190 to 230 mM, 200 to 220 mM, or 210 mM (7.18% w / v).

[0053] Pharmaceutical composition: Stabilizer The pharmaceutical composition may contain one or more stabilizers. The stabilizers may be methionine (Met), glutamic acid (Glu), arginine (Arg), N-alpha-acetylarginine, proline (Pro), glycine (Gly), lysine (Lys), glutamine (Gln), or combinations thereof such as Met and Glu.

[0054] The concentration of one stabilizer may be less than 50 mM, or 50 mM, 5 to 45 mM, 10 to 40 mM, 15 to 35 mM, 20 to 30 mM, 5 to 40 mM, 5 to 30 mM, 5 to 20 mM, 0 to 20 mM, or 10 mM.

[0055] The concentration of Met may be less than 20 mM, or 20 mM, 3 to 17 mM, 5 to 15 mM, 7 to 13 mM, 0 to 10 mM, or 10 mM.

[0056] The concentration of Arg may be less than 50 mM, or 50 mM, 0 to 50 mM, 5 to 40 mM, 10 to 30 mM, or 15 to 25 mM.

[0057] The concentration of N-alpha-acetylarginine may be less than 10 mM, 10 mM, 0 to 10 mM, or 3 to 7 mM.

[0058] The concentration of Pro may be less than 20 mM, or 20 mM, 0 to 20 mM, 3 to 17 mM, 5 to 15 mM, 7 to 13 mM, or 10 mM.

[0059] The concentration of Gly may be less than 40 mM, 40 mM, 0 to 40 mM, 5 to 35 mM, 5 to 30 mM, 5 to 25 mM, or 5 to 20 mM.

[0060] The concentration of Lys may be less than 20 mM, 20 mM, 0 to 20 mM, 3 to 17 mM, 5 to 15 mM, 7 to 13 mM, or 10 mM.

[0061] The concentration of Gln may be less than 20 mM, 20 mM, 0 to 20 mM, 3 to 17 mM, 5 to 15 mM, 7 to 13 mM, or 10 mM.

[0062] The concentration of Glu may be less than 20 mM, or 20 mM, 0 to 20 mM, 3 to 17 mM, 5 to 15 mM, 7 to 13 mM, or 10 mM.

[0063] Pharmaceutical composition: Organic cosolvent The pharmaceutical composition may contain one or more organic cosolvents, which may be polysorbate 80 (PS80), polysorbate 20 (PS20), poloxamer 188, polyethylene glycol (PEG), propylene glycol, or a combination thereof. In some embodiments, the organic cosolvent may refer to a surfactant.

[0064] The concentration of one organic cosolvent may be less than 0.05% (w / v), or 0.05% (w / v), 0 to 0.05% (w / v), 0.01 to 0.04% (w / v), 0.01 to 0.03% (w / v), or 0.02% (w / v).

[0065] Pharmaceutical composition: Other ingredients The pharmaceutical composition may contain histidine, citrate, phosphate, or a mixture thereof. In one embodiment, the pharmaceutical composition may contain histidine in the buffer of the liquid formulation.

[0066] The pharmaceutical composition may contain a salt. The salt may be, but is not limited to, NaCl, KCl, KBr, NaBr, Na2SO4, NaSCN, K2SO4, etc., or a mixture thereof. The salt concentration may be less than 155 mM or 150 mM, 10 to 145 mM, 20 to 135 mM, 30 to 125 mM, 40 to 115 mM, 50 to 100 mM, 60 to 90 mM, or 70 to 80 mM. Similarly, the salt concentration may be 0.7% (w / v) to 1.1% (w / v).

[0067] The pharmaceutical composition may contain preservatives. These preservatives may include, but are not limited to, octadecyldimethylbenzylammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol, benzyl alcohol, alkylparabens, catechol, resorcinol, cyclohexanol, 3-pentanol, m-cresol, or mixtures thereof.

[0068] The pharmaceutical composition may also contain additives known in the art, within a concentration range that does not substantially adversely affect antibody activity or the usability of the formulation.

[0069] Pharmaceutical composition: combination of ingredients The following is a list of possible component combinations for a formulation containing an anti-HER2 antibody, including trastuzumab, pertuzumab, or a mixture thereof. For example, the formulation may contain trastuzumab or pertuzumab at 120 mg / mL.

[0070] A formulation containing a combination of acetate buffer and sucrose. Preparations containing acetic acid, sucrose, and PS80; preparations containing acetic acid, sucrose, and a combination of methionine and glutamic acid; A preparation containing acetate, sucrose, PS80, and a combination of methionine and glutamic acid. A preparation containing acetate buffer, sucrose, PS80, and methionine. A preparation containing acetate buffer, sucrose, PS80, and glutamic acid. A preparation comprising acetate buffer, histidine, sucrose, methionine, and glutamic acid. A preparation comprising acetate buffer, histidine, sucrose, methionine, and glycine. A preparation containing histidine buffer, trehalose, PS20, and methionine. A formulation containing 20 mM acetate buffer at pH 5.2, 210 mM sucrose, 10 mM methionine, 10 mM glutamic acid, and 0.02% PS80.

[0071] Pharmaceutical composition: properties The following characteristics describe the pharmaceutical formulation of the present invention. Examples of techniques used to measure these characteristics are described in the Examples section below.

[0072] The following list describes liquid pharmaceutical formulations with specific HER2 binding affinity.

[0073] 5± A pharmaceutical liquid formulation exhibiting HER2-binding relative potency (%) of 93.9 to 107.9 when measured using an FcγRIII binding affinity assay after storage at 3°C ​​for 3 days.

[0074] Under accelerated thermal stress conditions 4 0± A liquid pharmaceutical formulation exhibiting HER2-binding relative potency (%) ranging from 90.0 to 118.0, as measured using an FcγRIII binding affinity assay after storage at 2°C for 3 days.

[0075] 5± A pharmaceutical liquid formulation having a relative HER2 binding potency (%) of 93.9 to 107.9 when measured using an FcγRIII binding affinity assay after storage for 3 days at a temperature of 3°C in 20 mM L-histidine buffer at pH 6.0 or 6.5, 20 mM citrate buffer at pH 5.5 or 6.0, or 20 mM acetate buffer at pH 5.0 or 5.5.

[0076] Under accelerated thermal stress conditions 4 0± A pharmaceutical liquid formulation having a relative HER2 binding potency (%) of 90.0 to 118.0 when measured using an FcγRIII binding affinity assay after storage for 3 days at a temperature of 2°C in 20 mM L-histidine buffer at pH 6.0 or 6.5, 20 mM citrate buffer at pH 5.5 or 6.0, or 20 mM acetate buffer at pH 5.0 or 5.5.

[0077] The following list describes pharmaceutical liquid formulations with specific active ingredient content (main peak / monomer %).

[0078] 5± A pharmaceutical liquid formulation containing 95 to 99% of the main component, as measured by SEC-HPLC after storage at 3°C ​​for 28 days.

[0079] In a 20 mM L-histidine buffer at pH 6.0 or 6.5, a 20 mM citrate buffer at pH 5.5 or 6.0, or a 20 mM acetate buffer at pH 5.0 or 5.5, 5 A pharmaceutical liquid formulation containing 95 to 99% of its main components, as measured by SEC-HPLC after storage at a temperature of ℃±3℃ for 28 days.

[0080] 4 0± A pharmaceutical liquid formulation containing 94 to 99% of its main component, as measured by SEC-HPLC after storage at 2°C for 28 days.

[0081] In a 20 mM L-histidine buffer at pH 6.0 or 6.5, a 20 mM citrate buffer at pH 5.5 or 6.0, or a 20 mM acetate buffer at pH 5.0 or 5.5, 4 0± A pharmaceutical liquid formulation containing 94 to 99% of its main component, as measured by SEC-HPLC after storage at 2°C for 28 days.

[0082] The following list describes pharmaceutical liquid formulations that contain a specific amount of high molecular weight component (their retention time is the peak located before the main peak).

[0083] 5± A pharmaceutical liquid formulation containing 0.78 to 2.17% high molecular weight components, as measured by SEC-HPLC after storage at 3°C ​​for 28 days.

[0084] In a 20 mM L-histidine buffer at pH 6.0 or 6.5, a 20 mM citrate buffer at pH 5.5 or 6.0, or a 20 mM acetate buffer at pH 5.0 or 5.5, 5± A pharmaceutical liquid formulation containing 0.78 to 2.17% high molecular weight components, as measured by SEC-HPLC after storage at 3°C ​​for 28 days.

[0085] 4 0± A pharmaceutical liquid formulation containing 0.78 to 4.11% high molecular weight components, as measured by SEC-HPLC after storage at 2°C for 28 days.

[0086] In a 20 mM L-histidine buffer at pH 6.0 or 6.5, a 20 mM citrate buffer at pH 5.5 or 6.0, or a 20 mM acetate buffer at pH 5.0 or 5.5, 4 0± A pharmaceutical liquid formulation containing 94 to 99% of its main component, as measured by SEC-HPLC after storage at 2°C for 28 days.

[0087] The following list describes pharmaceutical liquid formulations that contain specific amounts of low molecular weight components (their retention time is indicated by a peak located after the main peak).

[0088] 5± A pharmaceutical liquid formulation containing 0% low molecular weight components, as measured by SEC-HPLC after storage at 3°C ​​for 28 days.

[0089] In a 20 mM L-histidine buffer at pH 6.0 or 6.5, a 20 mM citrate buffer at pH 5.5 or 6.0, or a 20 mM acetate buffer at pH 5.0 or 5.5, 5± A pharmaceutical liquid formulation containing 0% low molecular weight components, as measured by SEC-HPLC after storage at 3°C ​​for 28 days.

[0090] 4 0± A pharmaceutical liquid formulation containing 1.61 to 2.20% low molecular weight components, as measured by SEC-HPLC after storage at 2°C for 28 days.

[0091] In a 20 mM L-histidine buffer at pH 6.0 or 6.5, a 20 mM citrate buffer at pH 5.5 or 6.0, or a 20 mM acetate buffer at pH 5.0 or 5.5, 4 0± A pharmaceutical liquid formulation containing 1.61 to 2.20% low molecular weight components, as measured by SEC-HPLC after storage at 2°C for 28 days.

[0092] The following list describes pharmaceutical liquid formulations containing specific amounts of major charge variant components.

[0093] 5± A pharmaceutical liquid formulation containing 55.06 to 64.58% of the major species charge variant components, as measured by CIX-HPLC after storage at 3°C ​​for 28 days.

[0094] In a 20 mM L-histidine buffer at pH 6.0 or 6.5, a 20 mM citrate buffer at pH 5.5 or 6.0, or a 20 mM acetate buffer at pH 5.0 or 5.5 5± A pharmaceutical liquid formulation containing 55.06 to 64.58% of the major species charge variant components, as measured by CIX-HPLC after storage at 3°C ​​for 28 days.

[0095] 4 0± A pharmaceutical liquid formulation containing 12.71% to 64.15% of the major species charge variant components, as measured by CIX-HPLC after storage at 2°C for 28 days.

[0096] In a 20 mM L-histidine buffer at pH 6.0 or 6.5, a 20 mM citrate buffer at pH 5.5 or 6.0, or a 20 mM acetate buffer at pH 5.0 or 5.5, 4 0±A pharmaceutical liquid formulation containing 12.71% to 64.15% of the major species charge variant components, as measured by CIX-HPLC after storage at 2°C for 28 days.

[0097] The following list describes pharmaceutical liquid formulations containing specific amounts of acidic species charge variant components (with peaks whose retention times are earlier than those of the dominant species charge variant).

[0098] 5± A pharmaceutical liquid formulation containing 25.98% to 59.69% acidic species charge variants, as measured by CIX-HPLC after storage at 3°C ​​for 28 days.

[0099] In a 20 mM L-histidine buffer at pH 6.0 or 6.5, a 20 mM citrate buffer at pH 5.5 or 6.0, or a 20 mM acetate buffer at pH 5.0 or 5.5, 5± A pharmaceutical liquid formulation containing 25.98% to 59.69% acidic species charge variant components, as measured by CIX-HPLC after storage at 3°C ​​for 28 days.

[0100] 4 0± A pharmaceutical liquid formulation containing 39.20% to 71.02% acidic species charge variants, as measured by CIX-HPLC after storage at 2°C for 28 days.

[0101] In a 20 mM L-histidine buffer at pH 6.0 or 6.5, a 20 mM citrate buffer at pH 5.5 or 6.0, or a 20 mM acetate buffer at pH 5.0 or 5.5, 4 0± A pharmaceutical liquid formulation containing 39.20% to 71.02% acidic species charge variant components, as measured by CIX-HPLC after storage at 2°C for 28 days.

[0102] The following list describes pharmaceutical liquid formulations that contain specific amounts of basic species charge variant components (with their retention times indicating peaks located after the major species charge variant).

[0103] 5±A pharmaceutical liquid formulation containing 8.47 to 12.56% of basic seed charge variant components, as measured by CIX-HPLC after storage at 3°C ​​for 28 days.

[0104] In a 20 mM L-histidine buffer at pH 6.0 or 6.5, a 20 mM citrate buffer at pH 5.5 or 6.0, or a 20 mM acetate buffer at pH 5.0 or 5.5, 5± A pharmaceutical liquid formulation containing 8.47 to 12.56% of basic seed charge variant components, as measured by CIX-HPLC after storage at 3°C ​​for 28 days.

[0105] 4 0± A pharmaceutical liquid formulation containing 16.27 to 34.50% basic seed charge variant components, as measured by CIX-HPLC after storage at 2°C for 28 days.

[0106] In a 20 mM L-histidine buffer at pH 6.0 or 6.5, a 20 mM citrate buffer at pH 5.5 or 6.0, or a 20 mM acetate buffer at pH 5.0 or 5.5, 4 0± A pharmaceutical liquid formulation containing 16.27 to 34.50% basic seed charge variant components, as measured by CIX-HPLC after storage at 2°C for 28 days.

[0107] The following list describes pharmaceutical liquid formulations with specific turbidity levels.

[0108] A pharmaceutical liquid formulation having an absorbance A350 of 0.23 to 0.338 when measured using a spectrophotometer (e.g., SpectraMax® iD3 multimode microplate reader) after being stored at a temperature of 5℃±3℃ for 28 days.

[0109] In a 20 mM L-histidine buffer at pH 6.0 or 6.5, a 20 mM citrate buffer at pH 5.5 or 6.0, or a 20 mM acetate buffer at pH 5.0 or 5.5, 5±A pharmaceutical liquid formulation having an absorbance of A350 ranging from 0.23 to 0.338 when measured using a spectrophotometer (e.g., SpectraMax® iD3 multimode microplate reader) after being stored at 3°C ​​for 28 days.

[0110] A pharmaceutical liquid formulation having an absorbance A350 of 0.232 to 0.397 when measured using a spectrophotometer (e.g., SpectraMax® iD3 multimode microplate reader) after being stored at a temperature of 40℃±2℃ for 28 days.

[0111] In a 20 mM L-histidine buffer at pH 6.0 or 6.5, a 20 mM citrate buffer at pH 5.5 or 6.0, or a 20 mM acetate buffer at pH 5.0 or 5.5, 4 0± A pharmaceutical liquid formulation having an absorbance A350 of 0.232 to 0.397 when measured using a spectrophotometer (e.g., SpectraMax® iD3 multimode microplate reader) after being stored at 2°C for 28 days.

[0112] The following list describes pharmaceutical liquid formulations with specific colloidal stability of APIs in buffer solutions.

[0113] Dynamic light scattering (DLS) was used to measure the colloidal stability, particle size, and aggregation of APIs in buffer solutions. 。

[0114] DLS is used to measure the positive gradient to determine API aggregation and colloidal stability in histidine buffer. Samples are measured by increasing the API concentration in histidine buffer at pH 6.0 or pH 6.5.

[0115] DLS is used to measure the negative gradient to determine API aggregation and colloidal stability in citrate buffer. Samples are measured by increasing the API concentration in citrate buffer at pH 5.5 or pH 6.0.

[0116] DLS is used to measure the positive gradient to determine API aggregation and colloidal stability in acetate buffer. Samples are measured by increasing the API concentration in acetate buffer at pH 5.0 or pH 5.5.

[0117] Pharmaceutical composition: preparation Such compositions are prepared by methods well known in pharmaceutical technology (see, for example, Remington's Pharmaceutical Sciences, Mace Publishing Co., Philadelphia, Pa. 17th Ed. (1985), and Modern Pharmaceutics, Marcel Dekker, Inc. 3rd Ed. (GS Banker & CT Rhodes, Eds.)).

[0118] Sterile injectable preparations or intravenous fluids may be prepared by incorporating the required amount of the compound according to the present invention into a suitable solvent, along with various other components as listed above, as needed, and then sterilized by filtration. Typically, dispersions are prepared by incorporating various sterile active ingredients into a sterile vehicle containing a basic dispersion medium and other components as needed from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation method is a vacuum drying and freeze-drying technique from which the active ingredient powder and any additional desired components are obtained from a solution that has been previously sterile filtered.

[0119] High-concentration trastuzumab liquid formulations can be produced from IV trastuzumab reprocessed by ultrafiltration (UF) and diffusion filtration (DF) using methods well known in the industry. Ultracentrifugal filtration and tangential flow filtration (TFF) are two approaches to achieve buffer exchange and mAb concentration. Buffer exchange can also be performed using dialysis tubing or dialysis cassettes.

[0120] Treatment method and route of administration The present invention provides a method for treating a target requiring the use of a therapeutically effective amount of the pharmaceutical composition described herein.

[0121] The present invention further provides a method for treating cancer in a subject requiring it, comprising administering a therapeutically effective amount of the pharmaceutical composition described herein to the subject.

[0122] The present invention further provides a method for binding HER2 to a subject requiring such binding, comprising administering a therapeutically effective amount of the pharmaceutical composition described herein to the subject.

[0123] The methods described herein further include identifying subjects having cancer before administering the pharmaceutical compositions described herein.

[0124] The present invention also provides the following: (1) Pharmaceutical compositions described herein for use in therapeutic methods for treating the target subject. (2) A pharmaceutical composition described herein for use in a method of treating cancer, wherein the method comprises administering a therapeutically effective amount of the pharmaceutical composition to a subject in need thereof. (3) A pharmaceutical composition described herein for use in binding HER2, wherein the binding of HER2 comprises administering a therapeutically effective amount of the composition to a subject requiring such binding. (4) Use of a therapeutically effective amount of the pharmaceutical composition described herein for treating a subject that requires it. (5) Use of the pharmaceutical composition described herein for the treatment of cancer in a subject requiring the use thereof, wherein the treatment comprises administering a therapeutically effective amount of the pharmaceutical composition to the subject. (6) Use of the pharmaceutical composition described herein for HER2 binding, wherein the HER2 binding comprises administering a therapeutically effective amount of the composition to a subject requiring it. (7) Use of the pharmaceutical compositions described herein for the manufacture of pharmaceuticals. (8) Use of the pharmaceutical composition described herein for the manufacture of a pharmaceutical for use in the treatment of cancer in a subject that requires it, wherein the treatment comprises administering a therapeutically effective amount of the composition to the subject. (9) Use of the pharmaceutical composition described herein for the manufacture of a pharmaceutical for use in binding HER2, wherein the binding of HER2 comprises administering a therapeutically effective amount of the composition to a subject requiring it.

[0125] In this specification, and unless otherwise specified, the terms “to treat,” “treating,” and “treatment” refer to actions taken while a subject is suffering from a particular disease, disorder, or condition that reduce the severity of the disease, disorder, or condition, or that prevent or slow the progression of the disease, disorder, or condition (“therapeutic action”). In this specification, disease, disorder, and condition are used interchangeably.

[0126] In this specification, the “subject” intended for management includes humans (i.e., males or females of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or elderly adults) and / or non-human animals, e.g., primates (e.g., cynomolgus macaques, rhesus macaques), mammals such as cattle, pigs, horses, sheep, goats, mice, cats, and / or dogs. In certain embodiments, the subject is human. In certain embodiments, the subject is a non-human animal. In this specification, the terms “human,” “patient,” and “subject” are used interchangeably.

[0127] In this specification, and unless otherwise specified, “therapeutic dose” means an amount sufficient to produce a therapeutic effect in the treatment of a disease, disorder, or condition, or to delay or minimize one or more symptoms associated with the disease, disorder, or condition. A therapeutic dose of a compound means the amount of therapeutic agent that produces a therapeutic effect in the treatment of a disease, disorder, or symptom. The term “therapeutic dose” may include an amount that improves the overall therapy or reduces or avoids the symptoms or causes of the disease or condition.

[0128] The pharmaceutical composition may be administered for the treatment of cancers such as breast cancer. Types of breast cancer include adjuvant breast cancer, metastatic breast cancer, advanced breast cancer, and early-stage breast cancer. The pharmaceutical composition may also be used for metastatic gastric cancer or gastroesophageal junction adenocarcinoma, as well as ovarian cancer, gastric cancer, colon cancer, and It may be administered for the treatment of cancers such as gastrointestinal cancer.

[0129] The pharmaceutical composition may be administered in single or multiple doses by any of the common methods of administration of drugs having similar efficacy, such as those described in the patents and patent applications incorporated by reference, including parenteral administration such as subcutaneous injection.

[0130] The amount of trastuzumab administered subcutaneously per dose is 100 mg to 1000 mg, 200 mg to 900 mg, 300 mg to 800 mg, 300 mg to 750 mg, or 400 mg to 700 mg, 480 mg to 700 mg, 500 mg to 700 mg, 550 mg to 650 mg, 500 mg to 600 mg, 600 mg, or 750 mg. Each subcutaneous dose can be 0.5 mL to 10 mL, 1 mL to 9 mL, 2 mL to 8 mL, 3 mL to 7 mL, or 4 mL to 6 mL, or 5 mL.

[0131] When administered subcutaneously, patients should receive treatment once every week, once every two weeks, once every three weeks, once every four weeks, or once every five weeks. The dose should be administered over 2 to 5 minutes and preferably injected into different sites on the thigh. Patients with early-stage breast cancer should receive trastuzumab subcutaneously for 52 weeks or until the first occurrence of disease recurrence or unacceptable cardiotoxicity. Patients with metastatic breast cancer (MBC) should receive trastuzumab subcutaneously until disease progression is observed.

[0132] The target may be administered subcutaneously using pharmaceutical compositions configured for subcutaneous administration, such as pre-filled syringes, microneedles, sustained-release delivery systems, and stimulus-responsive delivery systems.

[0133] Trastuzumab subcutaneous therapy solution may be supplied using a commercially available syringe infusion system such as KORU™ FreedomEdge® (available from KORU™ Medical Systems, Chester, NY, USA).

[0134] In one embodiment, the trastuzumab subcutaneous treatment solution may be administered using a pre-filled syringe containing 3 mL to 7 mL of the trastuzumab subcutaneous treatment solution.

[0135] In other embodiments, the trastuzumab subcutaneous therapy solution may be supplied through, but is not limited to, commercially available medical devices such as injection devices, infusion pumps, auto-injectors, needleless devices, or subcutaneous patch delivery systems. For example, the trastuzumab subcutaneous therapy solution may be supplied by a commercially available infusion pump such as the Crono S-PID pump (available from Canes SPA), as described in U.S. Patent No. 8,172,814,B2. The trastuzumab subcutaneous therapy solution may also be supplied by other commercially available infusion pumps such as the SCIg60 injector (available from EMED Technologies Corporation), as described in U.S. Patent No. 9,808,576,B2 and U.S. Patent Publication No. 2013,013,8075.

[0136] In one embodiment, the trastuzumab subcutaneous treatment solution may be administered using an infusion pump containing 10 mL to 15 mL of the trastuzumab subcutaneous treatment solution.

[0137] In other embodiments, the trastuzumab subcutaneous solution may be supplied in commercially available on-body syringes, such as large-volume syringes (LVI-V), LVI-P, LVI-U, dual-cartridge syringes (DCI), and automated reconstitution syringes (ART) (available from Sonceboz SA), as described in European Patent No. 3354303B1. The trastuzumab subcutaneous solution may also be supplied in commercially available on-body syringes such as SmartDose (available from West Pharmaceutical Services), as described in WIPO Patent Publication Nos. 2018222521A1 and WIPO Patent Publication Nos. 2019032395A1. The trastuzumab subcutaneous solution may be supplied in enFuse (登録商標) It may also be supplied in a commercially available on-body injector, such as an On-Body Infusor (available from Enable Injections Inc.), as described in U.S. Patent Publication 20180161497A1 and U.S. Patent Publication 20210353222A1.

[0138] In other embodiments, the trastuzumab subcutaneous therapeutic solution may be supplied in a commercially available auto-injector, such as an auto-injector device available from West Pharmaceutical Services, as described in U.S. Patent No. 8048029B2.

[0139] throw It is understood that the amount of compound administered is usually determined by a physician, taking into account relevant circumstances including the condition to be treated, the chosen route of administration, the compound actually administered and its relative activity, the individual patient's age, weight, and response, and the severity of the patient's symptoms.

[0140] Examples Sample preparation High-concentration trastuzumab This is IV transduction that has been reprocessed by ultrafiltration / diafiltration (UF / DF) and filtration. Bu Ra make be Concentration and buffer exchange are performed. High-concentration trastuzumabThis is a critical processing step in manufacturing. Centrifugal filtration and tangential flow filtration (TFF) are two approaches for achieving buffer exchange and mAb concentration. In early, small-scale development, due to limitations in sample availability, buffer exchange had to be performed using dialysis tubes or cassettes with 20 kDa cutoff porous membranes. High-concentration trastuzumab The samples were concentrated by repeated centrifugation at a spin speed of 4200 rpm for 20 minutes at 4°C using a 30 kDa cutoff centrifugal filter to achieve the target concentration. The centrifuge used was an Eppendorf 5804R with an S-4-72 swing rotor.

[0141] In large-scale laboratory development, High-concentration trastuzumab Sample preparation involves using TFF for mAb concentration and buffer exchange. TFF processing is performed by Merck Mi l The procedure was performed using the lipore Labscale TFF System®, Pellicon® 3, Ultracel® 30 kDa membrane, and D cassette. Trastuzumab was dialyzed with the test formulation buffer and then concentrated to the target concentration.

[0142] High concentrations of pertuzumab are prepared from pertuzumab treated by chromatography, ultrafiltration / diafiltration (UF / DF), and filtration. Concentration and buffer exchange are critical processing steps in the production of pertuzumab. Centrifugal filtration and tangential flow filtration (TFF) are two approaches for achieving buffer exchange and mAb concentration. In early small-scale development, due to limitations in sample availability, buffer exchange had to be performed using dialysis tubes or cassettes with 20 kDa cutoff porous membranes. Pertuzumab samples were concentrated by repeated centrifugation at a spin speed of 4200 rpm for 20 minutes at 4°C using a 30 kDa cutoff centrifugal filter to achieve the target concentration. The centrifuge used was an Eppendorf 5804R with an S-4-72 swing rotor.

[0143] In large-scale laboratory development, pertuzumab sample preparation involves the use of TFF for mAb concentration and buffer exchange. TFF processing was performed using the Merck Milipore Labscale TFF System®, Pellicon® 3 and Ultracel® 30 kDa membrane, and a D-cassette. Pertuzumab was dialyzed with the test formulation buffer and concentrated to the target concentration.

[0144] Stability testing methods Turbidity Turbidity is the detection of visible / invisible particles and therefore tests both the quality and stability of the formulation. The sample was gently inverted and mixed 10 times before loading into a 96-microwell plate (Cat. 269620). 200 μL of sample was loaded into each well in triplicates. The sample was measured three times using a SpectraMax® iD3 multimode microplate reader. Measurements were recorded as mean triplicate absorbance.

[0145] Purity, aggregation, and cutting Antibody purity, aggregation, and cleavage were measured by size exclusion high-performance liquid chromatography (SEC-HPLC) using an HPLC system HPLC-A6002 (Agilent 1260 Infinity II). A column (TOSOH TSK-gel G3000 SWxl, 7.8 x 300 mm, PN:0008541) was provided.

[0146] Charge variant Antibody charge variants were measured by cation exchange high-performance liquid chromatography (CIX-HPLC) using an HPLC system HPLC-A6002 (Agilent 1260 Infinity II series). A column (TOSOH TSK-gel CM-STAT, 4.6 x 100 mm, 7 μm, CN:0021966, Lot:082GA0017G) was provided. The main component (main peak), acidic variant, and basic variant of the antibody were detected from the samples.

[0147] Colloidal stability, aggregation, and particle size Colloidal stability, aggregation, and particle size were measured by dynamic light scattering (DLS) using a Dynamic Light Scattering DynaPro® Plate Reader III (Wyatt Technology Corp., CA) with a laser wavelength of 823.3 nm and a scattering angle of 150°.

[0148] Dynamic light scattering was used to measure the size distribution profile of vesicles in solution. Light passing through small particles in solution is scattered. The sample was gently mixed 10 times by inversion before being loaded onto an Auroa 384 microplate (Wyatt PN: P8806-38401). onset or T agg For this analysis, the initial and final temperatures were set to 25°C and 75°C, respectively. Each well of the microplate was read five times, and the average of the measurements for each well was recorded. The measured scattering constants and sample concentrations were plotted on the Y and X axes, respectively. A positive slope indicates light passage through the spaces between small particles in the concentrated sample and less aggregation. A negative slope indicates an increase in particle size due to aggregation in the concentrated sample.

[0149] Protein content assay Protein concentration was measured by a protein content assay using a Jasco v-730 spectrophotometer. The detection wavelength was set to 280 nm. Samples were diluted to appropriate concentrations, with an OD of 0.4–0.8. 110 μL of sample was loaded into a cuvette and measured at 280 nm. The average absorbance was recorded, and the protein (antibody) concentration was calculated.

[0150] Specifically, the method for measuring protein concentration included turning on the spectrophotometer and UV lamp at least 20 minutes before use.

[0151] The detection wavelength was 280 nm. A 1 cm quartz cuvette was washed with ultrapure water and dried. The cuvette was rinsed with formulation base buffer. Formulation base buffer was added to the cuvette. The cuvette was inserted into a spectrophotometer and recorded as a blank. The formulation base buffer was discarded, and the cuvette was rinsed with the test sample for equilibration. The test sample was discarded from the cuvette.

[0152] The test sample was loaded into a cuvette and measured. The sample was discarded, and the test sample was reloaded to measure it twice. The cuvette was washed with the basic formulation buffer, and the steps from blank measurement to measuring the test sample were repeated for the next test sample.

[0153] After completing the measurements, the cuvettes were washed with ultrapure water and dried. The absorbance of each sample was averaged. The concentration was calculated using the formula of Baer's Law.

[0154] Binding activity HER2-ECD binding ELISA The antibody binding activity was measured by a HER2-ECD binding ELISA assay.

[0155] FcγRIII binding assay The FcγRIII binding assay is a binding kinetics method that measures the interaction between trastuzumab and its receptor using Bio-Layer Interference (BLI) technology. The measurement is performed using a ForteBio OctetRed96 (Pall, instrument number BAZ-20002).

[0156] Stability Test Example 1: Comparison of Stability with Buffers Stability Test For the liquid pharmaceutical formulation used in Example 1, each buffer solution was prepared to match the corresponding pH and concentration, and antibodies were added to obtain the samples shown in Tables 1, 2, and 3 below. The specific content of each component is as described in Tables 1, 2, and 3 below. ru The total volume was, for example, 5 mL.

[0157] [Table 1]

[0158] [Table 2]

[0159] [Table 3]

[0160] The liquid pharmaceutical formulations prepared in Examples 1 to 3 were tested for stability at 0 days (initial), 3 days, 1 week, 2 weeks, and 4 weeks at temperatures of 5°C ± 3°C or 40°C ± 2°C. Example 1 is a histidine buffer system using a 20 mM L-histidine / L-histidine hydrochloride solution. Example 2 is a citrate system using a 20 mM citrate / sodium citrate solution. Example 3 is an acetic acid system using a 20 mM acetic acid / sodium acetate solution. The results are shown in Tables 1 to 3 above.

[0161] Figure 2 shows the colloidal stability of trastuzumab in different buffers. Scattering constants versus trastuzumab concentrations were plotted using DLS. Figure 2A shows the colloidal stability of trastuzumab in histidine buffer at pH 6.5. Figure 2B shows the colloidal stability of trastuzumab in histidine buffer at pH 6.0. Figure 2C shows the colloidal stability of trastuzumab in citrate buffer at pH 6.0. Figure 2D shows the colloidal stability of trastuzumab in citrate buffer at pH 5.5. Figure 2E shows the colloidal stability of trastuzumab in acetate buffer at pH 5.5. Figure 2F shows the colloidal stability of trastuzumab in acetate buffer at pH 5.0. The colloidal stability of trastuzumab in histidine and acetate buffers shows a positive slope. The colloidal stability of trastuzumab in citrate buffer exhibits a negative gradient. In one embodiment, histidine buffer, acetate buffer, or citrate buffer may be used as a buffer in the liquid pharmaceutical composition.

[0162] Stability Test Example 2: Stability of the composition with respect to the pH of acetate buffer solution Stability Test For the liquid pharmaceutical formulation used in Example 2, each buffer solution was prepared to match the corresponding pH and antibody concentration, and the samples shown in Table 4 below were obtained. The concentrations of each component are as described in Table 4 below. ru The total volume was, for example, 5 mL.

[0163] [Table 4] JPEG0007842895000005.jpg22362

[0164] The above formulations are 4 0± Stability was measured at 0 weeks (initial), 1 week, 1 month, 2 months, and 3 months at a temperature of 2°C. Examples 4, 5, and 6 show formulations using 20 mM acetic acid / sodium acetate solutions at pH 5.4, 5.0, and 4.6, respectively. The results are shown in Table 4 above.

[0165] Figure 3 shows the antibody profile measured by SEC-HPLC. Figure 3 shows the results over 28 days under accelerated thermal stress at 40°C. High-concentration trastuzumab The antibody profiles are shown below. Figure 3A shows the antibody profile under acetic acid at pH 5.4. Figure 3B shows the antibody profile under acetic acid at pH 5.0. Figure 3C shows the antibody profile under acetic acid at pH 4.6.

[0166] Figure 4 shows the antibodies measured by CIX-HPLC. Figure 4 shows the charge variant profiles of the antibodies over 28 days under accelerated thermal stress at 40°C. Figure 4A shows the charge variant profile under acetic acid at pH 5.4. Figure 4B shows the charge variant profile under acetic acid at pH 5.0. Figure 4C shows the charge variant profile under acetic acid at pH 4.6.

[0167] In one embodiment, the acetic acid may have a pH of 4.6 to 5.4.

[0168] Stability Test Example 3: Stability of the composition depending on the type of additive or pH in acetate buffer. Stability Test For the liquid pharmaceutical formulation used in Example 3, each buffer solution was prepared by adding an isotonic agent or salt to match the corresponding pH and antibody concentration, and the samples shown in Table 5 below were obtained. The concentrations of each component are as shown in Table 5 below. ru The total volume was, for example, 5 mL.

[0169] [Table 5] JPEG0007842895000007.jpg19984

[0170] The stability of the above formulations was measured at 0 weeks (initial), 1 week, 2 weeks, and 4 weeks at temperatures of 5°C ± 3°C or 40°C ± 2°C. Examples 7 and 8 show formulations with pH 5.6 or 5.2 containing 0.05% PS20, 150 mM trehalose, or 0.9% NaCl and 20 mM acetic acid / sodium acetate solution, respectively. The antibody concentration was 120 mg / mL. The results are shown in Table 5 above.

[0171] In one embodiment, the formulation may contain an acetic acid / sodium acetate solution with a pH between 5.6 and 5.2. In one embodiment, the formulation may contain an isotonic agent or a salt. The isotonic agent may be, but is not limited to, trehalose. The salt may be, but is not limited to, sodium chloride (NaCl).

[0172] Stability Test Example 4: Stability of the composition depending on the type of isotonic agent and buffering agent Stability Test For the liquid pharmaceutical formulation used in Example 4, each buffer solution was prepared by adding salt and further amino acids to match the corresponding buffer solution, isotonic agent, and antibody concentration, yielding the samples shown in Tables 6 to 13 below. The concentrations of each component are as described in Tables 6 to 13 below. ru The total volume was, for example, 5 mL.

[0173] [Table 6]

[0174] The stability of the above formulations was measured at 40°C ± 2°C for 0 days (initial) and after 4 days. The formulations represented by Samples 1 to 7 contain 20 mM histidine buffer at pH 6.0 and 20 mM or 300 mM trehalose. The antibody concentration is 120 mg / mL. The results are shown in Table 6 above.

[0175] In one embodiment, the formulation may, but is not limited to, a histidine buffer, trehalose, methionine, or NaCl, or a combination thereof.

[0176] [Table 7]

[0177] The stability of the above formulations was measured at 40°C ± 2°C for 0 days (initial) and after 4 days. The formulations represented by Samples 8 to 14 contain 20 mM histidine buffer at pH 6.0 and 20 mM or 300 mM mannitol. The antibody concentration is 120 mg / mL. The results are shown in Table 7 above. In one embodiment, the formulation may, but is not limited to, histidine buffer, mannitol, methionine, or NaCl, or a combination thereof.

[0178] [Table 8]

[0179] The stability of the above formulations was measured at 40°C ± 2°C for 0 days (initial) and after 4 days. The formulations represented by Samples 15 to 21 contain 20 mM histidine buffer at pH 6.0 and 20 mM or 300 mM sorbitol. The antibody concentration is 120 mg / mL. The results are shown in Table 8 above. In one embodiment, the formulation may, but is not limited to, histidine buffer, sorbitol, methionine, or NaCl, or a combination thereof.

[0180] [Table 9]

[0181] It is noteworthy that the formulation containing sucrose and methionine showed a high monomer percentage of 92.08% after exposure to 40°C ± 2°C for 4 days, exceeding the 79.87% observed in the formulation containing sorbitol and methionine.

[0182] The stability of the above formulations was measured at 40°C ± 2°C for 0 days (initial) and after 4 days. The formulations represented by Samples 22 to 28 contain 20 mM histidine buffer at pH 6.0 and 20 mM or 300 mM sucrose. The antibody concentration is 120 mg / mL. The results are shown in Table 9 above. In one embodiment, the formulation may, but is not limited to, histidine buffer, sucrose, methionine, or NaCl, or a combination thereof.

[0183] [Table 10]

[0184] The stability of the above formulations was measured at 40°C ± 2°C for 0 days (initial) and after 4 days. The formulations represented by Samples 29 to 35 contain 20 mM acetate buffer at pH 5.0 and 20 mM or 300 mM trehalose. The antibody concentration is 120 mg / mL. The results are shown in Table 10 above. In one embodiment, the formulation may, but is not limited to, acetate buffer, trehalose, methionine or NaCl, or a combination thereof.

[0185] [Table 11]

[0186] The stability of the above formulations was measured at 40°C ± 2°C for 0 days (initial) and after 4 days. The formulations represented by Samples 36 to 42 contain 20 mM acetate buffer at pH 5.0 and 20 mM or 300 mM mannitol. The antibody concentration is 120 mg / mL. The results are shown in Table 11 above. In one embodiment, the formulation may, but is not limited to, acetate buffer, mannitol, methionine, or NaCl, or a combination thereof.

[0187] [Table 12]

[0188] The stability of the above formulations was measured at 40°C ± 2°C for 0 days (initial) and after 4 days. The formulations represented by Samples 43 to 49 contain 20 mM acetate buffer at pH 5.0 and 20 mM or 300 mM sorbitol. The antibody concentration is 120 mg / mL. The results are shown in Table 12 above. In one embodiment, the formulation may, but is not limited to, acetate buffer, sorbitol, methionine, or NaCl, or a combination thereof.

[0189] [Table 13]

[0190] It is noteworthy that formulations containing sucrose consistently have higher monomer percentages (%) than formulations containing sorbitol at day 0 (initial stage).

[0191] The stability of the above formulations was measured at 40°C ± 2°C for 0 days (initial) and after 4 days. The formulations represented by samples 50 to 45 contain 20 mM acetate buffer at pH 5.0 and 20 mM or 300 mM sucrose. The antibody concentration is 120 mg / mL. The results are shown in Table 13 above. In one embodiment, the formulation may, but is not limited to, acetate buffer, sucrose, methionine, or NaCl, or a combination thereof.

[0192] Stability Test Example 5: Stability of Composition by Amino Acid Type Stability Test For the liquid pharmaceutical formulation used in Example 5, each buffer solution was prepared by adding sucrose and further organic cosolvents to match the corresponding amino acid and antibody concentrations, yielding the samples shown in Tables 14 to 16 below. The concentrations of each component are as described in Tables 14 to 16 below. ru The total volume was, for example, 4 mL.

[0193] [Table 14] JPEG0007842895000017.jpg19673

[0194] The liquid pharmaceutical formulations prepared in Examples 9 to 13 were tested for stability at 0 weeks (initial), 1 week, 2 weeks, 3 weeks, and 4 weeks at a temperature of 40°C ± 2°C. Examples 9 to 13 contain 150 mg / mL trastuzumab, 210 mM sucrose (or 7.18% w / v), and 0.02% PS80 in 20 mM acetate buffer at pH 5.2. Example 9 further contains 10 mM methionine, and Example 10 further contains 50 mM arginine. Example 11 further contains 20 mM glutamic acid. Example 12 further contains a combination of 10 mM methionine and 50 mM arginine. Example 13 further contains a combination of 10 mM methionine and 20 mM glutamic acid. The results are shown in Table 14 above. In one embodiment, the formulation may, but is not limited to, an acetate buffer, sucrose, PS80, methionine, or glutamic acid, or a combination thereof.

[0195] [Table 15]

[0196] The liquid pharmaceutical formulations prepared in Examples 9 and 13 were tested for stability at 0 hours (initially) and after 4 hours under steady-state vortexing during a stirring stress test. The results are shown in Table 15 above. The formulations remained stable even after stirring.

[0197] [Table 16]

[0198] The stability of the liquid pharmaceutical preparations prepared in Examples 9 and 13 was measured after 0 (initial), 1, 3, and 5 (cycle) freeze-thaw cycles. The results are shown in Table 16 above. The preparations remained stable even after repeated freeze-thaw cycles.

[0199] Stability Test Example 6: Stability of the composition under heat or ultraviolet stress Stability Test For the liquid pharmaceutical formulation used in Example 6, each buffer solution was prepared by adding methionine and further adding organic cosolvents to match the corresponding buffer concentration, pH, and isotonic agent, yielding the samples shown in Tables 17 to 18 below. The concentrations of each component are as described in Tables 17 to 18 below. ru The total volume was, for example, 5 mL.

[0200] [Table 17] JPEG0007842895000021.jpg22525

[0201] Heat stress The liquid pharmaceutical formulations prepared in Examples 14 to 17 were tested for stability at 0 days (initial), 3 days, 6 days, 10 days, and 2 weeks under conditions of a temperature of 50°C ± 2°C and a relative humidity of 75 ± 5%. The results are shown in Table 17 above. In one embodiment, the formulation may, but is not limited to, acetate buffer, sucrose or trehalose, methionine, or a combination thereof. In other embodiments, the formulation may, but is not limited to, histidine buffer, sucrose or trehalose, methionine, or a combination thereof.

[0202] [Table 18] JPEG0007842895000023.jpg22538

[0203] photostability For the liquid pharmaceutical formulations prepared in Examples 14 to 17, UV / visible light irradiation in the UV region (320 nm-400 nm) (≥200 watt-hours / m²) was performed. 2Under steady exposure to ), the stability was measured at 0 days (initially), 3 days, 6 days, 10 days, and 2 weeks. The results are shown in Table 18 above. In one embodiment, the formulation may, but is not limited to, acetate buffer, sucrose, methionine, or a combination thereof.

[0204] Stability Test Example 7: Stability of the composition depending on the type of buffer and isotonic agent. Stability Test For the liquid pharmaceutical formulation used in Example 7, each buffer solution was prepared by adding methionine and further organic cosolvents to suit the corresponding buffer type and isotonic agent, yielding the samples shown in Tables 19 to 20 below. The concentrations of each component are as described in Tables 19 to 20 below. ru The total volume was, for example, 5 mL or 4 mL for Table 19 or Table 20.

[0205] [Table 19]

[0206] [Table 20]

[0207] The liquid pharmaceutical formulations prepared in Examples 18 to 25 were tested for stability at 0 weeks (initial), 1 week, 2 weeks, 4 weeks, 8 weeks, and 12 weeks at a temperature of 40°C ± 2°C. Examples 18 to 21 are formulations containing an antibody concentration of 120 mg / mL. Examples 22 to 25 are formulations containing an antibody concentration of 150 mg / mL. The results are shown in Tables 19 and 20 above. In one embodiment, the formulation may, but is not limited to, acetate buffer, methionine, sucrose, or trehalose, or a combination thereof. In another embodiment, the formulation may, but is not limited to, histidine buffer, methionine, sucrose, or trehalose, or a combination thereof.

[0208] Stability Test Example 8: Stability of the composition depending on the type of organic cosolvent and amino acid. Stability Test: For the liquid pharmaceutical formulations used in Example 8, each buffer solution was prepared by adding an isotonic agent to suit the corresponding buffer type, organic cosolvent, and amino acid, yielding the samples shown in Tables 21 to 22 below. The concentrations of each component were as described in Tables 21 to 22 below. The total volume was, for example, 5 mL or 4 mL for Table 21 or Table 22.

[0209] [Table 21]

[0210] [Table 22]

[0211] The liquid pharmaceutical formulations prepared in Examples 26 to 32 were tested for stability at 0 months (initial), 0.5 months, 1 month, 3 months (or 3.36 months in Table 28), and 6 months at a temperature of 40°C ± 2°C. Examples 26 to 29 are formulations containing an antibody concentration of 120 mg / mL. Examples 30 to 32 are formulations containing an antibody concentration of 150 mg / mL. The results are shown in Tables 21 and 22 above. In one embodiment, the formulation may, but is not limited to, acetate buffer, sucrose, PS80, methionine, and glutamic acid. In another embodiment, the formulation may contain acetate buffer, sucrose, PS80, methionine, or glutamic acid, or a combination thereof. In yet another embodiment, the formulation may contain histidine buffer, trehalose, PS20, methionine, or a combination thereof.

[0212] Stability Test Example 9: Long-term Stability Stability Testing For the liquid pharmaceutical formulations used in Example 9, each buffer was prepared by adding an isotonic agent to suit the corresponding buffer type, organic cosolvent, and amino acid, yielding Sample Examples 33 to 36. The integrity stability was measured by SEC-HPLC at 5°C for 0, 0.25, 0.5, 0.75, 1, 2, 3.36, 6, 12, 18, and 24 months, and at 25°C and 40°C for up to 6 months. Example 33 is a formulation containing 150 mg / mL trastuzumab, 20 mM histidine buffer, pH 5.5, 210 mM trehalose, 10 mM methionine, and 0.04% polysorbate 20 (H / T / M / PS20 pH 5.5). Example 34 is a formulation containing 150 mg / mL trastuzumab, 20 mM acetate, pH 5.2, 210 mM sucrose, 10 mM methionine, and 0.02% polysorbate 80 (A / S / M / PS80 pH 5.2). Example 35 is a formulation containing 150 mg / mL trastuzumab, 20 mM acetate, pH 5.2, 210 mM sucrose, 10 mM methionine, 40 mM glycine, and 0.02% polysorbate 80 (A / S / M / 40Gly pH 5.2). Example 36 is a formulation containing 150 mg / mL trastuzumab, 20 mM acetate, pH 5.2, 210 mM sucrose, 10 mM methionine, 10 mM glutamic acid, and 0.02% polysorbate 80 (A / S / M / 10Glu pH 5.2). The integrity results at 5°C, 25°C, and 40°C are shown in Figures 5A, 5B, and 5C, respectively. The sample from Example 36 can maintain a high level of antibody purity for at least 24 months. For Examples 33 to 36, charge heterogeneity was measured by CEX analysis at 0, 0.25, 0.5, 0.75, 1, 2, 3.36, 6, 12, 18, and 24 months at 5°C (Figure 6A), 6 months at 25°C (Figure 6B), and 1 month at 40°C (Figure 6C).

[0213] Stability Test Example 10: Investigation of Isotonic Agents Stability Testing For the liquid pharmaceutical formulations used in Example 10, each buffer solution was prepared by adding an isotonic agent to suit the corresponding buffer type, organic cosolvent, and amino acid, yielding Sample Examples 37 to 40. The integrity stability was measured at 0, 0.5, 1, 2, and 3 months at temperatures of 5°C, 25°C, and 40°C. Example 37 is a formulation containing 120 mg / mL trastuzumab, 20 mM acetic acid, pH 5.2, 210 mM sucrose, 10 mM methionine, 10 mM glutamic acid, and 0.02% polysorbate 20. Example 38 is a formulation containing 120 mg / mL trastuzumab, 20 mM acetic acid, pH 5.2, 210 mM trehalose, 10 mM methionine, 10 mM glutamic acid, and 0.02% polysorbate 80. Example 39 is a formulation containing 120 mg / mL trastuzumab, 20 mM acetate, pH 5.2, 210 mM mannitol, 10 mM methionine, 10 mM glutamic acid, and 0.02% polysorbate 80. Example 40 is a formulation containing 120 mg / mL trastuzumab, 20 mM acetate, pH 5.2, 210 mM sorbitol, 10 mM methionine, 10 mM glutamic acid, and 0.02% polysorbate 80. The integrity results at 5°C, 25°C, and 40°C are shown in Figures 7A, 7B, and 7C, respectively.

[0214] The charge heterogeneity stability of the liquid pharmaceutical formulations prepared in Examples 37 to 40 was measured at temperatures of 5°C, 25°C, and 40°C after 0, 0.5, 1, 2, and 3 months. The results for charge heterogeneity at temperatures of 5°C, 25°C, and 40°C are shown in Figures 8A, 8B, and 8C, respectively.

[0215] Stability Test Example 11: Evaluation of the stability of pertuzumab Stability Test For the liquid pharmaceutical preparation used in Example 11, each buffer solution was prepared by adding an isotonic agent to be compatible with the corresponding buffer solution type, organic co-solvent, and amino acid, obtaining Sample Examples 41 to 44, and measuring the integrity stability after 0, 0.5, 1, 2, and 3 months at temperatures of 5°C, 25°C, and 40°C. Example 41 is a preparation containing 120 mg / mL of pertuzumab, 20 mM acetic acid, pH 5.2, 210 mM sucrose, 10 mM methionine, 10 mM glutamic acid, and 0.02% polysorbate 20. Example 42 is a preparation containing 120 mg / mL of pertuzumab, 20 mM acetic acid, pH 5.2, 210 mM trehalose, 10 mM methionine, 10 mM glutamic acid, and 0.02% polysorbate 80. Example 43 is a preparation containing 120 mg / mL of pertuzumab, 20 mM acetic acid, pH 5.2, 210 mM mannitol, 10 mM methionine, 10 mM glutamic acid, and 0.02% polysorbate 80. Example 44 is a preparation containing 120 mg / mL of pertuzumab, 20 mM acetic acid, pH 5.2, 210 mM sorbitol, 10 mM methionine, 10 mM glutamic acid, and 0.02% polysorbate 80. The results of integrity at temperatures of 5°C, 25°C, and 40°C are shown in Figure 9A, Figure 9B, and Figure 9C, respectively. Sample Examples 41 to 44 can maintain the antibody purity of pertuzumab at a high level.

[0216] For the liquid pharmaceutical preparations prepared from Sample Examples 41 to 44, the charge heterogeneity stability after 0, 0.5, 1, 2, and 3 months was measured at temperatures of 5°C, 25°C, and 40°C. The results of charge heterogeneity of Sample Examples 41 to 44 are shown in Figure 10A, Figure 10B, Figure 10C, and Figure 10D, respectively.

[0217] Animal Test Animal Test Example 1: Mouse Pharmacodynamic (PD) Test - Antitumor Activity of Trastuzumab under Different Buffer Conditions. Xenograft Model. All procedures were carried out in accordance with the appropriate laws, regulations, and guidelines of the National Institutes of Health (NIH), USA, and were approved by the Institutional Animal Care and Use Committee of Covance, USA. The human breast cancer cell line BT-474 (ATCC, USA) (1.0E+007 BT-474) was suspended in 200 μL of serum-free Dulbecco's Modified Eagle Medium (DMEM) medium and subcutaneously inoculated into NSG mice (NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ) mice (Jackson Laboratory, U.S.A.). Treatment was initiated 7 days after tumor inoculation. Tumor volume (mm 3 ) was determined using the formula (length × width 2 ) / 2, where the length was the longest axis and the width was the thickness measurement perpendicular to the length. Data were presented as the mean tumor volume ± SE for each treatment group. All data were analyzed for significant differences by Student's T-test. Six mice per group were used for each experiment.

[0218] Cell line and culture conditions The human breast cancer cell BT-474 was cultured in DMEM, 10% heat-inactivated fetal bovine serum (FBS), and 1% penicillin / streptomycin / L-glutamine (PSG) at 37 °C. The cell viability was 95% at the time of transplantation into mice.

[0219] Treatment of animals All mice were classified into test groups based on an estimated value by tumor volume calipers. Mice were treated according to Table 23.

[0220] Table 23: Treatment groups and dosing schedules according to FIG. 11.

Table 23

[0221] Note: ROA - route of administration, PBS - phosphate-buffered saline, Herceptin SC - commercial Roche EU-made trastuzumab 600 mg for SC injection, High-concentration trastuzumab and IV trastuzumabThe same active pharmaceutical ingredient (API) (trastuzumab) is used, administered subcutaneously (SC) or intravenously (IV), with Q7Dx once every 6-7 days for a total of 6 treatments. IV trastuzumab 150 mg of lyophilized trastuzumab was reconstituted in sterile water for injection and finally in a buffer of 4.4 mM L-histidine / L-histidine hydrochloride, pH 6.0, 1.71% trehalose dihydrate, and 0.01% PS20. High-concentration trastuzumab -3 was formulated with 20 mM acetate / sodium acetate, pH 5.2, 210 mM sucrose, 10 mM methionine, 10 mM glutamic acid, and 0.02% PS80.

[0222] The disease progression (PD) results of these treatments are shown in Figure 11.

[0223] Animal study Example 2: Mouse pharmacodynamic (PD) study to test the plasma concentration (μg / mL) versus time of trastuzumab in various formulations. The trastuzumab-containing test substances in various formulations for the PD study were prepared according to Table 24.

[0224] Table 24. Conditions for mouse pharmacokinetic (PK) studies. [Table 24]

[0225] Note: High-concentration trastuzumab -3 and IV trastuzumab It contains the same API (trastuzumab). Met-methionine, Ace-acetic acid / sodium acetate, PS-polysorbate.

[0226] The PK test was conducted in CD-1 male mice (Vital River, Pinghu, China). At the time of the test, the mice were 5 - 6 weeks old and weighed in the range of 25 - 38 g. The animals were housed in polycarbonate cages with bedding, in groups of a maximum of 5 animals / sex / cage. The animals were randomly assigned to each group. The mice were divided into 5 groups of 25 each. Groups 1 - 4 received SC bolus administration. Group 5 received IV bolus administration. The animals were treated according to the protocol in Table 25.

[0227] Table 25: Treatment groups according to Figure 12.

Table 25

[0228] Approximately 0.2 mL of blood was collected from the submandibular site at each time point. Samples were collected into serum separator tubes (SST) at 16 time points: pre - dose and 0.25, 1, 4, 8, 24 hours post - dose, and 48 (day 2), 72 (day 3), 96 (day 4), 168 (day 7), 336 (day 14), 504 (day 21), 672 (day 28), 840 (day 35), 1008 (day 42) and 1344 (day 56) hours. No anticoagulant was used. The blood was allowed to clot at room temperature for 30 minutes. Samples were centrifuged at approximately 2700 g for about 10 minutes at 2 - 8°C within 2 hours after collection, the serum was collected, and stored at - 60 to - 80°C until further analysis.

[0229] Bioanalysis. Pharmacokinetic samples were analyzed for trastuzumab in mouse serum using the ELISA method.

[0230] The results of the PK test are summarized in Table 26.

[0231] Table 26: Summary of the results of the PK test according to Figure 12.

Table 26

[0232] Note: SC: subcutaneous, IV: intravenous, T 1 / 2 : Half-life until the sample disappears from the animal body, C0: Initial concentration, T max : The time it takes for a drug to reach its peak concentration in the serum, C max : The highest (or peak) concentration of a drug in serum, AUC 0-last : Area under the curve from time 0 to the last measurable concentration, AUC 0-ifn : Area under the curve up to infinity, VD: Volume of distribution, CL: Clearance, MRT: Mean residence time, F%: Bioavailability percentage.

[0233] The results of the PK test are shown in Figures 12A and 12B.

[0234] Equivalents and scope of application In the claims, articles such as “a,” “an,” and “the” may mean one or more unless otherwise indicated or is clear from the context. Claims or descriptions containing “or” between one or more components of a group are deemed satisfied unless otherwise indicated or is clear from the context, if one, multiple, or all of the group are present in, used in, or related to the given object or process. The present invention includes embodiments in which exactly one component of a group is present in, used in, or otherwise related to the given object or process. The present invention includes embodiments in which multiple or all of the components of a group are present in, used in, or otherwise related to the given object or process.

[0235] Furthermore, the present invention encompasses all variations, combinations, and substitutions in which one or more limitations, elements, clauses, and descriptive terms from one or more of the described claims are introduced into another claim. For example, any claim that depends on another claim can be modified to include one or more limitations described in any other claim that depends on the same basic claim. Where elements are presented in list form, for example in Markush group form, subgroups of each element are also disclosed, and any element can be removed from a group. Where an invention or aspect of an invention is referred to as containing certain elements and / or features, it should be understood that certain embodiments of the invention or aspect of an invention contain, or essentially consist of, such elements and / or features. For brevity, these embodiments are not specifically shown in this specification in those exact words. Also, the terms “contains” and “includes” are intended to be open, allowing for the inclusion of additional elements or steps. Where a scope is given, both ends of that scope are included. Furthermore, unless otherwise specified or it is clear from the context and the understanding of those skilled in the art, the values ​​expressed as a range may take any specific value or subrange within the range defined in different embodiments of the present invention, up to one-tenth of the lower limit of the range, unless otherwise clearly specified in the context.

[0236] This application refers to various published patents, published patent applications, academic papers, and other publications, all of which are incorporated herein by reference. In the event of any inconsistency between the incorporated references and this specification, this specification shall prevail. Furthermore, any particular embodiment of the Invention that constitutes prior art may be expressly excluded from one or more claims. Such embodiments may be excluded even if the exclusion is not expressly stated herein, as they are considered to be well known to those skilled in the art. Any particular embodiment of the Invention may be excluded from any claim for any reason, whether or not it relates to the existence of prior art.

[0237] Those skilled in the art will recognize or confirm many equivalents to the specific embodiments described herein without further routine experimentation. The scope of the embodiments described herein is not intended to be limited to the above description, but rather as set forth in the appended claims. Those skilled in the art will understand that various changes and modifications to this specification may be made without departing from the spirit or scope of the invention, as defined in the following claims.

Claims

1. A pharmaceutical composition, (a) Active pharmaceutical ingredients containing anti-HER2 antibodies, (b) Methionine, (c) Glutamic acid, and (d) Acetate buffer solution with pH 4.6 to 6.5 Includes, The anti-HER2 antibody is selected from the group consisting of trastuzumab, pertuzumab, and mixtures thereof, and the concentration of the anti-HER2 antibody is 80 to 150 mg / mL, in a pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, wherein the methionine has a concentration of 5 to 15 mM, and the glutamic acid has a concentration of 5 to 15 mM.

3. The pharmaceutical composition according to claim 1, further comprising an isotonic agent selected from trehalose, sucrose, mannitol, and sorbitol.

4. The pharmaceutical composition according to claim 3, wherein the isotonic agent is sucrose.

5. The pharmaceutical composition according to claim 4, wherein the sucrose has a concentration of 150 to 300 mM.

6. The pharmaceutical composition according to claim 1, wherein the trastuzumab and pertuzumab are present in concentrations of 120 to 150 mg / mL.

7. The pharmaceutical composition according to claim 1, wherein the composition is configured for subcutaneous administration.

8. Use of the pharmaceutical composition according to claim 1 for the manufacture of a pharmaceutical for the treatment of cancer, wherein the pharmaceutical is for subcutaneous administration.

9. The use according to claim 8, wherein the cancer is breast cancer.

10. The use according to claim 8, wherein the cancer is metastatic gastric cancer or gastroesophageal junction adenocarcinoma.

11. The use of the drug according to claim 8, wherein the drug is administered once every three weeks.

12. A pre-filled syringe containing 3 mL to 7 mL of solution, wherein the solution is (a) anti-HER2 antibody, (b) Methionine, (c) Glutamic acid, and (d) Acetate buffer solution with pH 4.6 to 6.5 Includes, The anti-HER2 antibody is selected from the group consisting of trastuzumab, pertuzumab, and mixtures thereof, and the concentration of the anti-HER2 antibody is 80 to 150 mg / mL, in a pre-filled syringe.

13. The pre-filled syringe according to claim 12, wherein the methionine has a concentration of 5 to 15 mM, and the glutamic acid has a concentration of 5 to 15 mM.

14. The pre-filled syringe according to claim 12, wherein the solution further comprises sucrose.

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