Pharmaceutical composition containing anti-HER2 antibody for subcutaneous administration

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] Substances in the ASCII text file are incorporated by reference.

[0002] This application incorporates by reference the sequence listing contained in the following file: Filename: P4330-PCT_seq listing, 6 KB, Created: April 14, 2023.

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 333,930, filed April 22, 2022, and U.S. Provisional Application No. 63 / 333,935, both of which are incorporated by reference in their entireties. [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). The overexpression of the HER2 protein can potentially be exploited by targeting with 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 against tumor cells that overexpress the HER2 protein.

[0005] The pharmaceutical use of monoclonal antibodies has increased over the years. In many instances, such monoclonal antibodies have been infused by the intravenous (IV) route. Most therapeutic monoclonal antibodies (mAbs) have been administered by traditional intravenous (IV) administration. IV administration requires patients to travel long distances to medical facilities with medical personnel trained to administer the IV infusion and determine the appropriate dose and infusion rate. Clinical administration is expensive and inconvenient for patients.

[0006] Furthermore, administering appropriate amounts of low-concentration monoclonal antibodies via IV infusion is time-consuming (approximately 90 minutes) and inconvenient for the patient (requiring a visit to a well-staffed clinic).

[0007] Alternative routes of administration include subcutaneous (SC) administration, which allows patients to conveniently and economically self-administer therapeutics at home, leading to more convenience for patients, improved compliance, and reduced costs for healthcare providers.

[0008] However, the amount of monoclonal antibodies that can be injected via the subcutaneous route is limited. (less than 1 to 2 mL) and therefore liquid formulations with suitable solubility and stability are required. Therefore, a stable liquid pharmaceutical formulation containing a high concentration of an antibody such as trastuzumab is target A need arose to address the need for SC administration for therapy. Summary of the Invention

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

[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 invention is a pharmaceutical composition comprising an active pharmaceutical ingredient comprising an anti-HER2 antibody, methionine, and glutamic acid.

[0012] According to one embodiment of the 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 of the pharmaceutical composition has a concentration of 80 to 150 mg / mL.

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

[0015] According to one embodiment of the invention, the methionine of 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 invention, the pharmaceutical composition further comprises a tonicity agent selected from trehalose, sucrose, mannitol, or sorbitol.

[0017] According to one embodiment of the invention, the tonicity agent of the pharmaceutical composition is sucrose.

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

[0019] According to one embodiment of the invention, the pharmaceutical composition is substantially free of hyaluronidase.

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

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

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

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

[0024] Another embodiment of the present invention is a method of treating cancer in a subject in need thereof, comprising subcutaneously administering to the subject a therapeutically effective amount of the pharmaceutical composition.

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

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

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

[0028] Another embodiment of the invention is a pre-filled syringe containing 3 mL to 7 mL of a solution comprising: (a) trastuzumab 、 Pertuzumab, or mixtures thereof, (b) methionine, and (c) glutamic acid.

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

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

[0031] According to one embodiment of the 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 invention, the solution further comprises sucrose.

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

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

[0035] Although the embodiments are described herein in a manner that allows a clear and concise specification to be written, it is intended, and will be understood, that the 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 description of the drawings]

[0036] [Figure 1]FIG. 1 shows the amino acid sequences of the trastuzumab light chain (SEQ ID NO:1) and the trastuzumab heavy chain (SEQ ID NO:2) with corresponding domains. [Diagram 2] Figure 2. Colloidal stability of high concentration trastuzumab in different buffers. Scattering constant vs. 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. [Diagram 3] Figure 3. Stability of high concentration trastuzumab in acetate buffer as measured by size exclusion chromatography. Figure 3 shows the antibody profile of high concentration trastuzumab under accelerated heat stress at 40°C over 28 days. 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. 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 heat 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. [Diagram 5] FIG 5. Long-term stability of Trastuzumab formulations as measured by SEC-HPLC analysis. FIG 5 shows the antibody integrity of Trastuzumab up to 24 months under different temperature conditions. FIG 5A shows the antibody purity at 5°C. FIG 5B shows the antibody purity at 25°C. FIG 5C shows the antibody purity at 40°C. FIG 6. Long-term stability of Trastuzumab formulations as measured by CEX analysis. [Figure 6]Figure 6 shows the main peak profile of trastuzumab antibody under different temperatures up to 24 months. Figure 6A shows the main peak percentage at 5°C. Figure 6B shows the main peak percentage at 25°C. Figure 6C shows the main peak percentage at 40°C. [Figure 7] Figure 7. Stability of Trastuzumab formulations as measured by SEC-HPLC analysis. Figure 7 shows the antibody integrity of Trastuzumab up to 3 months at different temperatures. Figure 7A shows the antibody purity at 5°C. Figure 7B shows the antibody purity at 25°C. Figure 7C shows the antibody purity at 40°C. [Figure 8] Figure 8. Stability of Trastuzumab formulations measured by CEX analysis. Figure 8 shows the main peak profile of Trastuzumab antibody under different temperatures for up to 3 months. Figure 8A shows the main peak percentage at 5°C. Figure 8B shows the main peak percentage at 25°C. Figure 8C shows the main peak percentage at 40°C. [Figure 9] Figure 9. Stability of Pertuzumab formulations as measured by SEC-HPLC analysis. Figure 9 shows the antibody integrity of Pertuzumab up to 3 months under different temperatures. Figure 9A shows the antibody purity at 5°C. Figure 9B shows the antibody purity at 25°C. Figure 9C shows the antibody purity at 40°C. [Figure 10] Figure 10. Stability of Pertuzumab formulations measured by CEX analysis. Figure 10 shows the charge heterogeneity profile of Pertuzumab antibody 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 in different buffer conditions. Mean tumor volume + / - SE (mm3) is plotted on the y-axis and days after tumor implantation is plotted on the x-axis. PBS buffer control - filled squares, IV trastuzumab - filled circles, and high concentration trastuzumab-3 - filled triangles. [Figure 12] Figure 12. Pharmacokinetic (PK) study investigating plasma concentrations (μg / mL) of trastuzumab versus time in CD-1 mice following subcutaneous or intravenous administration of various formulations of trastuzumab at 10 mg / kg. Compounds high concentration trastuzumab 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 were from 0 to 96 hours. Figure 12B. Observation time points were from 0.25 to 1344 hours. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0038] Anti-HER2 antibody Human epidermal growth factor receptor 2 (HER2) is a tyrosine phosphorylating enzyme that binds to the surface of cell membranes. HER2 protein is known to be involved in signal transduction pathways that cause cell proliferation and cell differentiation, as well as cell malignancy associated with overexpression or activity. Overexpression of HER2 protein may be inhibited using targeted therapeutic agents, including anti-HER2 antibodies. Anti-HER2 antibodies include, but are not limited to, trastuzumab and pertuzumab. Applications for inhibiting overexpression of HER2 also 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 light chain of trastuzumab is (SEQ ID NO: 1).

[0040] The amino acid sequence of the heavy chain of Trastuzumab is (SEQ ID NO: 2). Figure 1 shows the amino acid sequences of the light chain of Trastuzumab (SEQ ID NO: 1) and the heavy chain of Trastuzumab (SEQ ID NO: 2) with 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 of is (SEQ ID NO:3). Pertuzumab The amino acid sequence of the heavy chain of Pertuzumab 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. Further, 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] As used herein, the term "pharmaceutical acceptable salt" refers to a salt that is suitable for use in contact with the tissues of humans and lower animals without causing undue toxicity, irritation, allergic reactions, etc., within the scope of sound medical judgment, and that is commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. provide a detailed description of pharmaceutical acceptable salts in J. Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutical acceptable, non-toxic acid addition salts include salts of amino groups formed using 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 other methods used in the art, such as ion exchange. Other pharma- ceutically acceptable salts include acetic acid, glutamic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, bisulfate, boric acid, butyric acid, camphoric acid, camphorsulfonic acid, citric acid, cyclopentanepropionic acid, digluconic acid, dodecylsulfate, 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 These include sulfuric acid, malic acid, maleic acid, malonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pectic 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, valeric acid, etc. Pharmaceutically acceptable salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N + (C 1-4Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Additionally, pharma- ceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed with counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkylsulfonates, and arylsulfonates.

[0046] Pharmaceutical Compositions The compounds provided according to the present invention are usually administered in the form of liquid pharmaceutical compositions. The present invention therefore provides pharmaceutical compositions containing one or more compounds described herein, or pharma- ceutically acceptable salts or esters, and one or more pharma- ceutically acceptable carriers. As used herein, "pharma- ceutically acceptable carriers" refer to non-toxic carriers, adjuvants, excipients, etc. that do not destroy the pharmacological activity of the formulated compounds. Pharmaceutically acceptable carriers that may be used for the compounds described herein include, but are not limited to, buffers, isotonicity 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 the penetration enhancer hyaluronidase. Hyaluronidase includes all commonly known various hyaluronidases or modified hyaluronidases, such as the enzyme protein 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 the composition contains no detectable hyaluronidase, less than 10 units / dose of hyaluronidase, less than 5 units / dose of hyaluronidase, or less than 1 unit / dose of hyaluronidase, as measured by techniques well known in the art, such as ELISA.

[0048] Pharmaceutical Compositions: Buffers The pharmaceutical compositions may include one or more buffers or buffering agents, which 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 L-histidine / histidine hydrochloride, sodium citrate / citric acid, L-histidine / acetic acid, phosphate, sodium acetate / acetic acid, acetic acid, or combinations thereof.

[0049] The concentration of one buffering agent 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 buffering agent may stabilize the pH of the pharmaceutical composition at 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: Tonicity Agent The pharmaceutical composition may include one or more isotonicity agents. The isotonicity agent may be, but is not limited to, a polyol or a sugar derivative. The isotonicity agent may be sucrose, trehalose, mannitol, sorbitol, or a combination thereof.

[0052] The concentration of one tonicity 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 Compositions: Stabilizers The pharmaceutical composition may include one or more stabilizers, which 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 stabilizing agent 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, 0-50 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 or 10 mM, between 0 and 10 mM, or between 3 and 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 or 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 or 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 or 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 Compositions: Organic Cosolvents The pharmaceutical composition may include one or more organic co-solvents, which may be the organic co-solvent polysorbate 80 (PS80), polysorbate 20 (PS20), poloxamer 188, polyethylene glycol (PEG), propylene glycol, or combinations thereof. In some embodiments, the organic co-solvent may refer to a surfactant.

[0064] The concentration of one organic co-solvent 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 mixtures thereof. In one embodiment, the pharmaceutical composition may include histidine in the buffer of the liquid formulation.

[0066] The pharmaceutical composition may include a salt. The salt may be, but is not limited to, NaCl, KCl, KBr, Na2SO4, NaSCN, K2SO4, etc., or mixtures thereof. The concentration of the salt 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 include a preservative, which may include, but is not limited to, octadecyldimethylbenzylammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol, benzyl alcohol, alkyl parabens, catechol, resorcinol, cyclohexanol, 3-pentanol, m-cresol, and the like, or mixtures thereof.

[0068] The pharmaceutical composition may also contain additives known in the art in concentration ranges that do not substantially adversely affect antibody activity or the usability of the formulation.

[0069] Pharmaceutical Compositions: Combinations of Ingredients The following is a list of contemplated combinations of components for a formulation containing an anti-HER2 antibody containing trastuzumab, pertuzumab, or a mixture thereof. For example, the formulation may contain trastuzumab or pertuzumab at 120 mg / mL.

[0070] A formulation comprising a combination of acetate buffer and sucrose; formulations comprising acetate, sucrose and PS80; formulations comprising acetate, sucrose, and a combination of methionine and glutamic acid; A formulation comprising acetic acid, sucrose, PS80, and a combination of methionine and glutamic acid; A formulation comprising acetate buffer, sucrose, PS80, and methionine; A formulation comprising acetate buffer, sucrose, PS80, and glutamic acid; A formulation comprising acetate buffer, histidine, sucrose, methionine, and glutamic acid; A formulation comprising acetate buffer, histidine, sucrose, methionine, and glycine; A formulation comprising 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 Compositions: Properties The following properties describe the pharmaceutical formulations of the present invention. Examples of techniques used to measure these properties are described in the Examples section below.

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

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

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

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

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

[0077] The following list describes pharmaceutical liquid formulations with specific main component contents (Main Peak / Monomer %):

[0078] 5± A pharmaceutical liquid formulation containing 95 to 99% of the active ingredient as determined by SEC-HPLC after storage at a temperature of 3°C for 28 days.

[0079] in a 20 mM L-histidine buffer of pH 6.0 or 6.5, a 20 mM citrate buffer of pH 5.5 or 6.0, or a 20 mM acetate buffer of pH 5.0 or 5.5, 5 A pharmaceutical liquid formulation containing 95 to 99% of the active ingredient as determined by SEC-HPLC after storage for 28 days at a temperature of 3°C ± 3°C.

[0080] 4 0± A pharmaceutical liquid formulation containing 94 to 99% of the active ingredient as determined by SEC-HPLC after storage for 28 days at a temperature of 2°C.

[0081] 4 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 0± A pharmaceutical liquid formulation containing 94 to 99% of the active ingredient as determined by SEC-HPLC after storage for 28 days at a temperature of 2°C.

[0082] The following list describes pharmaceutical liquid formulations with specific amounts of high molecular weight components (peaks whose retention times lie before the main peak):

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

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

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

[0086] 4 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 0± A pharmaceutical liquid formulation containing 94 to 99% of the active ingredient as determined by SEC-HPLC after storage for 28 days at a temperature of 2°C.

[0087] The following list describes pharmaceutical liquid formulations with specific amounts of low molecular weight components (peaks whose retention times lie after the main peak):

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

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

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

[0091] 4 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 0± A pharmaceutical liquid formulation containing 1.61 to 2.20% of low molecular weight components as determined by SEC-HPLC after storage for 28 days at a temperature of 2°C.

[0092] The following list describes pharmaceutical liquid formulations having specific amounts of major species of charge variant components:

[0093] 5± After storage at a temperature of 3° C. for 28 days, the pharmaceutical liquid formulation contained 55.06 to 64.58% of the major species charge variant component as determined by CIX-HPLC.

[0094] 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 5± After storage at a temperature of 3° C. for 28 days, the pharmaceutical liquid formulation contained 55.06 to 64.58% of the major species charge variant component as determined by CIX-HPLC.

[0095] 4 0± After storage at a temperature of 2°C for 28 days, the pharmaceutical liquid formulation contained 12.71 to 64.15% of the major species charge variant component as determined by CIX-HPLC.

[0096] 4 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 0±After storage at a temperature of 2°C for 28 days, the pharmaceutical liquid formulation contained 12.71 to 64.15% of the major species charge variant component as determined by CIX-HPLC.

[0097] The following list describes pharmaceutical liquids that contain specific amounts of acidic species charge variant components (peaks whose retention times lie before the main species charge variant):

[0098] 5± After storage at a temperature of 3°C for 28 days, the pharmaceutical liquid formulation contained 25.98 to 59.69% of the acidic species charge variant component as determined by CIX-HPLC.

[0099] in a 20 mM L-histidine buffer of pH 6.0 or 6.5, a 20 mM citrate buffer of pH 5.5 or 6.0, or a 20 mM acetate buffer of pH 5.0 or 5.5, 5± After storage at a temperature of 3° C. for 28 days, the pharmaceutical liquid formulation contained 25.98 to 59.69% of the acidic species charge variant component as determined by CIX-HPLC.

[0100] 4 0± After storage at a temperature of 2°C for 28 days, the pharmaceutical liquid formulation contained 39.20 to 71.02% of the acidic species charge variant component as determined by CIX-HPLC.

[0101] 4 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 0± After storage at a temperature of 2° C. for 28 days, the pharmaceutical liquid formulation contained 39.20 to 71.02% of the acidic species charge variant component as determined by CIX-HPLC.

[0102] The following list describes pharmaceutical liquid formulations with specific amounts of basic species charge variant components (peaks whose retention times lie after the main species charge variant):

[0103] 5±After storage at a temperature of 3° C. for 28 days, the pharmaceutical liquid formulation contained 8.47 to 12.56% of the basic species charge variant component as measured by CIX-HPLC.

[0104] in a 20 mM L-histidine buffer of pH 6.0 or 6.5, a 20 mM citrate buffer of pH 5.5 or 6.0, or a 20 mM acetate buffer of pH 5.0 or 5.5, 5± After storage at a temperature of 3° C. for 28 days, the pharmaceutical liquid formulation contained 8.47 to 12.56% of the basic species charge variant component as measured by CIX-HPLC.

[0105] 4 0± After storage at a temperature of 2° C. for 28 days, the pharmaceutical liquid formulation contained 16.27 to 34.50% of the basic species charge variant component as measured by CIX-HPLC.

[0106] 4 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 0± After storage at a temperature of 2° C. for 28 days, the pharmaceutical liquid formulation contained 16.27 to 34.50% of the basic species charge variant component as measured by CIX-HPLC.

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

[0108] A pharmaceutical liquid formulation having an absorbance A350 of 0.23 to 0.338 as measured using a spectrophotometer (e.g., a SpectraMax® iD3 multi-mode microplate reader) after storage for 28 days at a temperature of 5° C.±3° C.

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

[0110] A pharmaceutical liquid formulation having an absorbance A350 of 0.232 to 0.397 as measured using a spectrophotometer (e.g., a SpectraMax® iD3 multi-mode microplate reader) after storage for 28 days at a temperature of 40° C.±2° C.

[0111] 4 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 0± A pharmaceutical liquid formulation having an absorbance A350 of 0.232 to 0.397 when measured using a spectrophotometer (e.g., a SpectraMax® iD3 multimode microplate reader) after storage for 28 days at a temperature of 2°C.

[0112] The following list describes pharmaceutical liquid formulations with particular colloidal stability of the API in a buffer:

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

[0114] DLS is used to determine API aggregation and colloidal stability in histidine buffers by measuring the positive gradient. Samples are run at increasing concentrations of API in histidine buffers at pH 6.0 or pH 6.5.

[0115] DLS is used to determine API aggregation and colloidal stability in citrate buffers by measuring the negative slope. Samples are run at increasing concentrations of API in citrate buffers at pH 5.5 or pH 6.0.

[0116] DLS is used to determine API aggregation and colloidal stability in acetate buffers by measuring the positive gradient. Samples are run at increasing concentrations of API in acetate buffers at pH 5.0 or pH 5.5.

[0117] Pharmaceutical Compositions: Preparation Such compositions are prepared according to methods well known in the pharmaceutical art (see, e.g., 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 injectable fluids can be prepared by incorporating the compound according to the present invention in the required amount in a suitable solvent with various other ingredients as listed above, if necessary, and then sterilized by filtration. Typically, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and other ingredients required from those listed above. In the case of sterile powders for preparing sterile injectable solutions, the preparation method is vacuum drying and freeze-drying technology, which yields a powder of active ingredient and any additional desired ingredient from the solution that has previously been sterile-filtered.

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

[0120] Treatment Methods and Routes of Administration The present invention provides a method of treating a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein.

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

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

[0123] The methods described herein further include identifying a subject with cancer prior to administration of a pharmaceutical composition described herein.

[0124] The present invention also provides the following: (1) A pharmaceutical composition as described herein for use in a method of treating a subject by therapy. (2) A pharmaceutical composition as described herein for use in a method for treating cancer, wherein the method comprises administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition. (3) A pharmaceutical composition as described herein for use in binding HER2, wherein binding HER2 comprises administering to a subject in need thereof a therapeutically effective amount of the composition. (4) Use of a therapeutically effective amount of a pharmaceutical composition described herein to treat a subject in need thereof. (5) Use of a pharmaceutical composition described herein for the treatment of cancer in a subject in need thereof, wherein the treatment comprises administering to the subject a therapeutically effective amount of the pharmaceutical composition. (6) Use of a pharmaceutical composition described herein for binding HER2, wherein binding HER2 comprises administering to a subject in need thereof a therapeutically effective amount of the composition. (7) Use of a pharmaceutical composition described herein for the manufacture of a medicament. (8) Use of a pharmaceutical composition as described herein for the manufacture of a medicament for use in treating cancer in a subject in need thereof, wherein treating comprises administering to the subject a therapeutically effective amount of the composition. (9) Use of a pharmaceutical composition as described herein for the manufacture of a medicament for use in binding HER2, wherein binding HER2 comprises administering to a subject in need thereof a therapeutically effective amount of the composition.

[0125] As used herein, and unless otherwise specified, the terms "treat," "treating," and "treatment" refer to treatment that occurs while a subject is afflicted with a particular disease, disorder, or condition, which reduces the severity of the disease, disorder, or condition, or prevents or slows the progression of the disease, disorder, or condition ("therapeutic treatment"). Disease, disorder, and condition are used interchangeably herein.

[0126] As used herein, a "subject" contemplated for management includes a human (i.e., male or female of any age, e.g., a pediatric subject (e.g., infant, child, adolescent) or an adult subject (e.g., young adult, middle-aged adult, or elderly adult) and / or a non-human animal, e.g., a mammal such as a primate (e.g., cynomolgus monkey, rhesus monkey), cow, pig, horse, sheep, goat, mouse, cat, and / or dog. In certain embodiments, the subject is a human. In certain embodiments, the subject is a non-human animal. As used herein, the terms "human," "patient," and "subject" are used interchangeably.

[0127] As used herein, and unless otherwise specified, a "therapeutically effective amount" is an amount sufficient to provide a therapeutic benefit in the treatment of a disease, disorder, or condition, or to delay or minimize one or more symptoms associated with a disease, disorder, or condition. A therapeutically effective amount of a compound refers to an amount of a therapeutic agent that provides a therapeutic benefit in the treatment of a disease, disorder, or condition. The term "therapeutically effective amount" can include an amount that improves overall therapy or reduces or avoids the symptoms or causes of a disease or condition.

[0128] The pharmaceutical compositions may be administered for the treatment of cancer, 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 compositions may be administered for the treatment of metastatic gastric or gastroesophageal junction adenocarcinoma, as well as ovarian cancer, gastric cancer, colon cancer, and It may be administered for the treatment of cancer, such as gastrointestinal cancer.

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

[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 in a volume of 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] For subcutaneous administration, patients should receive treatment once per week, once per 2 weeks, once per 3 weeks, once per 4 weeks, or once per 5 weeks. Doses are administered over 2-5 minutes and are preferably injected at different sites in the thigh. Patients with early stage breast cancer should receive trastuzumab subcutaneously for 52 weeks or until disease recurrence or unacceptable cardiac toxicity, whichever occurs first. Patients with metastatic breast cancer (MBC) should receive trastuzumab subcutaneously until disease progression is observed.

[0132] The subject may be administered the drug subcutaneously using a pharmaceutical composition configured for subcutaneous administration, such as a pre-filled syringe, a microneedle, a sustained release delivery system, and a stimuli-responsive delivery system.

[0133] The trastuzumab subcutaneous therapeutic solution may be delivered with a commercially available syringe injection system, such as KORU™ FreedomEdge® (available from KORU™ Medical Systems, Chester, NY, USA).

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

[0135] In other embodiments, the trastuzumab subcutaneous therapeutic solution may be delivered through commercially available medical devices, such as, but not limited to, injection devices, infusion pumps, self-injection devices, needleless devices, or subcutaneous patch delivery systems. For example, the trastuzumab subcutaneous therapeutic solution may be delivered by commercially available infusion pumps, such as the Crono S-PID pump (available from Canes SPA), described in U.S. Patent No. 8,172,814 B2. The trastuzumab subcutaneous therapeutic solution may be delivered by other commercially available infusion pumps, such as the SCIg60 injector (available from EMED Technologies Corporation), described in U.S. Patent No. 9,808,576 B2 and U.S. Patent Publication No. 20130138075.

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

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

[0138] In other embodiments, the trastuzumab subcutaneous therapeutic solution may be supplied in a commercially available autoinjector, such as the autoinjector device available from West Pharmaceutical Services and described in US Pat. No. 8,048,029 B2.

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

[0140] Working Example Sample preparation High-concentration trastuzumab The IV trastuzumab was reprocessed by ultrafiltration / diafiltration (UF / DF) and filtration. Ugh Made by Can Concentration and buffer exchange are performed High-concentration trastuzumabIt is a critical processing step in manufacturing. Centrifugal filtration and tangential flow filtration (TFF) are two approaches to achieve buffer exchange and concentration of mAbs. In early small-scale development, limited sample availability required buffer exchange to be performed with dialysis tubing or dialysis cassettes with 20 kDa cutoff porous membranes. High-concentration trastuzumab The samples were concentrated by repeated centrifugation at a spin speed of 4200 rpm in a 30 kDa cutoff centrifugal filter for 20 min at 4 °C to achieve the target concentration. The centrifuge used was an Eppendorf 5804R equipped with an S-4-72 swinging rotor.

[0141] In large-scale laboratory development, High-concentration trastuzumab Sample preparation included the use of TFF for concentration and buffer exchange of the mAbs. TFF processing was performed using a Merck Microchip product. l It was performed using a lipore Labscale TFF System® with Pellicon® 3 and Ultracel® 30 kDa membrane, D cassette. Trastuzumab was diafiltered against test formulation buffer and concentrated to the target concentration.

[0142] Highly concentrated Pertuzumab is produced from processed Pertuzumab by chromatography, ultrafiltration / diafiltration (UF / DF), and filtration. Concentration and buffer exchange are key processing steps in the manufacture of Pertuzumab. Centrifugal filtration and tangential flow filtration (TFF) are two approaches to achieve buffer exchange and concentrate the mAb. In early small-scale development, limited sample availability required buffer exchange to be performed with dialysis tubing or dialysis cassettes with a 20 kDa cutoff porous membrane. Pertuzumab samples were concentrated by repeated centrifugation at a spin speed of 4200 rpm in 30 kDa cutoff centrifugal filters for 20 min at 4°C to achieve the target concentration. The centrifuge used was an Eppendorf 5804R with an S-4-72 swing rotor.

[0143] For large-scale laboratory development, Pertuzumab sample preparation involved the use of TFF for concentration and buffer exchange of the mAb. TFF processing was performed using a Merck Milipore Labscale TFF System® with Pellicon® 3 and Ultracel® 30 kDa membranes, D-cassettes. Pertuzumab was diafiltered against test formulation buffer and concentrated to the target concentration.

[0144] Stability Testing Method Turbidity Turbidity is the detection of visible / subvisible particles and therefore tests both the quality and stability of the formulation. Samples were mixed by gentle inversion 10 times before loading into a 96-microwell plate (Cat. No. 269620). 200 μL of sample was loaded in triplicate into each well. Samples were measured in triplicate on a SpectraMax® iD3 multimode microplate reader. Measurements were recorded as the average triplicate absorbance.

[0145] Purity, Aggregation, and Cleavage 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) provided with a column (TOSOH TSK-gel G3000 SWxl, 7.8 x 300 mm, PN:0008541).

[0146] Charge variants The 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). The column (TOSOH TSK-gel CM-STAT, 4.6 x 100 mm, 7 um, CN: 0021966, Lot: 082GA0017G) was provided. The main component (main peak), acidic variants, and basic variants 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. Samples were gently mixed by inversion 10 times before loading into an Auroa 384 microplate (Wyatt PN:P8806-38401). onset Or T agg For the 1000-mL 1000-mL 1000-mL 15 ...

[0149] Protein content assay Protein concentration was measured by protein content assay using Spectrophotometer Jasco v-730. Detection wavelength was set at 280 nm. Samples were diluted to appropriate concentrations to give OD 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 involved turning on the spectrophotometer and UV lamp at least 20 minutes prior to use.

[0151] 280 nm was the detection wavelength. 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 the spectrophotometer and recorded as a blank. The formulation base buffer was discarded and the cuvette was rinsed with test sample for equilibration. The test sample was discarded from the cuvette.

[0152] The test sample was loaded into the cuvette and measured. The sample was discarded and the test sample was reloaded and measured in duplicate. The cuvette was washed with formulation base buffer and the steps from taking the 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 by Beer's law formula.

[0154] Binding activity HER2-ECD binding ELISA The binding activity of the antibodies 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 of trastuzumab with its receptor by Bio-Layer Interference (BLI) technology. The measurement is performed using ForteBio OctetRed96 (Pall, instrument number BAZ-20002).

[0156] Stability Test Example 1: Comparison of Stability with Buffer Solution Stability Test For the liquid pharmaceutical formulation used in Example 1, each buffer solution was prepared to match the corresponding pH and concentration, and the antibody was added to obtain the samples shown in Tables 1, 2, and 3 below. The specific contents of each component are as shown in Tables 1, 2, and 3 below. R The total volume was, for example, 5 mL.

[0157] [Table 1]

[0158] [Table 2]

[0159] [Table 3]

[0160] The stability of the liquid pharmaceutical formulations prepared in Examples 1 to 3 was measured after 0 days (referred to as "initial"), 3 days, 1 week, 2 weeks, and 4 weeks at a temperature 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 citric acid system using a 20 mM citric acid / 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] FIG. 2 shows the colloidal stability of trastuzumab in different buffers. DLS was used to plot the scattering constant versus trastuzumab concentration. FIG. 2A shows the colloidal stability of trastuzumab in histidine buffer at pH 6.5. FIG. 2B shows the colloidal stability of trastuzumab in histidine buffer at pH 6.0. FIG. 2C shows the colloidal stability of trastuzumab in citrate buffer at pH 6.0. FIG. 2D shows the colloidal stability of trastuzumab in citrate buffer at pH 5.5. FIG. 2E shows the colloidal stability of trastuzumab in acetate buffer at pH 5.5. FIG. 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 slope. In one embodiment, histidine buffer, acetate buffer or citrate buffer may be used as a buffer in the liquid pharmaceutical composition.

[0162] Stability Testing Example 2: Stability of the Compositions Depending on the pH of the Acetate Buffer Stability Test For the liquid pharmaceutical formulation used in Example 2, each buffer solution was prepared to match the corresponding pH and antibody concentration to obtain the samples shown in Table 4 below. The concentrations of each component are as shown in Table 4 below. R The total volume was, for example, 5 mL.

[0163] [Table 4] JPEG2025513655000072.jpg22362

[0164] The above formulation is 0± Stability was measured after 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, or 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 antibody profile measured by SEC-HPLC over 28 days under accelerated heat stress at 40°C. High-concentration trastuzumab Figure 3 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 antibody measured by CIX-HPLC. Figure 4 shows the charge variant profile of the antibody under accelerated heat stress at 40°C over 28 days. 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 adjusted by adding an isotonicity agent or salt to match the corresponding pH and antibody concentration, to obtain the samples shown in Table 5 below. The concentrations of each component are as shown in Table 5 below. R The total volume was, for example, 5 mL.

[0169] [Table 5] JPEG2025513655000074.jpg19984

[0170] The above formulations were tested for stability after 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 containing 0.05% PS20, 150 mM trehalose, or 0.9% NaCl, and 20 mM acetic acid / sodium acetate solution at pH 5.6 or 5.2, respectively. The antibody concentration is 120 mg / mL. The results are shown in Table 5 above.

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

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

[0173] [Table 6]

[0174] The above formulations were tested for stability after 0 days (initial) and 4 days at a temperature of 40° C.±2° C. 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 include, but is not limited to, histidine buffer, trehalose, methionine, or NaCl, or a combination thereof.

[0176] [Table 7]

[0177] The above formulations were tested for stability after 0 days (initial) and 4 days at a temperature of 40° C.±2° C. The formulations represented by samples 8 through 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 contain, but is not limited to, histidine buffer, mannitol, methionine, or NaCl, or a combination thereof.

[0178] [Table 8]

[0179] The above formulations were tested for stability after 0 days (initial) and 4 days at a temperature of 40° C.±2° C. 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 contain, 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 exhibits a higher % monomer (%) of 92.08% after exposure at 40° C.±2° C. for 4 days, compared to 79.87% for the formulation containing sorbitol and methionine.

[0182] The above formulations were tested for stability after 0 days (initial) and 4 days at a temperature of 40° C.±2° C. 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 contain, but is not limited to, histidine buffer, sucrose, methionine, or NaCl, or a combination thereof.

[0183] [Table 10]

[0184] The above formulations were tested for stability after 0 days (initial) and 4 days at a temperature of 40° C.±2° C. 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 contain, but is not limited to, acetate buffer, trehalose, methionine, or NaCl, or a combination thereof.

[0185] [Table 11]

[0186] The above formulations were tested for stability after 0 days (initial) and 4 days at a temperature of 40° C.±2° C. 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 contain, but is not limited to, acetate buffer, mannitol, methionine, or NaCl, or a combination thereof.

[0187] [Table 12]

[0188] The above formulations were tested for stability after 0 days (initial) and 4 days at a temperature of 40° C.±2° C. 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 contain, but is not limited to, acetate buffer, sorbitol, methionine, or NaCl, or a combination thereof.

[0189] [Table 13]

[0190] It is noteworthy that the formulations containing sucrose consistently had a higher % monomer after 0 days (initial) than the formulations containing sorbitol.

[0191] The above formulations were tested for stability after 0 days (initial) and 4 days at a temperature of 40° C.±2° C. 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 contain, but is not limited to, acetate buffer, sucrose, methionine, or NaCl, or a combination thereof.

[0192] Stability Test Example 5: Stability of Compositions Depending on the Type of Amino Acid Stability Test For the liquid pharmaceutical formulation used in Example 5, each buffer solution was prepared by adding sucrose and further adding organic co-solvent to match the corresponding amino acid and antibody concentration, to obtain the samples shown in Tables 14 to 16 below. The concentrations of each component are as shown in Tables 14 to 16 below. R The total volume was, for example, 4 mL.

[0193] [Table 14] JPEG2025513655000084.jpg19673

[0194] The stability of the liquid pharmaceutical formulations prepared in Examples 9 to 13 was measured after 0 weeks (denoted as initial), 1 week, 2 weeks, 3 weeks, and 4 weeks at a temperature of 40°C ± 2°C. Examples 9 to 13 include 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 includes 10 mM methionine, and Example 10 further includes 50 mM arginine. Example 11 further includes 20 mM glutamic acid. Example 12 further includes a combination of 10 mM methionine and 50 mM arginine. Example 13 further includes 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 include, but is not limited to, acetate buffer, sucrose, PS80, methionine or glutamic acid, or combinations thereof.

[0195] [Table 15]

[0196] The stability of the liquid pharmaceutical formulations prepared in Examples 9 and 13 was measured at 0 hours (denoted as initial) and after 4 hours under constant vortexing in the stirring stress test. The results are shown in Table 15 above. The formulations are stable even after stirring.

[0197] [Table 16]

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

[0199] Stability Testing Example 6: Stability of the Compositions Subjected to Heat or UV Stress Stability Test For the liquid pharmaceutical formulation used in Example 6, each buffer solution was prepared by adding methionine and further adding organic co-solvent to match the corresponding buffer concentration, pH, and isotonicity agent to obtain the samples shown in Tables 17 to 18 below. The concentrations of each component are as described in Tables 17 to 18 below. R The total volume was, for example, 5 mL.

[0200] [Table 17] JPEG2025513655000088.jpg22525

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

[0202] [Table 18] JPEG2025513655000090.jpg22538

[0203] photostability For the liquid pharmaceutical formulations prepared in Examples 14 to 17, UV / visible light irradiation in the UV range (320 nm-400 nm) (≥ 200 watt-hours / m 2The stability was measured after 0 days (designated initial), 3 days, 6 days, 10 days, and 2 weeks under constant exposure to 1000 mM NaCl, 1 ...

[0204] Stability Test Example 7: Stability of the composition depending on the type of buffer and isotonicity agent Stability Test For the liquid pharmaceutical formulation used in Example 7, each buffer solution was prepared by adding methionine and further adding organic co-solvents to match the corresponding buffer type and tonicity agent to obtain the samples shown in Tables 19 to 20 below. The concentrations of each component are as shown in Tables 19 to 20 below. R 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 stability of the liquid pharmaceutical formulations prepared in Examples 18 to 25 was measured after 0 weeks (referred to as 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 include, but is not limited to, acetate buffer, methionine, sucrose, or trehalose, or a combination thereof. In other embodiments, the formulation may include, but is not limited to, histidine buffer, methionine, sucrose, or trehalose, or a combination thereof.

[0208] Stability Testing Example 8: Stability of Compositions Depending on Organic Co-solvent and Amino Acid Type For the liquid pharmaceutical formulations used in Example 8, each buffer solution was prepared by adding an isotonicity agent to match the corresponding buffer type and organic co-solvent and amino acid to obtain the samples shown in Tables 21 to 22 below. The concentration of each component was 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 measured for stability at 40°C ± 2°C after 0 months (referred to as initial), 0.5 months, 1 month, 3 months (or 3.36 months in Table 28), and 6 months. 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 include, but is not limited to, acetate buffer, sucrose, PS80, methionine, and glutamic acid. In other embodiments, the formulation may include acetate buffer, sucrose, PS80, methionine, or glutamic acid, or a combination thereof. In yet other embodiments, the formulation may include histidine buffer, trehalose, PS20, methionine, or a combination thereof.

[0212] Stability Study Example 9: Long-Term Stability Stability Test For the liquid pharmaceutical formulations used in Example 9, each buffer was prepared by adding an isotonicity agent to match the corresponding buffer type and organic co-solvent and amino acid, and sample examples 33 to 36 were obtained and their integrity stability was measured by SEC-HPLC after 0, 0.25, 0.5, 0.75, 1, 2, 3.36, 6, 12, 18, and 24 months at 5° C., and up to 6 months at temperatures of 25° C. and 40° C. 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 of Example 36 is able to maintain high levels of antibody purity for at least 24 months. Examples 33 to 36 were measured for charge heterogeneity by CEX analysis after 0, 0.25, 0.5, 0.75, 1, 2, 3.36, 6, 12, 18 and 24 months at 5°C (Figure 6A), after 6 months at a temperature of 25°C (Figure 6B) and after 1 month at 40°C (Figure 6C).

[0213] Stability test Example 10: Examination of isotonicity agents Stability Test For the liquid pharmaceutical formulation used in Example 10, each buffer was prepared by adding an isotonicity agent to match the type of the corresponding buffer and the organic co-solvent and amino acid, and sample examples 37 to 40 were obtained to measure the integrity stability after 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. Integrity results at 5° C., 25° C., and 40° C. are shown in Figures 7A, 7B, and 7C, respectively.

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

[0215] Stability Study Example 11: Stability Evaluation of Pertuzumab For the liquid pharmaceutical formulation used in Example 11, each buffer was prepared by adding an isotonicity agent to match the type of the corresponding buffer and the organic co-solvent and amino acid, and sample examples 41 to 44 were obtained to measure 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 formulation containing 120 mg / mL 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 formulation containing 120 mg / mL 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 formulation containing 120 mg / mL pertuzumab, 20 mM acetate, pH 5.2, 210 mM mannitol, 10 mM methionine, 10 mM glutamic acid, and 0.02% polysorbate 80. Example 44 is a formulation containing 120 mg / mL pertuzumab, 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 temperatures of 5° C., 25° C., and 40° C. are shown in Figures 9A, 9B, and 9C, respectively. Sample Examples 41 to 44 can maintain the antibody purity of pertuzumab at a high level.

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

[0217] Animal testing Animal Studies Example 1: Mouse Pharmacodynamic (PD) Study-Antitumor Activity of Trastuzumab under Different Buffer Conditions. Xenograft model. All procedures were performed in accordance with the appropriate laws, regulations, and guidelines of the National Institutes of Health (NIH) and approved by the Animal Care and Use Committee of Covance, USA. 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's medium (DMEM) medium and transfected into NSG mice (NOD.Cg-Prkdc scid Il2rg tm1Wjl Tumors were subcutaneously inoculated into mice (Jackson Laboratory, USA). Treatment was started 7 days after tumor inoculation. Tumor volume (mm 3 ) is the formula (length x width 2 ) / 2, where length is the longest axis and width is the thickness measurement perpendicular to the length. Data are expressed as the mean tumor volume for each treatment group ± SE. All data were analyzed for significance by Student's T-test. Six mice per group were used for each study.

[0218] Cell lines and culture conditions Human breast cancer cells, BT-474, were cultured in DMEM, 10% non-heat-inactivated fetal bovine serum (FBS), and 1% penicillin / streptomycin / L-glutamine (PSG) at 37°C. Upon implantation into mice, cell viability was 95%.

[0219] Animal treatment All mice were assigned to study groups based on caliper estimates of tumor burden. Mice were treated according to Table 23.

[0220] Table 23: Treatment groups and dosing schedule according to Figure 11. [Table 23]

[0221] Note: ROA - Route of administration, PBS - Phosphate buffered saline, Herceptin SC - Commercial Roche EU made trastuzumab for injection SC 600 mg, High-concentration trastuzumab and IV trastuzumabThe study consisted of 6 doses of the same active pharmaceutical ingredient (API) (trastuzumab), SC-subcutaneous, IV-intravenous, Q7Dx6-one treatment every 7 days for a total of 6 doses. IV trastuzumab 150 mg of lyophilized trastuzumab was reconstituted with water for injection and finally in a buffer of 4.4 mM L-histidine / L-histidine hydrochloride, pH 6.0, 1.71% trehalose dihydrate, 0.01% PS20. High-concentration trastuzumab -3 was formulated in 20 mM acetic acid / sodium acetate, pH 5.2, 210 mM sucrose, 10 mM methionine, 10 mM glutamic acid, and 0.02% PS80.

[0222] The PD results of these treatments are depicted in FIG.

[0223] Animal Studies Example 2: Mouse Pharmacodynamics (PD) Study to Examine Plasma Concentrations (μg / mL) vs. Time of Trastuzumab in Various Formulations Test articles containing trastuzumab in various formulations for PD studies 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 Contains the same API (Trastuzumab). Met-Methionine, Ace-Acetic acid / Sodium acetate, PS-Polysorbate.

[0226] PK studies were performed in CD-1 male mice (Vital River, Pinghu, China). At the time of study, the mice were 5-6 weeks old and weighed between 25-38 g. The animals were housed in polycarbonate cages with bedding in groups of up to 5 mice / sex / cage. Animals were randomly assigned to each group. The mice were divided into 5 groups of 25 mice each. Groups 1-4 were administered SC bolus doses. Group 5 was administered IV bolus doses. 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 per time point. Samples were collected into serum separator tubes (SSTs) at 16 time points: pre-dose and 0.25, 1, 4, 8, 24 hours post-dose, as well as 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. Blood was allowed to clot for 30 minutes at room temperature. Samples were centrifuged at approximately 2700 g for approximately 10 minutes at 2 to 8°C within 2 hours of collection, 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 an ELISA method.

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

[0231] Table 26: Summary of PK study results 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 : Time it takes for the drug to reach its maximum concentration in serum, C max : Maximum (or peak) concentration of 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 to infinity, VD: volume of distribution, CL: clearance, MRT: mean residence time, F%: fractional bioavailability.

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

[0234] Equivalents and Scope In the claims, articles such as "a," "an," and "the" may mean one or more, unless otherwise indicated or clear from the context. A claim or description containing "or" between one or more members of a group is considered to be satisfied if one, more than one, or all of the group are present in, used in, or otherwise relevant to a given product or process, unless otherwise indicated or clear from the context. The invention includes embodiments in which exactly one member of a group is present in, used in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one or all of the members of a group are present in, used in, or otherwise relevant to a given product or process.

[0235] Furthermore, the present invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the claims set forth are introduced into another claim. For example, any claim that is dependent on another claim can be amended to include one or more limitations set forth in any other claim that is dependent on the same base claim. When elements are presented in list form, e.g., in Markush group form, subgroups of each element are also disclosed, and any element can also be removed from the group. When an invention, or an aspect of an invention, is referred to as including certain elements and / or features, it should be understood that a particular embodiment of the invention or aspect of an invention includes or consists essentially of such elements and / or features. For the sake of brevity, these embodiments have not been specifically set forth in that language herein. Additionally, the terms "comprising" and "containing" are intended to be open to permit the inclusion of additional elements or steps. When ranges are presented, both ends are included. Furthermore, unless otherwise specified or clear from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges can take any particular value or subrange within the ranges defined in different embodiments of the invention, down to one-tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0236] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. In the event of a conflict between an incorporated reference and this specification, this specification will control. Furthermore, any particular embodiment of the present invention that falls within the prior art may be expressly excluded from one or more claims. Such embodiments may be excluded even if the exclusion is not expressly set forth herein, since they are deemed to be well known to those of skill in the art. Any particular embodiment of the present invention may be excluded from any claim for any reason, whether or not related to the existence of prior art.

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

Claims

1. 1. A pharmaceutical composition comprising: (a) an active pharmaceutical ingredient comprising an anti-HER2 antibody; (b) methionine, and (c) Glutamic acid 13. A pharmaceutical composition comprising:

2. The pharmaceutical composition of claim 1 , wherein the anti-HER2 antibody comprises trastuzumab or pertuzumab.

3. 2. The pharmaceutical composition of claim 1, wherein the anti-HER2 antibody is trastuzumab or pertuzumab, and the trastuzumab or pertuzumab has a concentration of 80 to 150 mg / mL.

4. 10. The pharmaceutical composition of claim 1, wherein the composition further comprises glycine, lysine, glutamine, or a combination thereof.

5. 2. The pharmaceutical composition of 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.

6. 10. The pharmaceutical composition of claim 1, further comprising a tonicity agent selected from trehalose, sucrose, mannitol, and sorbitol.

7. 7. The pharmaceutical composition of claim 6, wherein the tonicity agent is sucrose.

8. 8. The pharmaceutical composition of claim 7, wherein the sucrose has a concentration of 150 to 300 mM.

9. 10. The pharmaceutical composition of claim 1, wherein the composition is substantially free of hyaluronidase.

10. The pharmaceutical composition of claim 1 , wherein the composition is adapted for subcutaneous administration.

11. 10. The pharmaceutical composition of claim 1, further comprising an acetate buffer at pH 4.6 to 6.

5.

12. 13. A method for treating cancer in a subject in need thereof, comprising subcutaneously administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 1.

13. 13. The method of claim 12, wherein the cancer is breast cancer.

14. 13. The method of claim 12, wherein the cancer is metastatic gastric cancer or gastroesophageal junction adenocarcinoma.

15. 13. The method of claim 12, wherein the pharmaceutical composition is administered once every three weeks.

16. A pre-filled syringe containing 3 mL to 7 mL of a solution, the solution comprising: (a) Anti-HER2 antibody, (b) methionine, and (c) Glutamic acid A prefilled syringe comprising:

17. 17. The pre-filled syringe of claim 16, wherein the anti-HER2 antibody is trastuzumab or pertuzumab and has a concentration of 80 to 150 mg / mL.

18. 18. The pre-filled syringe of claim 17, wherein the methionine has a concentration of 5 to 15 mM and the glutamic acid has a concentration of 5 to 15 mM.

19. 17. The pre-filled syringe of claim 16, wherein the solution further comprises sucrose.

20. 17. A method of treating cancer in a subject in need thereof, comprising subcutaneously administering to the subject a therapeutically effective amount of the solution from the prefilled syringe of claim 16.

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