Pharmaceutical compositions comprising anti-HER2 antibodies for subcutaneous administration

Abstract

The present invention relates to a stable liquid pharmaceutical formulation comprising a high concentration of trastuzumab, pertuzumab, or a combination thereof for subcutaneous administration. The formulations of the invention may be administered to treat cancers such as breast cancer and metastatic gastric cancer or gastroesophageal nodal adenocarcinoma.

Description

ASCII files incorporated into this article for reference

[0001] This article incorporates the sequence listing file prepared on April 14, 2023 (file name: P4330-PCT_seqlisting, size 6B) for reference. Related Applications

[0002] This application claims the entire contents of U.S. Provisional Application Nos. 63 / 333,930 and 63 / 333,935 filed on April 22, 2022, the contents of which are hereby incorporated by reference. Background Art

[0003] Human epidermal growth factor receptor 2 (HER2) is a proto-oncogene that encodes a transmembrane protein. HER2 protein is known to be overexpressed in various cancers, such as breast cancer (e.g., breast cancer), gastric cancer (e.g., gastric adenocarcinoma), and gastroesophageal cancer (e.g., gastroesophageal junction adenocarcinoma). HER2-expressing breast and gastrointestinal cancers can be treated by targeting the overexpressed HER2 protein with a monoclonal antibody (MAb) specific for HER2. Trastuzumab is a recombinant humanized monoclonal antibody that binds to HER2. After binding to HER2 on the surface of tumor cells, trastuzumab induces antibody-dependent cell-mediated cytotoxicity and inhibits tumor cells that overexpress HER2 protein.

[0004] Over the past few years, the medical use of monoclonal antibodies has gradually increased. In many cases, these monoclonal antibodies are injected into the body of an individual via the intravenous (IV) route. Most therapeutic monoclonal antibodies are delivered to the human body via the conventional IV route. IV administration requires patients to travel long distances to medical institutions, where medical professionals must be trained to perform IV infusions and determine the appropriate dose and infusion rate. Clinical administration is not only expensive, but also quite inconvenient for patients.

[0005] In addition, when injecting an appropriate dose of low-concentration monoclonal antibody, IV infusion often takes a long time (about 90 minutes) and is not convenient for patients (needing to go to a fully staffed clinic).

[0006] Alternative routes of administration include subcutaneous (SC) administration, which allows patients to self-administer therapeutic agents at home, which is more convenient and economical to implement. This is more convenient for patients, improves compliance, and reduces costs for healthcare providers.

[0007] However, based on the solubility and stability in appropriate liquid formulations, as well as the volume of SC injections, the number of monoclonal antibodies that can be injected via the subcutaneous route is limited. SC administration is usually administered via a SC injection device, which is limited by the injection volume (<1 to 2 ml). Therefore, the related field is in urgent need of a stable liquid pharmaceutical formulation containing a high concentration of an antibody (e.g., trastuzumab) to solve the problem of SC administration during monoclonal antibody therapy. Summary of the invention

[0008] One embodiment of the present invention relates to a pharmaceutical composition comprising an anti-HER2 protein, a buffer having a pH of 4.6 to 6.5, one or more stabilizers, and a tensile agent.

[0009] In one embodiment of the present disclosure, the pharmaceutical composition comprises an anti-HER2 antibody, sucrose and methionine.

[0010] In another embodiment of the present disclosure, a pharmaceutical composition comprises an active pharmaceutical ingredient, methionine, and glutamic acid, wherein the active pharmaceutical ingredient comprises an anti-HER2 antibody.

[0011] According to one embodiment of the present disclosure, the anti-HER2 antibody comprises trastuzumab or pertuzumab.

[0012] According to one embodiment of the present disclosure, the anti-HER2 antibody is trastuzumab or pertuzumab, and the concentration of trastuzumab or pertuzumab in the pharmaceutical composition is 80 to 150 mg per milliliter.

[0013] According to one embodiment of the present disclosure, the stabilizer comprises methionine, glutamic acid, arginine, N-α-acetylarginine, proline, glycine, lysine, glutamine or a combination thereof.

[0014] According to one embodiment of the present disclosure, the concentration of methionine in the pharmaceutical composition is 5 to 15 mM, and the concentration of glutamate is 5 to 15 mM.

[0015] According to one embodiment of the present disclosure, the pharmaceutical composition further comprises a tension agent selected from the group consisting of trehalose, sucrose, mannitol and sorbitol.

[0016] According to one embodiment of the present disclosure, the tonicity agent of the pharmaceutical composition is sucrose.

[0017] According to one embodiment of the present disclosure, the concentration of sucrose is 150 to 300 mM.

[0018] According to one embodiment of the present disclosure, the pharmaceutical composition is substantially free of hyaluronidase.

[0019] According to one embodiment of the present disclosure, the pharmaceutical composition is for subcutaneous administration.

[0020] According to one embodiment of the present disclosure, after storage at 5°C±3°C for 3 days, the relative potency (%) of the pharmaceutical composition for binding to FcγRIII is 93.9 to 107.9.

[0021] According to one embodiment of the present disclosure, the pharmaceutical composition comprises: (a) 5 to 150 mg of trastuzumab or pertuzumab per milliliter; (b) 0 to 15 mM methionine; (c) 0 to 15 mM glutamate; (d) 0 to 0.05% (w / v) polysorbate 80; (e) 0 to 300 mM sucrose; and (f) 5 to 40 mM acetate buffer at a pH of 4.6 to 6.5.

[0022] According to one embodiment of the present disclosure, the pharmaceutical composition comprises: (a) 80 to 150 mg of trastuzumab or pertuzumab per milliliter; (b) 5 to 15 mM methionine; (c) 5 to 15 mM glutamate; (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 a pH of 4.6 to 6.5.

[0023] Another embodiment of the present disclosure is directed to a method for treating cancer in a subject in need of such treatment, comprising subcutaneously administering to the subject a therapeutically effective amount of a pharmaceutical composition.

[0024] According to one embodiment of the present disclosure, the cancer is breast cancer.

[0025] According to one embodiment of the present disclosure, the cancer is metastatic gastric cancer or gastroesophageal junction adenocarcinoma.

[0026] According to one embodiment of the present disclosure, the pharmaceutical composition is administered once every three weeks.

[0027] Another embodiment of the present disclosure is a prefilled syringe comprising 3 ml to 7 ml of a solution comprising: (a) trastuzumab or pertuzumab; (b) methionine; and (c) glutamic acid.

[0028] Another embodiment of the present disclosure is a prefilled syringe comprising 3 ml to 7 ml of a solution comprising: (a) trastuzumab or pertuzumab; (b) methionine; (c) glutamic acid; (d) an organic cosolvent; (e) sucrose; and (f) an acetate buffer.

[0029] According to one embodiment of the present disclosure, the concentration of trastuzumab or pertuzumab is 80 to 150 mg per milliliter.

[0030] According to one embodiment of the present disclosure, the concentration of methionine is 5 to 15 mM, and the concentration of glutamate is 5 to 15 mM.

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

[0032] Another embodiment of the present disclosure is directed to a method for treating cancer in a subject in need of such treatment, the method comprising subcutaneously administering to the subject a therapeutically effective amount of a solution from a prefilled syringe.

[0033] Another embodiment of the present disclosure is a method for stabilizing trastuzumab. The method comprises combining trastuzumab, methionine and glutamic acid in a solution. After storage at 5°C ± 3°C for 3 days, the relative potency (%) of the composition for binding to FcγRIII is 93.9 to 107.9.

[0034] The present disclosure is written in a clear and concise manner, and it is understood that different embodiments can be combined or disassembled without departing from the principles and spirit of the present invention. For example, it is understood that all preferred features described in the present disclosure are applicable to various inventive aspects of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The amino acid sequence of the light chain of trastuzumab (SEQ ID NO: 1) and the amino acid sequence of the heavy chain of trastuzumab (SEQ ID NO: 2) with corresponding domain tags are shown.

[0036] Figure 2 is about the colloidal stability of EG13074 in different buffers and the correlation between the scattering constant and the concentration of trastuzumab. Figure 2A Contains histidine buffer, pH 6.5. Figure 2B Contains histidine buffer, pH 6.0. Figure 2C Contains citrate buffer, pH 6.0. Figure 2D Contains citrate buffer, pH 5.5. Figure 2E Contains acetate buffer, pH 5.5. Figure 2F Contains acetate buffer, pH 5.0.

[0037] Figure 3 is about the stability of EG13074 in acetate buffer measured by size-selection chromatography. Figure 3 shows the antibody profile of EG13074 after being placed in accelerated thermal stress at 40°C for 28 days. Figure 3A Antibody profiles in acetic acid, pH 5.4. Figure 3B Antibody profiles in acetic acid, pH 5.0. Figure 3C Antibody profiles in acetic acid, pH 4.6.

[0038] Figure 4 is about the stability of EG13074 in acetate buffer measured by cation exchange chromatography. Figure 4 shows the antibody charge variant profile of EG13074 after being placed in accelerated heat stress at 40°C for 28 days. Figure 4A Shown is the charge variant profile of acetic acid at pH 5.4. Figure 4B Shown is the charge variant profile of acetic acid at pH 5.0. Figure 4C Shown is the charge variant profile of acetic acid at pH 4.6.

[0039] Figure 5 is about the long-term stability of the trastuzumab formulation measured by size exclusion high performance liquid chromatography (SEC-HPLC) analysis. Figure 5 shows the antibody integrity of trastuzumab after being placed at different temperatures for up to 24 months. Figure 5A Antibody purity at 5°C is shown. Figure 5B Antibody purity at 25°C is shown. Figure 5C Antibody purity at 40°C is shown.

[0040] Figure 6 is about the long-term stability of the trastuzumab formulation measured by cation exchange liquid chromatography. Figure 6 shows the antibody main peak profile of trastuzumab after being placed at different temperatures for up to 24 months. Fig. 6A The percentage of the main peak at 5°C is plotted. Figure 6B The percentage of the main peak at 25°C is plotted. Figure 6CThe percentage of the main peak at 40°C is plotted.

[0041] Figure 7 is about the stability of the trastuzumab formulation measured by molecular sizing HPLC analysis. Figure 7 shows the antibody integrity of trastuzumab after being placed at different temperatures for up to 3 months. Fig. 7A Antibody purity at 5°C is shown. Figure 7B Antibody purity at 25°C is shown. Figure 7C Antibody purity at 40°C is shown.

[0042] Figure 8 is about the stability of the trastuzumab formulation measured by cation exchange liquid chromatography. Figure 8 shows the main peak profile of trastuzumab antibody after being placed at different temperatures for up to 3 months. Fig. 8A The percentage of the main peak at 5°C is plotted. Figure 8B The percentage of the main peak at 25°C is plotted. Figure 8C The percentage of the main peak at 40°C is plotted.

[0043] Figure 9 is about the stability of the Pertuzumab formulation measured by molecular sizing HPLC analysis. Figure 9 shows the antibody integrity of Pertuzumab after being placed at different temperatures for up to 3 months. Fig.9A Antibody purity at 5°C is shown. Fig. 9B Antibody purity at 25°C is shown. Fig. 9C Antibody purity at 40°C is shown.

[0044] Figure 10 is about the stability of Pertuzumab formulation measured by cation exchange liquid chromatography analysis. Figure 10 shows the antibody charge heterogeneity profile of Pertuzumab after storage at different temperatures for up to 3 months. Fig. 10A The charge heterogeneity profile of Example 41 samples is shown. Fig. 10B The charge heterogeneity profile of Example 42 samples is shown. Fig. 10C The charge heterogeneity profile of Example 43 samples is shown. Fig. 10D The charge heterogeneity profile of Example 44 samples is shown.

[0045] Fig.11 The antitumor activity of trastuzumab in different buffer conditions. The mean + / - SE of tumor volume (cubic millimeters) is plotted on the y-axis, and the number of days after tumor implantation is plotted on the x-axis. PBS buffer control group – solid squares, EG12014 – solid circles, EG13074-3 – solid triangles.

[0046] FIG12 is a pharmacokinetic (PK) study of the relationship between the concentration of trastuzumab (μg / mL) in mouse plasma and time after subcutaneous or intravenous administration of 10 mg / kg of different trastuzumab formulations to CD-1 mice. Compounds EG13074 and EG12014 contain the same active pharmaceutical ingredient (trastuzumab). Concentrations are calculated based on median values. G3: Group 3, EG13074 SC. G5: Group 5, EG12014 IV. Fig. 12A : The observation time points are from 9 to 96 hours. Fig. 12B : The observation time points are from 0.25 to 1344 hours. DETAILED DESCRIPTION

[0047] The present invention relates to a stable liquid pharmaceutical formulation comprising a high concentration of an anti-HER2 antibody (including trastuzumab, pertuzumab or a mixture thereof) to facilitate subcutaneous administration of a small volume of the formulation.

[0048] Anti-HER2 antibodies

[0049] Human epidermal growth factor receptor 2 (HER2) is a tyrosine phosphatase that binds to the cell membrane surface. It is known that HER2 protein is involved in the information transmission pathway that promotes cell growth and cell differentiation. When it is overexpressed or activated, it will cause cell canceration. HER2 protein can be inhibited by targeted therapeutic agents such as anti-HER2 antibodies. Anti-HER2 antibodies include trastuzumab and pertuzumab, but are not limited to them. A mixture of trastuzumab and pertuzumab can also be used to inhibit the overexpression of HER2.

[0050] Trastuzumab

[0051] Trastuzumab is a recombinant humanized monoclonal antibody that targets HER2. The amino acid sequence of the light chain of trastuzumab is: (SEQ ID NO: 1).

[0052] The amino acid sequence of the heavy chain of trastuzumab is: (SEQ ID NO: 2). Figure 1 The amino acid sequence of the light chain of trastuzumab (SEQ ID NO: 1) and the amino acid sequence of the heavy chain of trastuzumab (SEQ ID NO: 2) with corresponding domain tags are shown.

[0053] Trastuzumab has the CAS number 180288-69-1.

[0054] Pertuzumab

[0055] Pertuzumab is a recombinant humanized monoclonal antibody that targets HER2. The amino acid sequence of the light chain of trastuzumab is: (sequence number: 3). The amino acid sequence of the heavy chain of trastuzumab is: (sequence number: 4). Pertuzumab has a CAS number of 380610-27-5.

[0056] 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. In addition, 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.

[0057] The concentration of trastuzumab or pertuzumab can be freely adjusted within a range without causing substantial adverse effects on the stability of the liquid pharmaceutical formulation of the present invention.

[0058] In the present disclosure, "pharmaceutically acceptable salts" means salts that are suitable for use in human and lower animal tissues without excessive toxicity, irritation, or allergic reaction within the scope of sound medical judgment, and have a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known to those skilled 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 salts derived from suitable inorganic or organic acids and bases. Exemplary pharmaceutically acceptable, non-toxic acidic addition salts are salts formed by amino groups with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, or perchloric acid), or salts formed with organic acids (e.g., acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, glutamic acid, or malonic acid), or formed by methods known in the art such as ion exchange. Other pharmaceutically acceptable salts include acetate, glutamine, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfonate, ethanesulfonate, formate, butyrate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydrogen-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, hydroxynaphthoate, jelly salt, peroxysulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate and the like. Pharmaceutically acceptable salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N + (C 1-4 Representative alkali metals or alkaline earth metals include sodium, lithium, potassium, calcium and magnesium. Other pharmaceutically acceptable salts include non-toxic ammonium salts, quaternary ammonium salts and amine cations formed using counter ions, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates and aryl sulfonates, as appropriate.

[0059] Pharmaceutical composition

[0060] The compounds provided by the present invention can be administered in the form of a liquid pharmaceutical composition. The present invention therefore provides a pharmaceutical composition comprising one or more compounds described in the present disclosure as active ingredients, or pharmaceutically acceptable salts or esters thereof, and one or more pharmaceutically acceptable carriers. In the present disclosure, the term "pharmaceutically acceptable carrier" refers to a non-toxic carrier, adjuvant, excipient, etc., which does not destroy the pharmaceutical activity of the compound prepared therewith. Pharmaceutically acceptable carriers that can be used in the compositions of the present invention include, but are not limited to, buffers, tonicity agents, stabilizers, organic co-solvents, penetration enhancers, solubilizers, fillers, diluents and combinations thereof.

[0061] A pharmaceutical composition of the present invention may be substantially free of hyaluronidase (a penetration enhancer). Hyaluronidase includes various known hyaluronidases or modified hyaluronidases, such as a human enzyme protein having an amino acid sequence of GenBank: AAC70915.1. Hyaluronidase may be a glycoprotein, for example, rHuPH20. The term "substantially free of hyaluronidase" means that there is no detectable hyaluronidase in the composition, less than 10 units of hyaluronidase per dose, less than 5 units of hyaluronidase per dose, or less than 1 unit of hyaluronidase per dose when measured by conventional techniques such as ELISA.

[0062] Pharmaceutical composition: buffer

[0063] The pharmaceutical composition may include one or more buffers or buffering agents. The buffer may be L-histidine / histidine-hydrochloride, sodium citrate / citric acid, L-histidine / acetic acid, phosphate, sodium acetate / acetic acid, acetate, or a combination thereof, at a concentration 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.

[0064] The concentration of one of the buffers can be from 2 to 120 mM, from 5 to 100 mM, from 5 to 90 mM, from 5 to 80 mM, from 5 to 70 mM, from 5 to 60 mM, from 5 to 50 mM, from 5 to 40 mM, from 5 to 30 mM, from 5 to 20 mM, from 5 to 10 mM, from 5 to 35 mM, from 10 to 30 mM, from 15 to 25 mM, or 20 mM.

[0065] The buffer can stabilize the pH of the pharmaceutical composition at a pH of from 4.6 to 6.5, from 4.7 to 6.3, from 4.8 to 6.1, from 4.9 to 5.9, from 5.0 to 5.7, from 5.0 to 5.4, from 5.1 to 5.5, from 5.1 to 5.3, from 4.6 to 5.4, from 4.6 to 5.0, from 5.0 to 5.4, from 5.2 to 5.6, or at a pH of 5.2.

[0066] Pharmaceutical composition: tonicity agent

[0067] The pharmaceutical composition may include one or more tonicity agents. The tonicity agent may be a polyol or a sugar derivative, but is not limited thereto. The tonicity agent may be sucrose, trehalose, mannitol, sorbitol or a combination thereof.

[0068] The concentration of one of the tonicity agents can be from 20 to 300 mM, from 50 to 290 mM, from 80 to 280 mM, from 110 to 270 mM, from 140 to 260 mM, from 170 to 250 mM, from 180 to 240 mM, from 190 to 230 mM, from 200 to 220 mM, or 210 mM (7.18% w / v).

[0069] Pharmaceutical compositions: stabilizers

[0070] The pharmaceutical composition may include one or more stabilizers. The stabilizer may be methionine (Met), glutamic acid (Glu), arginine (Arg), N-α-acetylarginine, proline (Pro), glycine (Gly), lysine (Lys), glutamine (Gln), or a combination thereof (e.g., methionine and glutamic acid).

[0071] The concentration of one of the stabilizers can be less than or equal to 50 mM, from 5 to 45 mM, from 10 to 40 mM, from 15 to 35 mM, from 20 to 30 mM, from 5 to 40 mM, from 5 to 30 mM, from 5 to 20 mM, from 0 to 20 mM, or 10 mM.

[0072] The concentration of methionine can be less than or equal to 20 mM, from 3 to 17 mM, from 5 to 15 mM, from 7 to 13 mM, from 0 to 10 mM, or 10 mM.

[0073] The concentration of arginine can be less than or equal to 50 mM, from 0 to 50 mM, from 5 to 40 mM, from 10 to 30 mM, or from 15 to 25 mM.

[0074] The concentration of N-α-acetylarginine may be less than or equal to 10 mM, from 0 to 10 mM, or from 3 to 7 mM.

[0075] The concentration of proline can be less than or equal to 20 mM, from 0 to 20 mM, from 3 to 17 mM, from 5 to 15 mM, from 7 to 13 mM, or 10 mM.

[0076] The concentration of glycine can be less than or equal to 40 mM, from 0 to 40 mM, from 5 to 35 mM, from 5 to 30 mM, from 5 to 25 mM, or from 5 to 20 mM.

[0077] The concentration of lysine can be less than or equal to 20 mM, from 0 to 20 mM, from 3 to 17 mM, from 5 to 15 mM, from 7 to 13 mM, or 10 mM.

[0078] The concentration of Gln can be less than or equal to 20 mM, from 0 to 20 mM, from 3 to 17 mM, from 5 to 15 mM, from 7 to 13 mM, or 10 mM.

[0079] The concentration of glutamate can be less than or equal to 20 mM, from 0 to 20 mM, from 3 to 17 mM, from 5 to 15 mM, from 7 to 13 mM, or 10 mM.

[0080] Pharmaceutical composition: organic co-solvent

[0081] The pharmaceutical composition may include one or more organic cosolvents, which may be organic cosolvents polysorbate 80 (PS80), polysorbate 20 (PS20), poloxamer 188 (poloxamer 188), polyethylene glycol (PEG), propylene glycol, or a combination thereof. In certain embodiments, the organic cosolvent may also refer to a surfactant.

[0082] The concentration of one of the organic co-solvents can be less than or equal to 0.05% (w / v), from 0 to 0.05% (w / v), from 0.01 to 0.04% (w / v), from 0.01 to 0.03% (w / v), or 0.02% (w / v).

[0083] Pharmaceutical composition: other ingredients

[0084] The pharmaceutical composition may comprise histidine, citrate, phosphate or a mixture thereof. In one embodiment, the pharmaceutical composition may comprise histidine in a buffer of a liquid formulation.

[0085] The pharmaceutical composition may include salts. The salt may be, but is not limited to, sodium chloride, KCl, KBr, NaBr, Na2SO4, NaSCN, K2SO4, etc., or a mixture thereof. The concentration of the salt may be less than or equal to 155 mM, less than or equal to 150 mM, from 10 to 145 mM, from 20 to 135 mM, from 30 to 125 mM, from 40 to 115 mM, from 50 to 100 mM, from 60 to 90 mM, or from 70 to 80 mM. Similarly, the salt concentration may be from 0.7% (w / v) to 1.1% (w / v).

[0086] The pharmaceutical composition may contain a preservative. The preservative may be, but is not limited to, octadecyldimethylbenzyl ammonium chloride, benzalkonium chloride, benzathonine chloride, phenol, butyl alcohol, benzyl alcohol, methylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, m-cresol, etc., or a mixture thereof.

[0087] The pharmaceutical composition may also contain additives known in the art within a concentration range that does not substantially adversely affect the activity of the antibody or the usability of the formulation.

[0088] Pharmaceutical composition: combination of ingredients

[0089] The following is a contemplated combination of ingredients for a formulation comprising an anti-HER2 antibody, wherein the anti-HER2 antibody comprises trastuzumab, pertuzumab, or a mixture thereof. For example, the formulation may comprise trastuzumab or pertuzumab at a concentration of 120 mg per ml.

[0090] A formulation comprising a combination of acetate buffer and sucrose;

[0091] a preparation comprising acetic acid, sucrose and polysorbate 80;

[0092] a formulation comprising acetic acid, sucrose, and a combination of methionine and glutamate;

[0093] a formulation comprising acetic acid, sucrose, polysorbate 80, and a combination of methionine and glutamate;

[0094] A formulation comprising acetate buffer, sucrose, polysorbate 80 and methionine;

[0095] A formulation comprising acetate buffer, sucrose, polysorbate 80 and glutamic acid;

[0096] A preparation comprising acetate buffer, histidine, sucrose, methionine and glutamic acid;

[0097] A formulation comprising acetate buffer, histidine, sucrose, methionine and glycine;

[0098] A preparation comprising histidine buffer, trehalose, polysorbate 20 and methionine;

[0099] A formulation comprising 20 mM acetate buffer (pH 5.2), 210 mM sucrose, 10 mM methionine, 10 mM glutamic acid and 0.02% polysorbate 80.

[0100] Pharmaceutical composition: properties

[0101] The following describes the properties of the pharmaceutical preparations of the present invention. The examples illustratively disclose techniques that can be used to measure these properties.

[0102] The following describes a pharmaceutical liquid formulation having a specific HER2 binding affinity.

[0103] A pharmaceutical liquid preparation having a binding relative potency (%) of 93.9 to 107.9 to HER2 measured by an FcγRIII binding affinity assay after storage at 5°C±3°C for 3 days.

[0104] A pharmaceutical liquid preparation having a binding relative potency (%) of 90.0 to 118.0 to HER2 measured by an FcγRIII binding affinity test after being stored at a temperature of 40°C±2°C and in an accelerated thermal stress environment for 3 days.

[0105] A pharmaceutical liquid preparation, which is placed in 20 mM L-histidine buffer (pH 6.0 or 6.5), 20 mM citrate buffer (pH 5.5 or 6.0) or 20 mM acetate buffer (pH 5.0 or 5.5) and has a binding relative potency (%) of 93.9 to 107.9 for HER2 measured by an FcγRIII binding affinity test after being stored at 5°C±3°C for 3 days.

[0106] A pharmaceutical liquid preparation, which is placed in 20 mM L-histidine buffer (pH 6.0 or 6.5), 20 mM citrate buffer (pH 5.5 or 6.0) or 20 mM acetate buffer (pH 5.0 or 5.5) and stored at a temperature of 40°C±2°C and an accelerated heat stress environment for 3 days, has a binding relative potency (%) of 90.0 to 118.0 for HER2 measured by an FcγRIII binding affinity test.

[0107] Pharmaceutical liquid preparations having a specific amount of main component content (main peak / monomer %) are shown below.

[0108] A pharmaceutical liquid preparation comprises 95 to 99% of a major ingredient as measured by molecular sieving high performance liquid chromatography after storage at 5°C ± 3°C for 28 days.

[0109] A pharmaceutical liquid preparation comprises 95 to 99% of a main component, which is placed in 20 mM L-histidine buffer (pH 6.0 or 6.5), 20 mM citrate buffer (pH 5.5 or 6.0) or 20 mM acetate buffer (pH 5.0 or 5.5) and is stored at 4°C ± 3°C for 28 days and then measured by molecular sieving high performance liquid chromatography.

[0110] A pharmaceutical liquid preparation comprises 94 to 99% of a main ingredient as measured by molecular sieving high performance liquid chromatography after storage at 40°C ± 2°C for 28 days.

[0111] A pharmaceutical liquid preparation comprising 94 to 99% of a main ingredient, which is placed in 20 mM L-histidine buffer (pH 6.0 or 6.5), 20 mM citrate buffer (pH 5.5 or 6.0) or 20 mM acetate buffer (pH 5.0 or 5.5) and stored at 40°C ± 2°C for 28 days and measured by molecular sieving high performance liquid chromatography.

[0112] A pharmaceutical liquid preparation having a specific amount of a high molecular weight component (a peak having a retention time before the main peak) is shown below.

[0113] A pharmaceutical liquid preparation comprises 0.78 to 2.17% of a high molecular weight component, as measured by molecular sieving HPLC after storage at 5°C ± 3°C for 28 days.

[0114] A pharmaceutical liquid preparation comprising 0.78 to 2.17% of a high molecular weight component is placed in 20 mM L-histidine buffer (pH 6.0 or 6.5), 20 mM citrate buffer (pH 5.5 or 6.0) or 20 mM acetate buffer (pH 5.0 or 5.5) and stored at 5°C ± 3°C for 28 days and then measured by molecular sieving high performance liquid chromatography.

[0115] A pharmaceutical liquid preparation comprises 0.78 to 4.11% of a high molecular weight component, which is measured by molecular sieving high performance liquid chromatography after storage at 40°C ± 2°C for 28 days.

[0116] A pharmaceutical liquid preparation comprising 94 to 99% of a main ingredient, which is placed in 20 mM L-histidine buffer (pH 6.0 or 6.5), 20 mM citrate buffer (pH 5.5 or 6.0) or 20 mM acetate buffer (pH 5.0 or 5.5) and stored at 40°C ± 2°C for 28 days and measured by molecular sieving high performance liquid chromatography.

[0117] A pharmaceutical liquid preparation having a specific amount of low molecular weight components (a peak having a retention time after the main peak) is shown below.

[0118] A pharmaceutical liquid preparation comprises 0% of low molecular weight components as measured by molecular sieving HPLC after storage at 5°C ± 3°C for 28 days.

[0119] A pharmaceutical liquid preparation comprising 0% of a low molecular weight component is placed in a 20 mM L-histidine buffer (pH 6.0 or 6.5), a 20 mM citrate buffer (pH 5.5 or 6.0) or a 20 mM acetate buffer (pH 5.0 or 5.5) and stored at 5°C ± 3°C for 28 days before being measured by molecular sieving high performance liquid chromatography.

[0120] A pharmaceutical liquid preparation comprises 1.61 to 2.20% of a low molecular weight component as measured by molecular sieving HPLC after storage at 40°C ± 2°C for 28 days.

[0121] A pharmaceutical liquid preparation comprising 1.61 to 2.20% of a low molecular weight component, which is placed in 20 mM L-histidine buffer (pH 6.0 or 6.5), 20 mM citrate buffer (pH 5.5 or 6.0) or 20 mM acetate buffer (pH 5.0 or 5.5) and stored at 40°C ± 2°C for 28 days and then measured by molecular sieving high performance liquid chromatography.

[0122] The following is a pharmaceutical liquid formulation having a specific amount of the main species of the charged variant components.

[0123] A pharmaceutical liquid preparation comprises 55.06 to 64.58% of a main substance with charge variation, which is measured by cation exchange high performance liquid chromatography (CIX-HPLC) after storage at 5°C ± 3°C for 28 days.

[0124] A pharmaceutical liquid preparation comprising 55.06 to 64.58% of a main substance charged variable component, which is placed in 20 mM L-histidine buffer (pH 6.0 or 6.5), 20 mM citrate buffer (pH 5.5 or 6.0) or 20 mM acetate buffer (pH 5.0 or 5.5) and stored at 5°C ± 3°C for 28 days and measured by cation exchange high performance liquid chromatography.

[0125] A pharmaceutical liquid preparation comprises 12.71 to 64.15% of a major material charge variable component, which is measured by cation exchange high performance liquid chromatography after storage at 40°C ± 2°C for 28 days.

[0126] A pharmaceutical liquid preparation comprising 12.71 to 64.15% of a main substance charged variable component, which is placed in 20 mM L-histidine buffer (pH 6.0 or 6.5), 20 mM citrate buffer (pH 5.5 or 6.0) or 20 mM acetate buffer (pH 5.0 or 5.5) and stored at 40°C ± 2°C for 28 days and measured by cation exchange high performance liquid chromatography.

[0127] The following is a pharmaceutical liquid preparation having a specific amount of acidic species of the charged variant components (acidic species of the charged variant components, with a peak whose retention time is located before the charged variant of the main species).

[0128] A pharmaceutical liquid preparation comprises 25.98 to 59.69% of an acidic substance with a charge variation component, which is measured by cation exchange high performance liquid chromatography after storage at a temperature of 5°C ± 3°C for 28 days.

[0129] A pharmaceutical liquid preparation comprising 25.98 to 59.69% of an acidic charged variable component, which is placed in 20 mM L-histidine buffer (pH 6.0 or 6.5), 20 mM citrate buffer (pH 5.5 or 6.0) or 20 mM acetate buffer (pH 5.0 or 5.5) and stored at 5°C ± 3°C for 28 days and then measured by cation exchange high performance liquid chromatography.

[0130] A pharmaceutical liquid preparation comprises 39.20 to 71.02% of an acidic substance with charge variation, which is measured by cation exchange high performance liquid chromatography after storage at 40°C ± 2°C for 28 days.

[0131] A pharmaceutical liquid preparation comprising 39.20 to 71.02% of an acidic charged variable component, which is placed in 20 mM L-histidine buffer (pH 6.0 or 6.5), 20 mM citrate buffer (pH 5.5 or 6.0) or 20 mM acetate buffer (pH 5.0 or 5.5) and stored at 40°C ± 2°C for 28 days and then measured by cation exchange high performance liquid chromatography.

[0132] The following is a pharmaceutical liquid preparation having a specific amount of basic species of the charged variant components (the retention time is located at the peak after the charged variant of the main substance).

[0133] A pharmaceutical liquid preparation comprises 8.47 to 12.56% of a basic substance charge variation component, which is measured by cation exchange high performance liquid chromatography after storage at 5°C ± 3°C for 28 days.

[0134] A pharmaceutical liquid preparation contains 8.47 to 12.56% of charged variant components of alkaline substances, which are placed in 20mM L-histidine buffer (pH 6.0 or 6.5), 20mM citrate buffer (pH 5.5 or 6.0) or 20mM acetate buffer (pH 5.0 or 5.5) and stored at 5°C±3°C for 28 days and then measured by cation exchange high performance liquid chromatography.

[0135] A pharmaceutical liquid preparation comprises 16.27 to 34.50% of basic substance charge variation component, which is measured by cation exchange high performance liquid chromatography after storage at 40°C ± 2°C for 28 days.

[0136] A pharmaceutical liquid preparation contains 16.27 to 34.50% of a charged variant component of a basic substance, which is placed in 20 mM L-histidine buffer (pH 6.0 or 6.5), 20 mM citrate buffer (pH 5.5 or 6.0) or 20 mM acetate buffer (pH 5.0 or 5.5) and is stored at 40°C ± 2°C for 28 days and then measured by cation exchange high performance liquid chromatography.

[0137] Pharmaceutical liquid preparations having a specific turbidity (turbidity) are listed below.

[0138] A pharmaceutical liquid preparation having an absorbance A350 of 0.23 to 0.338, which is measured by spectrophotometry (for example, iD3 multi-mode microplate reader) for measurements.

[0139] A pharmaceutical liquid preparation having an absorbance A350 of 0.23 to 0.338, which is placed in 20 mM L-histidine buffer (pH 6.0 or 6.5), 20 mM citrate buffer (pH 5.5 or 6.0) or 20 mM acetate buffer (pH 5.0 or 5.5) and stored at 5°C ± 3°C for 28 days, and the absorbance A350 is measured by spectrophotometer (for example, iD3 multi-mode microplate reader) for measurements.

[0140] A pharmaceutical liquid preparation having an absorbance A350 of 0.232 to 0.397, which is measured by spectrophotometry (for example, iD3 multi-mode microplate reader) for measurements.

[0141] A pharmaceutical liquid preparation having an absorbance A350 of 0.232 to 0.397, which is placed in 20 mM L-histidine buffer (pH 6.0 or 6.5), 20 mM citrate buffer (pH 5.5 or 6.0) or 20 mM acetate buffer (pH 5.0 or 5.5) and stored at 40°C ± 2°C for 28 days, and the absorbance A350 is measured by spectrophotometer (for example, iD3 multi-mode microplate reader) for measurements.

[0142] Listed below are pharmaceutical liquid formulations of active pharmaceutical ingredients (API) having a specified colloidal stability in buffer.

[0143] Dynamic Light Scattering (DLS) is used to measure the colloidal stability, particle size, and aggregation of API in buffer. A pharmaceutical liquid formulation with a positive slope in the scattering constant ((K*c) / R(θ)(1 / Da)) vs. concentration (mg / mL) plot indicates that the scattering value increases due to an increase in the number of API particles as the concentration increases. Therefore, the API is dispersed in the buffer. A negative slope indicates that the number of API particles decreases as the concentration increases. Therefore, the API accumulates in the buffer.

[0144] Aggregation and colloidal stability of the active pharmaceutical ingredient in histidine buffer were determined by the positive slope of the dynamic light scattering measurement. Samples were measured at increasing concentrations of the active pharmaceutical ingredient in histidine buffer (pH 6.0 or pH 6.5).

[0145] Aggregation and colloidal stability of the active pharmaceutical ingredient in citrate buffer were determined by the negative slope of the dynamic light scattering measurement. Samples were measured at increasing concentrations of the active pharmaceutical ingredient in citrate buffer (pH 5.5 or pH 6.0).

[0146] Aggregation and colloidal stability of the active pharmaceutical ingredient in acetate buffer were determined by the positive slope of the dynamic light scattering measurement. Samples were measured at increasing concentrations of the active pharmaceutical ingredient in acetate buffer (pH 5.0 or pH 5.5).

[0147] Pharmaceutical compositions: preparation

[0148] The compositions of the present invention are prepared by methods well known in the pharmaceutical art, for example, see Remington's Pharmaceutical Sciences, Mace Publishing Co., Philadelphia, Pa. 17th Ed. (1985), and Modern Pharmaceutics, Marcel Dekker, Inc. 3rd Ed. (GS Banker & C.T. Rhodes, Eds.).

[0149] According to the needs of use, the required amount of the compound of the present invention and other ingredients described in the present disclosure can be mixed in an appropriate solvent, and then filtered and sterilized to prepare sterile injection water or intravenous injection. In general, various sterilized active ingredients can be added to a sterile carrier containing a basic dispersion medium and other ingredients described in the present disclosure to prepare a dispersant. When sterile powders are used to prepare injectable solutions, the preparation method can be vacuum drying and freeze drying techniques to produce powders of active ingredients and additional required ingredients from sterile filtered solutions.

[0150] A high concentration of trastuzumab in liquid form can be prepared from IV trastuzumab by ultrafiltration (UF) and diafiltration (DF) as known in the art. Ultracentrifugal filters and tangential flow filtration (TFF) are two ways to achieve buffer exchange and concentrate mAb. Buffer exchange can also be performed using dialysis tubing or dialysis cassettes.

[0151] Treatment methods and administration routes

[0152] The present invention provides a method for treating a subject in need of treatment; the method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition described in the present disclosure.

[0153] The present invention further provides a method for treating cancer in a subject in need of treatment; the method comprises administering to the subject a therapeutically effective amount of the pharmaceutical composition described in the present disclosure.

[0154] The present invention further provides a method for binding HER2 in an individual in need thereof; the method comprises administering to the individual a therapeutically effective amount of the pharmaceutical composition described in the present disclosure.

[0155] The methods described in the present disclosure further comprise confirming that the individual suffers from cancer prior to administering the pharmaceutical composition of the present invention.

[0156] The present invention also provides:

[0157] (1) A pharmaceutical composition according to the present disclosure, which is used in a method of treating an individual.

[0158] (2) A pharmaceutical composition according to the present disclosure is used in a method for treating cancer, wherein the method comprises administering a therapeutically effective amount of the pharmaceutical composition to a subject in need of treatment.

[0159] (3) A pharmaceutical composition according to the present disclosure is used for binding to HER2, wherein binding to HER2 comprises administering a therapeutically effective amount of the composition to a subject in need thereof.

[0160] (4) Use of a therapeutically effective amount of the pharmaceutical composition described in the present disclosure to treat an individual in need of treatment.

[0161] (5) Use of the pharmaceutical composition described in the present disclosure for treating cancer in a subject in need of treatment, wherein the treatment comprises administering a therapeutically effective amount of the pharmaceutical composition to the subject.

[0162] (6) Use of the pharmaceutical composition described in the present disclosure for binding to HER2, wherein the binding to HER2 comprises administering a therapeutically effective amount of the composition to a subject in need thereof.

[0163] (7) Use of the pharmaceutical composition described in the present disclosure in preparing a drug.

[0164] (8) Use of the pharmaceutical composition described in the present disclosure for preparing a medicament, wherein the medicament is used to treat cancer in a subject in need of treatment, and the treatment comprises administering a therapeutically effective amount of the composition to the subject.

[0165] (9) Use of the pharmaceutical composition described in the present disclosure for preparing a drug for binding to HER2, wherein the binding to HER2 comprises administering a therapeutically effective amount of the composition to a subject in need thereof.

[0166] In the present disclosure, unless otherwise specified, the term "treat", "treating" or "treatment" refers to an action performed on an individual suffering from a specific disease, condition or symptom to reduce the severity of the disease, condition or symptom, or to delay or slow the progression of the disease, condition or symptom ("therapeutic treatment"). In the present disclosure, disease, condition and symptom are interchangeable terms.

[0167] In the present disclosure, the "subject" to be administered includes, but is not limited to, humans (i.e., males or females of any age group, such as non-adult subjects such as infants, children or adolescents, or adult subjects such as young people, middle-aged people or the elderly), and / or non-human animals, such as mammals (e.g., stone crab macaques, rhesus monkeys), cattle, pigs, horses, sheep, goats, cats and / or dogs. In certain embodiments, the subject is a human. In certain embodiments, the subject is a non-human animal. "Human", "patient" and "subject" are interchangeable terms in the present disclosure.

[0168] In this disclosure, unless otherwise indicated, a "therapeutically effective amount" of a compound is a dose sufficient to provide a therapeutic benefit in treating a disease, condition, or symptom, or a dose sufficient to delay or minimize symptoms associated with the disease, condition, or symptom. A therapeutically effective amount of a compound is a dose of a therapeutic agent that provides a therapeutic benefit in treating a disease, condition, or symptom. The term "therapeutically effective amount" includes a dose that improves overall treatment, or a dose that reduces or avoids the symptoms or causes of a disease or condition.

[0169] The pharmaceutical composition can be administered to treat cancers such as breast cancer. Types of breast cancer include adjuvant breast cancer, metastatic breast cancer, advanced breast cancer, and early breast cancer. The pharmaceutical composition can also be administered to treat cancers such as metastatic gastric cancer or gastroesophageal node adenocarcinoma, ovarian cancer, gastric cancer, colon cancer, and gastrointestinal cancer.

[0170] Single or multiple doses of the pharmaceutical composition may be administered by any of the accepted modes of administration of pharmaceutical agents having similar efficacy, for example, by the modes described in the patents and patent applications cited in this disclosure, including parenteral modes of administration such as subcutaneous administration.

[0171] The dose of trastuzumab per subcutaneous administration can be from 100 mg to 1000 mg, from 200 mg to 900 mg, from 300 mg to 800 mg, from 300 mg to 750 mg, from 400 mg to 700 mg, from 480 mg to 700 mg, from 500 mg to 700 mg, from 550 mg to 650 mg, from 500 mg to 600 mg, 600 mg, or 750 mg. The volume of each subcutaneous administration can be from 0.5 ml to 10 ml, from 1 ml to 9 ml, from 2 ml to 8 ml, from 3 ml to 7 ml, from 4 ml to 6 ml, or 5 ml.

[0172] When administered subcutaneously, patients should be treated once a week, once every two weeks, once every three weeks, once every four weeks, or once every five weeks. Doses should be administered over 2-5 minutes and preferably injected into different locations on the thigh. Patients with early-stage breast cancer should receive subcutaneous trastuzumab for 52 weeks or until disease recurrence or unacceptable cardiac toxicity, whichever occurs first. Patients with metastatic breast cancer (MBC) should receive subcutaneous trastuzumab until disease progression.

[0173] Pharmaceutical compositions formulated for subcutaneous administration can be administered subcutaneously to a subject, such as pre-filled syringes, microneedles, sustained release delivery systems, and stimuli-responsive delivery systems.

[0174] Trastuzumab subcutaneous therapeutic solution can be loaded into commercially available syringe infusion systems, such as KORUTM (Available from KORU™ Medical Systems, Chester, NY, USA).

[0175] In one embodiment, the trastuzumab subcutaneous therapeutic solution is administered using a prefilled syringe, wherein the prefilled syringe contains 3 ml to 7 ml of the trastuzumab subcutaneous therapeutic solution.

[0176] In another embodiment, the trastuzumab subcutaneous treatment solution is loaded in a commercial medical device, such as an injection device, an infusion device, an infusion pump, an automatic injection device, a needle-free device, or an unrestricted subcutaneous patch delivery system. For example, the trastuzumab subcutaneous treatment solution can be loaded in a commercial infusion pump, such as a Crono S-PID pump (available from Canes SPA), and an infusion pump as described in US Patent No. 8172814B2. The trastuzumab subcutaneous treatment solution can also be loaded in another commercial infusion pump, such as, for example, SCIg60 Infuser (available from EMED Technologies Corporation), and an infusion pump as described in US Patent No. 9808576B2 and US Patent Publication No. 20130138075.

[0177] In one embodiment, the trastuzumab subcutaneous therapeutic solution is administered using an infusion pump containing 10 ml to 15 ml of the trastuzumab subcutaneous therapeutic solution.

[0178] In another embodiment, the trastuzumab subcutaneous treatment solution is loaded into a commercial human syringe, such as a large volume syringe (LVI-V), LVI-P, LVI-U, a dual cartridge syringe (DCI), an automatic reconstitution syringe (ART) (available from Sonceboz SA), and a syringe as described in European Patent No. 3354303B1. The trastuzumab subcutaneous treatment solution can be loaded into a commercial human syringe, such as SmartDose (available from West Pharmaceutical Services), and a syringe as described in WIPO Patent Publication No. 2018222521A1 and WIPO Patent Publication No. 2019032395A1. The trastuzumab subcutaneous treatment solution can be loaded into a commercial human syringe, such as Human syringe( On-Body Infusor) (available from Enable Injections Inc.), and injectors as described in US Patent Publication No. 20180161497A1 and US Patent Publication No. 20210353222A1.

[0179] In other embodiments, the trastuzumab subcutaneous treatment solution is loaded into a commercially available automatic injector, such as the automatic injection device obtained from West Pharmaceutical Services, and the automatic injection device described in US Patent No. 8048029B2.

[0180] As can be expected, medical professionals usually determine the actual dosage of the compound to be administered based on relevant circumstances, including the condition to be treated, the selected administration route, the actual compound administered or its relevant activity, the patient's age, weight and response, and the severity of the patient's symptoms. Example

[0181] Sample preparation

[0182] EG13074 is a highly concentrated liquid formulation of trastuzumab prepared from IV trastuzumab (EG12014) and processed by ultrafiltration / diafiltration (UF / DF) and filtration reprocessing. Concentration and buffer exchange are key processing steps in the preparation of EG13074. Centrifugal filtration and tangential flow filtration (TFF) are two ways to achieve buffer exchange and concentration of mAb. In early small-scale development, buffer exchange can be performed using dialysis tubing or dialysis cassettes (with 20 kDa cutoff membranes). EG13074 samples can be concentrated using a 30 kDa cutoff centrifugal filter at 4200 rpm for 20 minutes at 4°C and repeated several times to achieve the target concentration. The centrifuge used was an Eppendorf 5804R with an S-4-72 swing-out rotor.

[0183] During the large-scale laboratory development phase, EG13074 sample preparation included the use of TFF to concentrate mAb and exchange buffers. 30kDa membrane, D cassette 3. Merck Milipore Labscale TFF Trastuzumab was diafiltered into test formulation buffer and concentrated to the target concentration.

[0184] High concentrations of Pertuzumab were prepared by chromatography, ultrafiltration / diafiltration (UF / DF), and filtration. Concentration and buffer exchange are key processing steps in the preparation process. Centrifugal filtration and tangential flow filtration (TFF) are two ways to achieve buffer exchange and concentration of mAb. In early small-scale development, buffer exchange can be performed using dialysis tubing or dialysis cassettes (with a 20 kDa cutoff membrane). Samples of Pertuzumab can be concentrated using a 30 kDa cutoff centrifugal filter at 4200 rpm for 20 minutes at 4°C and repeated several times to achieve the desired concentration. The centrifuge used was an Eppendorf 5804R with an S-4-72 swing-out rotor.

[0185] During the large-scale laboratory development phase, the sample preparation of Pertuzumab involved the use of TFF to concentrate the mAb and exchange buffers. 30kDa membrane, D cassette 3Merck Milipore LabscaleTFF Pertuzumab was diafiltered against the test formulation buffer and concentrated to the target concentration.

[0186] Methods for testing stability

[0187] Turbidity

[0188] Turbidity is analyzed by detecting visible / subvisible particles to monitor the quality and stability of the formulation. Before injecting into a 96-well microplate (Cat No. 269620), gently invert the sample 10 times. 200 μl of the sample is injected into each well in triplicate. The samples were measured three times by iD3 multi-mode microplate reader. The three absorbance values ​​were averaged to record the reading.

[0189] Purity, aggregation and lysis

[0190] The purity, aggregation and cleavage of the antibody were measured by size exclusion high performance liquid chromatography (SEC-HPLC) using a high performance liquid chromatography system HPLC-A6002 (Agilent 1260 Infinity II) using a column (TOSOH TSK-gel G3000SWxl, 7.8 x 300 mm, PN: 0008541).

[0191] Charged variant

[0192] The charged variants of the antibody were measured by cation exchange high performance liquid chromatography (CIX-HPLC) using a high performance liquid chromatography 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: 082GA00017G) was used. The main component (main peak), acidic variants and basic variants of the antibody were detected from the sample.

[0193] Colloidal stability, aggregation and particle size

[0194] Using dynamic light scattering with 823.3 nm laser light and 150° scattering angle Colloidal stability, aggregation, and particle size were measured using a plate reader III (Wyatt Technology Corp., CA) with dynamic light scattering (DLS).

[0195] Dynamic light scattering is used to measure the size and distribution of particles in a solution. Light is scattered when it passes through small particles in a solution. Before injecting into an Auroa 384 microplate (Wyatt PN: P8806-38401), the sample was gently inverted 10 times. The starting temperature (T onset ) and end temperature (T agg ) were set to 25°C and 75°C, respectively. Each well of the microplate was read 5 times and the average measurement value of each well was recorded. The scattering constant and concentration of the sample were plotted as the Y-axis and X-axis, respectively. A positive slope indicates that the concentrated sample has less aggregation, so light can penetrate the space between small particles. A negative slope indicates that aggregation in the concentrated sample results in a larger particle volume.

[0196] Protein content detection

[0197] Protein content analysis was performed using a spectrophotometer Jasco v-730 to measure protein concentration. The detection light wave was set to 280 nm. The sample was diluted to an appropriate concentration, including OD 0.4-0.8. 110 μl of the sample was injected into a colorimetric tube and then measured at a wavelength of 280 nm. The average absorbance was recorded and the concentration of the protein (antibody) was calculated.

[0198] Specifically, the method for measuring protein concentration includes turning on the spectrophotometer and UV lamp at least 20 minutes before use.

[0199] The detection wavelength is 280 nm. Wash a 1 cm quartz cuvette with ultrapure water and dry it. Rinse the cuvette with the formulation base buffer. Add the formulation base buffer to the cuvette. Place the cuvette in the spectrophotometer and record the value as the blank. After removing the formulation base buffer, rinse the cuvette with the sample to be tested to achieve equilibrium. Remove the sample to be tested from the cuvette.

[0200] Add the sample to be tested to the colorimetric tube and measure. After removing the sample, add the sample to be tested again and repeat the measurement three times. Rinse the colorimetric tube with the formulation base buffer, and then repeat the steps of measuring the blank value and measuring the sample to be tested for the next sample to be tested.

[0201] After the measurement is completed, wash the colorimetric tube with ultrapure water and dry it. Average the absorbance values ​​obtained from repeated measurements of each sample. Calculate the concentration using the formula of Beer's law.

[0202] Binding activity

[0203] HER2-ECD binding ELISA

[0204] The binding activity of the antibodies was measured using a HER2-ECD binding ELISA assay.

[0205] FcγRIII binding assay

[0206] The FcγRIII binding assay is a method to measure the binding kinetics of the interaction between trastuzumab and its receptor using the Bio-Layer Interference (BLI) technique. The measurement was performed using ForteBio OctetRed96 (Pall, equipment number BAZ-20002).

[0207] Stability test

[0208] Stability Test Example 1: Comparison of Stability Based on Buffer Solutions

[0209] Regarding the liquid pharmaceutical preparation used in the stability test Example 1, buffer solutions with corresponding pH and concentration were prepared, and antibodies were added to obtain the samples listed in Tables 1, 2 and 3 below. The specific content of each component is recorded in Tables 1, 2 and 3 below. For example, the total volume is 5 ml.

[0210]

[0211]

[0212]

[0213] The stability of the liquid pharmaceutical preparations prepared in Examples 1 to 3 was measured after being placed at 5°C ± 3°C or 40°C ± 2°C for 0 days (indicated as the start), 3 days, 1 week, 2 weeks and 4 weeks. Example 1 is a histidine buffer system using a 20mM L-histidine / L-histidine hydrochloride solution. Example 2 is a citric acid system using a 20mM citric acid / sodium citrate solution. Example 3 is an acetic acid system using a 20mM acetic acid / sodium acetate solution. The analytical results are described in Tables 1 to 3 above, respectively.

[0214] Figure 2 shows the colloidal stability of trastuzumab in different buffers. Dynamic light scattering was used for analysis, and a graph showing the relationship between the scattering constant and the trastuzumab concentration was plotted. Figure 2A The colloidal stability of trastuzumab in histidine buffer (pH 6.5) is shown. Figure 2B The colloidal stability of trastuzumab in histidine buffer (pH 6.0) is shown. Figure 2C The colloidal stability of trastuzumab in citrate buffer (pH 6.0) is shown. Figure 2D The colloidal stability of trastuzumab in citrate buffer (pH 5.5) is shown. Figure 2E The colloidal stability of trastuzumab in acetate buffer (pH 5.5) is shown. Figure 2F The colloidal stability of trastuzumab in acetate buffer (pH 5.0) is shown. The colloidal stability of trastuzumab in histidine and acetate buffers has a positive slope. The colloidal stability of trastuzumab in citrate buffer has a negative slope. In one embodiment, histidine buffer, acetate buffer or citrate buffer can be used as a buffer in the liquid pharmaceutical composition.

[0215] Stability Test Example 2: The stability of the composition depends on the pH value of the acetate buffer

[0216] Regarding the liquid pharmaceutical preparation used in Stability Test Example 2, buffer solutions having corresponding pH and antibody concentrations were prepared to obtain the samples listed in Table 4 below. The concentration of each component is recorded in Table 4 below. For example, the total volume is 5 ml.

[0217]

[0218] The stability of the above formulations was measured after 0 days (marked as the start), 1 week, 1 month, 2 months and 3 months at a temperature of 40°C ± 2°C. Examples 4, 5 and 6 are formulations using 20 mM acetic acid / sodium acetate solution (pH 5.4, 5.0 or 4.6), respectively. The analysis results are described in Table 4 above.

[0219] Figure 3 shows the antibody profile measured by molecular sieving HPLC. Figure 3 shows the antibody profile of EG13074 after being placed in accelerated thermal stress at 40°C for 28 days. Figure 3A The antibody profiles in acetic acid at pH 5.4 are shown. Figure 3B Antibody profiles in acetic acid at pH 5.0 are shown. Figure 3C The antibody profiles in acetic acid at pH 4.6 are shown.

[0220] Figure 4 shows the antibody analysis data obtained by cation exchange high performance liquid chromatography. Figure 4 shows the profile of charged variants of antibodies after being placed in accelerated heat stress at 40°C for 28 days. Figure 4A The profile of charged variants in acetic acid at pH 5.4 is shown. Figure 4B Depicted are the profiles of charged variants exposed to acetic acid at pH 5.0. Figure 4C Depicted is the profile of charged variants exposed to acetic acid at pH 4.6.

[0221] In one embodiment, the pH of the acetic acid is 4.6 to 5.4.

[0222] Stability Test Example 3: The stability of the composition depends on the type of additive or pH value in the acetate buffer

[0223] Regarding the liquid pharmaceutical preparation used in Stability Test Example 3, buffer solutions having corresponding pH values ​​and antibody concentrations were prepared, and tonicity agents or salts were added to obtain the samples listed in Table 5 below. The concentrations of each component are recorded in Table 5 below. For example, the total volume is 5 ml.

[0224]

[0225] The stability of the above formulations was measured after 0 weeks (initial), 1 week, 2 weeks and 4 weeks at 5°C ± 3°C or 40°C ± 2°C. Examples 7 and 8 are formulations comprising 0.05% polysorbate 20, 150 mM trehalose or 0.9% sodium chloride, and 20 mM acetic acid / sodium acetate solution (pH 5.6 or 5.2), respectively. The concentration of the antibody was 120 mg per ml. The results of the analysis are described in Table 5 above.

[0226] In one embodiment, the formulation may include an acetic acid / sodium acetate solution with a pH between 5.6 and 5.2. In one embodiment, the formulation may include a tonicity agent or a salt. The tonicity agent may be trehalose, but is not limited thereto. The salt may be sodium chloride (NaCl), but is not limited thereto.

[0227] Stability Test Example 4: The stability of the composition depends on the type of tonicity agent and buffer

[0228] Regarding the liquid pharmaceutical preparation used in Stability Test Example 4, buffer solutions having corresponding buffer, tonicity agent and antibody concentrations were prepared, and salts and amino acids were added to obtain the samples listed in Tables 6 to 13 below. The concentration of each component is recorded in Tables 6 to 13 below. For example, the total volume is 5 ml.

[0229]

[0230] The stability of the above formulations was measured after 0 days (initial) and 4 days at 40°C ± 2°C. The formulations designated as samples 1 to 7 contained 20 mM histidine buffer (pH 6.0) and 20 mM or 300 mM trehalose. The concentration of the antibody was 120 mg per ml. The results of the analysis are described in Table 6 above.

[0231] In one embodiment, the formulation may include histidine buffer, trehalose, methionine or sodium chloride, or a combination thereof, but is not limited thereto.

[0232]

[0233] The stability of the above formulations was measured after 0 days (starting) and 4 days at a temperature of 40°C ± 2°C. The formulations designated as samples 8 to 14 comprised 20 mM histidine buffer (pH 6.0) and 20 mM or 300 mM mannitol. The concentration of the antibody was 120 mg per ml. The results of the analysis are described in Table 7 above. In one embodiment, the formulation may comprise histidine buffer, mannitol, methionine or sodium chloride, or a combination thereof, but is not limited thereto.

[0234]

[0235] The stability of the above formulations was measured after 0 days (starting) and 4 days at a temperature of 40°C ± 2°C. The formulations designated as samples 15 to 21 comprised 20 mM histidine buffer (pH 6.0) and 20 mM or 300 mM sorbitol. The concentration of the antibody was 120 mg per ml. The results of the analysis are described in Table 8 above. In one embodiment, the formulation may comprise histidine buffer, sorbitol, methionine or sodium chloride, or a combination thereof, but is not limited thereto.

[0236]

[0237] It is noteworthy that after 4 days of exposure to a temperature of 40°C ± 2°C, the formulation containing sucrose and methionine had a higher monomer content (92.08%), compared to only 79.87% for the formulation containing sorbitol and methionine.

[0238] The stability of the above formulations was measured after 0 days (start) and 4 days at a temperature of 40°C ± 2°C. The formulations designated as samples 22 to 28 comprised 20 mM histidine buffer (pH 6.0) and 20 mM or 300 mM sucrose. The concentration of the antibody was 120 mg per ml. The results of the analysis are described in Table 9 above. In one embodiment, the formulation may comprise histidine buffer, sucrose, methionine or sodium chloride, or a combination thereof, but is not limited thereto.

[0239]

[0240] The stability of the above formulations was measured after 0 days (starting) and 4 days at a temperature of 40°C ± 2°C. The formulations designated as samples 29 to 35 comprised 20 mM acetate buffer (pH 5.0) and 20 mM or 300 mM trehalose. The concentration of the antibody was 120 mg per ml. The results of the analysis are described in Table 10 above. In one embodiment, the formulation may comprise acetate buffer, trehalose, methionine or sodium chloride, or a combination thereof, but is not limited thereto.

[0241]

[0242] The stability of the above formulations was measured after 0 days (starting) and 4 days at a temperature of 40°C ± 2°C. The formulations designated as samples 36 to 42 comprised 20 mM acetate buffer (pH 5.0) and 20 mM or 300 mM mannitol. The concentration of the antibody was 120 mg per ml. The results of the analysis are described in Table 11 above. In one embodiment, the formulation may comprise acetate buffer, mannitol, methionine or sodium chloride, or a combination thereof, but is not limited thereto.

[0243]

[0244] The stability of the above formulations was measured after 0 days (starting) and 4 days at a temperature of 40°C ± 2°C. The formulations designated as samples 43 to 49 comprised 20 mM acetate buffer (pH 5.0) and 20 mM or 300 mM sorbitol. The concentration of the antibody was 120 mg per ml. The results of the analysis are described in Table 12 above. In one embodiment, the formulation may comprise acetate buffer, sorbitol, methionine or sodium chloride, or a combination thereof, but is not limited thereto.

[0245]

[0246] It is noteworthy that the formulations containing sucrose had a consistently higher amount of monomer (%) after day 0 (start) compared to the formulations containing sorbitol.

[0247] The stability of the above formulations was measured after 0 days (starting) and 4 days at a temperature of 40°C ± 2°C. The formulations designated as samples 50 to 45 contained 20 mM acetate buffer (pH 5.0) and 20 mM or 300 mM sucrose. The concentration of the antibody was 120 mg per ml. The results of the analysis are described in Table 13 above. In one embodiment, the formulation may contain acetate buffer, sucrose, methionine or sodium chloride, or a combination thereof, but is not limited thereto.

[0248] Stability Test Example 5: The stability of the composition depends on the type of amino acid

[0249] Regarding the liquid pharmaceutical preparation used in Stability Test Example 5, buffer solutions having corresponding amino acid and antibody concentrations were prepared, and sucrose and an organic co-solvent were added to obtain the samples listed in Tables 14 to 16 below. The concentration of each component is recorded in Tables 14 to 16 below. For example, the total volume is 4 ml.

[0250]

[0251] The stability of the liquid pharmaceutical formulations prepared in Examples 9 to 13 was measured after 0 weeks (indicated as start), 1 week, 2 weeks, 3 weeks and 4 weeks at a temperature of 40°C ± 2°C. Examples 9 to 13 comprised 150 mg of trastuzumab per ml, 210 mM sucrose (or 7.18% w / v) and 0.02% polysorbate 80 in 20 mM acetate buffer (pH 5.2). Example 9 further comprised 10 mM methionine. Example 10 further comprised 50 mM arginine. Example 11 further comprised 20 mM glutamic acid. Example 12 further comprised a combination of 10 mM methionine and 50 mM arginine. Example 13 further comprised a combination of 10 mM methionine and 20 mM glutamic acid. The results of the analysis are described in Table 14 above. In one embodiment, the formulation may include acetate buffer, sucrose, polysorbate 80, methionine or glutamic acid, or a combination thereof, but is not limited thereto.

[0252] Table 15

[0253] In the stirring stress test, the liquid pharmaceutical formulations prepared in Examples 9 and 13 were placed in a constant vortex and their stability was measured after 0 hours (marked as the start) and 4 hours. The results of the analysis are described in Table 15 above. The formulations were in a stable state during the stirring process.

[0254] Table 16

[0255] The stability of the liquid pharmaceutical formulations prepared in Examples 9 and 13 was analyzed after being frozen and thawed 0 times (marked as start), 1 time, 3 times and 5 times (marked as cycle). The results of the analysis are described in Table 16 above. The formulations were in a stable state during the repeated freezing and thawing process.

[0256] Stability Test Example 6: Stability of the Composition Depends on Thermal Stress and UV Light Pressure

[0257] Regarding the liquid pharmaceutical preparation used in Stability Test Example 6, buffer solutions having corresponding buffer concentrations, pH values ​​and tonicity agents were prepared, and methionine and an organic co-solvent were added to obtain the samples listed in Tables 17 and 18 below. The concentration of each component is recorded in Tables 17 and 18 below. For example, the total volume is 5 ml.

[0258]

[0259] Heat stress

[0260] The stability of the liquid pharmaceutical formulations prepared in Examples 14 to 17 was measured after being placed at a temperature of 50°C ± 2°C and a room humidity of 75 ± 5% for 0 days (marked as the start), 3 days, 6 days, 10 days and 2 weeks. The analysis results are described in Table 17 above. In one embodiment, the formulation may include acetate buffer, sucrose or trehalose, methionine, or a combination thereof, but is not limited thereto. In another embodiment, the formulation may include histidine buffer, sucrose or trehalose, methionine, or a combination thereof, but is not limited thereto.

[0261]

[0262] UV radiation pressure

[0263] The liquid pharmaceutical formulations prepared in Examples 14 to 17 were continuously exposed to UV / visible radiation (≥200 watt-hours / meter2, UV range of 320 nm-400 nm) at 0 days (marked as start), 3 days, 6 days, 10 days and 2 weeks, and their stability was measured. The results of the analysis are described in Table 18 above. In one embodiment, the formulation may include acetate buffer, sucrose, methionine, or a combination thereof, but is not limited thereto.

[0264] Stability Test Example 7: The stability of the composition depends on the type of buffer and tonicity agent

[0265] Regarding the liquid pharmaceutical preparation used in Stability Test Example 7, buffer solutions having corresponding buffer types and tonicity agents were prepared, and methionine and an organic co-solvent were added to obtain the samples listed in Tables 19 and 20 below. The concentration of each component is recorded in Tables 19 and 20 below. For example, the total volume of Table 19 or 20 is 5 ml or 4 ml.

[0266]

[0267]

[0268] The stability of the liquid pharmaceutical formulations prepared in Examples 18 to 25 was measured after being placed at a temperature of 40°C ± 2°C for 0 weeks (marked as the start), 1 week, 2 weeks, 4 weeks, 8 weeks and 12 weeks. Examples 18 to 21 are formulations comprising an antibody concentration of 120 mg per ml. Examples 22 to 25 are formulations comprising an antibody concentration of 150 mg per ml. The results of the analysis are described in Tables 19 and 20 above, respectively. In one embodiment, the formulation may comprise an acetate buffer, methionine, sucrose or trehalose, or a combination thereof, but is not limited thereto. In another embodiment, the formulation may comprise a histidine buffer, methionine, sucrose or trehalose, or a combination thereof, but is not limited thereto.

[0269] Stability Test Example 8: The stability of the composition depends on the type of buffer, organic co-solvent and amino acid

[0270] Regarding the liquid pharmaceutical preparation used in Stability Test Example 8, buffer solutions having corresponding buffer types, organic co-solvents and amino acids were prepared, and tonicity agents were added to obtain the samples listed in Tables 21 and 22 below. The concentration of each component is recorded in Tables 21 and 22 below. For example, the total volume of Table 21 or 22 is 5 ml or 4 ml.

[0271]

[0272]

[0273] After being placed at a temperature of 40°C ± 2°C for 0 months (marked as the start), 0.5 months, 1 month, 3 months (or 3.36 months in Table 28) and 6 months, the qualitative properties of the liquid pharmaceutical formulations prepared in Examples 26 to 32 were measured. Examples 26 to 29 are formulations comprising an antibody concentration of 120 mg per ml. Examples 30 to 32 are formulations comprising an antibody concentration of 150 mg per ml. The analytical results are described in Tables 21 and 22 above, respectively. In one embodiment, the formulation may comprise acetate buffer, sucrose, polysorbate 80, methionine and glutamic acid, but are not limited thereto. In another embodiment, the formulation may comprise acetate buffer, sucrose, polysorbate 80, methionine or glutamic acid, or a combination thereof, but are not limited thereto. In another embodiment, the formulation may comprise histidine buffer, trehalose, polysorbate 20, methionine, or a combination thereof.

[0274] Stability Test Example 9: Long-term Stability

[0275] Regarding the liquid pharmaceutical formulation used in the stability test Example 9, buffer solutions having corresponding buffer types, organic co-solvents and amino acids were prepared, and tonicity agents were added to obtain samples of Examples 33 to 36, which were placed at a temperature of 5° C. for 0, 0.25, 0.5, 0.75, 1, 2, 3.36, 6, 12, 18 and 24 months, or at a temperature of 25° C. and 40° C. for up to 6 months, and their intact stability was measured by molecular sieving high performance liquid chromatography. Example 33 is a formulation containing 150 mg of trastuzumab per ml, 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 comprising 150 mg of trastuzumab per ml, 20 mM acetic acid (pH 5.2), 210 mM sucrose, 10 mM methionine, and 0.02% polysorbate 80 (A / S / M / PS80 pH5.2). Example 35 is a formulation comprising 150 mg of trastuzumab per ml, 20 mM acetic acid (pH 5.2), 210 mM sucrose, 10 mM methionine, 40 mM glycine, and 0.02% polysorbate 80 (A / S / M / 40Gly pH5.2). Example 36 is a formulation comprising 150 mg of trastuzumab per ml, 20 mM acetic acid (pH 5.2), 210 mM sucrose, 10 mM methionine, 10 mM glutamic acid, and 0.02% polysorbate 80 (A / S / M / 10Glu pH5.2). Figure 5A , Figure 5B and Figure 5CThe integrity results at 5°C, 25°C and 40°C are described respectively. The sample of Example 36 can maintain high antibody purity for at least 24 months. After 0, 0.25, 0.5, 0.75, 1, 2, 3.36, 6, 12, 18 and 24 months at 5°C ( Fig. 6A ), after being placed at 25℃ for 6 months ( Figure 6B ), or after being placed at 40℃ for 1 month ( Figure 6C ), the charge heterogeneity of Examples 33 to 36 was measured using CEX analysis.

[0276] Stability Test Example 10: Tonicity Agent Analysis

[0277] Regarding the liquid pharmaceutical formulation used in the stability test Example 10, buffer solutions having corresponding buffer types, organic co-solvents and amino acids were prepared, and tonicity agents were added to obtain samples of Examples 37 to 40, which were placed at 5° C., 25° C. or 40° C. for 0, 0.5, 1, 2 and 3 months, and their complete stability was measured. Example 37 is a formulation comprising 120 mg of trastuzumab per ml, 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 comprising 120 mg of trastuzumab per ml, 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 comprising 120 mg of trastuzumab per ml, 20 mM acetic acid (pH 5.2), 210 mM mannitol, 10 mM methionine, 10 mM glutamic acid, and 0.02% polysorbate 80. Example 40 is a formulation comprising 120 mg of trastuzumab per ml, 20 mM acetic acid (pH 5.2), 210 mM sorbitol, 10 mM methionine, 10 mM glutamic acid, and 0.02% polysorbate 80. Fig. 7A , Figure 7B and Figure 7C The integrity results at 5°C, 25°C and 40°C are described separately.

[0278] The charge heterogeneity stability of the liquid pharmaceutical formulations prepared in Examples 37 to 40 was measured after being placed at 5° C., 25° C., or 40° C. for 0, 0.5, 1, 2, and 3 months. Fig. 8A , Figure 8B and Figure 8C The charge heterogeneity results are described for exposure to temperatures of 5°C, 25°C and 40°C, respectively.

[0279] Stability Test Example 11: Evaluation of the Stability of Pertuzumab

[0280] Regarding the liquid pharmaceutical formulation used in the stability test Example 11, buffer solutions having the corresponding buffer species, organic co-solvents and amino acids were prepared, and tonicity agents were added to obtain samples of Examples 41 to 44, which were placed at 5° C., 25° C. or 40° C. for 0, 0.5, 1, 2 and 3 months, and their intact stability was measured. Example 41 is a formulation comprising 120 mg of Pertuzumab per ml, 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 comprising 120 mg of Pertuzumab per ml, 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 comprising 120 mg of Pertuzumab per ml, 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 formulation comprising 120 mg of Pertuzumab per ml, 20 mM acetic acid (pH 5.2), 210 mM sorbitol, 10 mM methionine, 10 mM glutamic acid, and 0.02% polysorbate 80. Fig.9A , Fig. 9B and Fig. 9C The integrity results at 5°C, 25°C and 40°C are described respectively. The samples of Examples 41 to 44 can maintain a high level of purity of the Pertuzumab antibody.

[0281] The charge heterogeneity stability of the liquid pharmaceutical formulations prepared in Experiments 41 to 44 was measured after storage at 5°C, 25°C and 40°C for 0, 0.5, 1, 2 and 3 months. Fig. 10A , Fig. 10B , Fig. 10C and Fig. 10D The charge heterogeneity results of Examples 41 to 44 are described separately.

[0282] Animal testing

[0283] Animal Test Example 1: Mouse pharmacodynamics (PD) test - anti-tumor activity of trastuzumab under different buffer conditions.

[0284] Xenogeneic implantation mode

[0285] All procedures were performed in accordance with applicable laws, regulations, and guidelines provided by the National Institutes of Health (NIH) and were approved by the Animal Care and Use Committee. BT-474 human breast cancer cell line (ATCC, USA) (1.0E+007 BT-474) in 200 μl of serum-free Dulbecco's Modified Eagle Medium (DMEM) was implanted into NSG mice (NOD.Cg-Prkdc scid Iq tm1Wjl / SzJ mice, Jackson Laboratory, USA). Treatment was started 7 days after tumor implantation. The formula (length x width 2 ) / 2 Calculate tumor volume (cubic millimeters, mm 3 ), where length is the longest axis and width is the thickness measurement at right angles to the length. The values ​​for each treatment group are expressed as mean tumor volume ± SE. The Student's T test was used to analyze whether the values ​​were statistically significant. In each experiment, the number of mice per group was 6.

[0286] Cell lines and culture conditions

[0287] Human breast cancer cell line BT-474 was cultured in DMEM containing 10% non-heat-inactivated fetal bovine serum (FBS) and 1% penicillin / streptomycin / L-Glutamine (PSG) at 37°C. When implanted into mice, the cell survival rate was 95%.

[0288] Therapy Animals

[0289] Mice were divided into study groups based on tumor size measured by vernier caliper. Mice were treated according to Table 23:

[0290] Table 23: Basis Fig.11 Treatment groups and dosage courses

[0291] Notes: ROA-route of administration; PBS-Phosphate-Buffered Saline; Herceptin SC-600 mg SC injection solution of trastuzumab obtained from Roche EU, EG13074 and EG12014 are the same active pharmaceutical ingredient (API) (trastuzumab); SC-subcutaneous; IV-intravenous; Q7Dx6-treatment once every 7 days, a total of 6 treatments. EG12014 (150 mg of freeze-dried trastuzumab) was completely reconstituted with water for injection and finally placed in a buffer of 4.4 mM L-histidine / L-histidine hydrochloride (pH 6.0), 1.71% trehalose dihydrate, and 0.01% polysorbate 20. EG13074-3 was prepared in 20 mM acetic acid / sodium acetate (pH 5.2), 210 mM sucrose, 10 mM methionine, 10 mM glutamic acid, and 0.02% polysorbate 80.

[0292] Fig.11 The PD results of these treatments are plotted.

[0293] Animal Test Example 2: Mouse pharmacodynamics (PD) test to detect the relationship between the plasma concentration (μg / mL) of trastuzumab in different formulations and time.

[0294] Different formulations containing trastuzumab were prepared according to Table 24 for PD analysis.

[0295] Table 24. Mouse pharmacokinetic (PK) analysis conditions Note: EG13074-3 and EG12014 contain the same active pharmaceutical ingredient (trastuzumab); Met – methionine; Ace – acetic acid / sodium acetate; PS – polysorbate.

[0296] PK studies were performed in CD-1 male mice (Vital River, Pinghu, China), where each mouse was 5-6 weeks old and weighed between 25-38 grams. A maximum of 5 animals / sex / cage were included in each group and housed in polycarbonate cages with bedding. Animals were randomly assigned to groups. Mice were divided into 5 groups of 25 mice each. Groups 1-4 were given SC bolus doses. Group 5 was given IV bolus doses. Animals were treated according to the protocol described in Table 25.

[0297] Table 25: Treatment groups as described in Figure 12

[0298] At each time point, approximately 0.2 ml of blood was collected from the submandibular area. The specimens were collected into serum separation tubes at 16 time points: before administration, 0.25, 1, 4, 8, and 24 hours after administration, 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 after administration. No anticoagulant was used. The blood was allowed to coagulate for 30 minutes at room temperature. Within 2 hours after collection, the specimens were centrifuged at approximately 2700 g for approximately 10 minutes at 2 to 8°C, and the serum was collected and stored at -60 to -80°C for subsequent analysis.

[0299] Biological analysis

[0300] The pharmacokinetic analysis of trastuzumab in mouse serum was performed using ELISA.

[0301] Table 26 summarizes the analysis results of the PK studies.

[0302]

[0303] The analysis results of the PK test are shown in Fig. 12A and Fig. 12B middle.

[0304] Equality and scope

[0305] Unless otherwise indicated, the words "a" or "an" and "the" in the claims may mean one or more. Unless otherwise indicated, the claims or description may use the word "or" to connect one, more, or all of a group of components when the components are present in, included in, or related to a particular product or process. The present disclosure includes certain embodiments in which a component of the group is present in, included in, or related to a particular product or process. The present disclosure includes certain embodiments in which more or all of the components of the group are present in, included in, or related to a particular product or process (manufacturing process).

[0306] In addition, the present invention encompasses all variations, combinations and permutations, wherein one or more limitations, components, clauses and descriptive words recited in one or more claims may be referenced in another claim. For example, any dependent claim attached to a claim may be modified to include one or more limitations recited in another dependent claim, as long as the dependent claims are attached to the same basic claim. When components are listed in a group description form such as Markush, and the specification discloses each subgroup of components, any component can be removed from the group. It is understood that, in general, when the present invention or the aspects of the present invention include specific components and / or features, then certain embodiments of the present invention or the aspects of the present invention are composed of these components and / or features, or are essentially composed of these components and / or features. For the sake of brevity, the present disclosure does not specifically describe these embodiments. It should also be noted that "comprising" or "containing" should be understood as open-ended and may include additional components or steps. When a range is recorded, the range also includes the endpoints. In addition, in different embodiments of the present disclosure, unless otherwise specified, or the present disclosure and those of ordinary skill in the art to which the invention belongs have contrary teachings or understandings, the values ​​expressed as ranges may be any specific values ​​or sub-ranges within the range, up to one tenth of the unit of the lower limit of the range.

[0307] The present disclosure relates to various announced patents, published patent applications, journal documents and other public contents, which are incorporated into the present disclosure for reference. If any incorporated reference conflicts with the present disclosure, the present disclosure shall prevail. In addition, any specific implementation of the present disclosure that falls into the prior art may be explicitly excluded in one or more claims. Based on these implementations, it can be considered that the technical field to which the present invention belongs has the skills known to the ordinary knowledgeable person, even if the present disclosure does not explicitly set forth. Regardless of any factors, whether or not related to the prior art, specific implementations of the present disclosure may be excluded from the scope of the patent application.

Claims

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

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

3. The pharmaceutical composition of claim 1, wherein the anti-HER2 antibody is trastuzumab or pertuzumab, and the concentration of trastuzumab or pertuzumab is 80 to 150 mg per ml.

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

5. The pharmaceutical composition of claim 1, wherein the concentration of methionine is 5 to 15 mM, and the concentration of glutamate is 5 to 15 mM.

6. The pharmaceutical composition of claim 1, further comprising a tension agent selected from the group consisting of trehalose, sucrose, mannitol and sorbitol.

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

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

9. The pharmaceutical composition of claim 1, wherein the composition is substantially free of hyaluronidase. Page 1 of 2 (Claims) 10. The pharmaceutical composition of claim 1, wherein the composition is for subcutaneous administration.

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

5.

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

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

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

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

16. A prefilled syringe comprising 3 ml to 7 ml of a solution, wherein the solution comprises: (a) anti-HER2 antibodies, (b) methionine, and (c) Glutamate.

17. The prefilled syringe of claim 16, wherein the anti-HER2 antibody is trastuzumab or pertuzumab and is present in a concentration of 80 to 150 mg per ml.

18. The prefilled syringe of claim 17, wherein the concentration of the methionine is 5 to 15 mM, and the concentration of the glutamate is 5 to 15 mM.

19. The prefilled syringe of claim 16, wherein the solution further comprises sucrose.

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

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