CEACAM5 antibody-drug conjugate preparation

Abstract

Provided are stabilized formulations of antibody-drug conjugates (huMAb2-3-SPDB-DM4) targeted to human carcinoembryonic antigen-related cell adhesion molecule 5 (hCEACAM5), as well as methods for making and using the same.

Description

[Technical Field]

[0001] Sequence List This application is filed electronically in ASCII format and includes an array listing which is incorporated in its entirety by reference herein. The ASCII copy prepared on 31 July 2020 is named 707628_SA9-297-1_ST25.txt and has a size of 9,175 bytes.

[0002] field This disclosure relates to the field of treatment for cancer, such as non-squamous non-small cell lung cancer expressing CEACAM5. Specific aspects of the present invention relate to formulations and uses of CEACAM5 antagonists, such as anti-CEACAM5 immunoconjugates, for treating cancer. [Background technology]

[0003] Carcinoembryonic antigens (CEAs) are glycoproteins involved in cell adhesion. CEAs were first identified in 1965 as proteins normally expressed in the fetal intestine during the first six months of pregnancy (Non-Patent Literature 1), and have also been found in pancreatic, liver, and colon cancers. CEAs are part of a family belonging to the immunoglobulin superfamily. The CEA family, consisting of 18 genes, is subdivided into two protein subgroups: the carcinoembryonic antigen-associated cell adhesion molecule (CEACAM) subgroup and the pregnancy-specific glycoprotein subgroup (Non-Patent Literature 2).

[0004] Numerous studies have revealed that CEACAM5, a subgroup of CEACAM and identical to the initially identified CEA, is highly expressed on the surface of colorectal, gastric, lung, breast, prostate, ovarian, cervical, and bladder tumor cells, while being weakly expressed in some normal epithelial tissues, such as columnar epithelium and goblet cells in the colon, gastric glandular mucinous cells in the stomach, and squamous epithelial cells in the esophagus and cervix (Non-Patent Literature 3). Therefore, CEACAM5 constitutes a suitable therapeutic target for tumor-specific targeting approaches, such as immunoconjugates.

[0005] huMAb2-3-SPDB-DM4 is an immunoconjugate (antibody-drug conjugate, ADC) containing a humanized anti-CEACAM5 antibody linked to meitansinoid derivative 4 (DM4), a potent antimitotic agent that inhibits microtubule assembly. DM4 is stable in plasma and covalently binds to the antibody via an optimized linker SPDB [N-succinimidyl-4-(2-pyridyldithio)butanoic acid] that is cleavable inside cells. After binding and internalizing into targeted cancer cells, huMAb2-3-SPDB-DM4 is degraded, releasing cytotoxic DM4 metabolites.

[0006] Currently, huMAb2-3-SPDB-DM4 is undergoing several clinical trials and is expected to be useful in the treatment of various types of CEACAM5-expressing cancers. For example, recent clinical trials have shown that huMAb2-3-SPDB-DM4 is effective in treating non-squamous non-small cell lung cancer (NSQ NSCLC), a subtype that accounts for approximately 60% of lung cancers (see Patent Document 1). However, ADCs, such as huMAb2-3-SPDB-DM4, are difficult to formulate to have long-term stability and shelf life. Therefore, the development of a novel formulation of huMAb2-3-SPDB-DM4 is of great interest. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] International Publication No. 2020 / 161214 [Non-Patent Document]

[0008] [Non-Patent Document 1] Gold and Freedman, J Exp Med, 121, 439, 1965 [Non-Patent Document 2] Kammerer & Zimmermann, BMC Biology 2010, 8:12 [Non-Patent Document 3] Hammarstrom et al., 2002, in “Tumor Markers, Physiology, Pathobiology, Technology and Clinical Applications” Eds. Diamandis E.P. et al., AACC Press, Washington pp375ff [Summary of the Invention] [Problems to be Solved by the Invention]

[0009] The present disclosure particularly provides an improved formulation of huMAb2-3-SPDB-DM4, which is an antibody-drug conjugate (ADC) consisting of a monoclonal antibody that specifically binds to human carcinoembryonic antigen-related cell adhesion molecule 5 (hCEACAM5) conjugated with N2’-deacetyl-N-2’(4-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4). The formulations disclosed herein are different and optimized compared to the formulations of huMAb2-3-SPDB-DM4 that have been used in clinical trials to date. [Means for Solving the Problems]

[0010] Aspects of the present disclosure are at a pH of about 5.0 to about 6.5, i) an anti-human carcinoembryonic antigen-related cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Buffering agent, iii) Isotonic agents, and iv) Surfactants A pharmaceutical formulation containing [a specific compound], wherein the ADC consists of huMAb2-3-SPDB-DM4.

[0011] In a particular embodiment, the pharmaceutical formulation is a lyophilized formulation.

[0012] In a particular embodiment, the pharmaceutical formulation is a liquid formulation.

[0013] The aspects of this disclosure are as follows: i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Buffering agent, iii) tonicity agent; iv) Surfactants, and v) Chelating agents A pharmaceutical formulation containing [a specific compound], wherein the ADC consists of huMAb2-3-SPDB-DM4.

[0014] In a particular embodiment, the pharmaceutical formulation is a lyophilized formulation.

[0015] In a particular embodiment, the pharmaceutical formulation is a liquid formulation.

[0016] In certain embodiments, the buffer is selected from the group consisting of acetates, arginine, histidine, citrates, and any combination thereof. In some embodiments, the buffer is an acetate, such as sodium acetate.

[0017] In certain embodiments, the isotonic agent is a polyol. In certain embodiments, the polyol is selected from the group consisting of erythritol, glycerin, lactitol, maltitol, mannitol, sorbitol, sucrose, threitol, xylitol, and any combination thereof. In certain embodiments, the isotonic agent is sorbitol.

[0018] In a particular embodiment, the surfactant is selected from the group consisting of polysorbate 20, polysorbate 80, poloxamer 188, and any combination thereof.

[0019] In a particular embodiment, the surfactant is polysorbate 80.

[0020] In certain embodiments, the chelating agent is selected from the group consisting of diethylenetriaminepentaacetic acid (DTPA), ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), ethylenediaminetetraacetic acid (EDTA), hydroxyethylenediaminetriacetic acid (HEDTA), nitrilotriacetic acid, and any combination thereof. In certain embodiments, the chelating agent is EDTA.

[0021] In a particular embodiment, the pharmaceutical formulation is at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) For example, a buffer selected from the group consisting of acetates, arginine, histidine, citrates, and any combination thereof, or for example, an acetate, iii) Polyols, for example, sorbitol, iv) Polysorbates, for example, polysorbate 80, and v) Chelatives may be used depending on the circumstances The ADC includes huMAb2-3-SPDB-DM4.

[0022] In a particular embodiment, the pharmaceutical formulation is at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) For example, a buffer selected from the group consisting of acetates, arginine, histidine, citrates, and any combination thereof, or for example, an acetate, iii) Sorbitol, iv) Polysorbates, and v) Chelatives may be used depending on the circumstances The ADC includes huMAb2-3-SPDB-DM4.

[0023] In a particular embodiment, the pharmaceutical formulation is at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) For example, a buffer selected from the group consisting of acetates, arginine, histidine, citrates, and any combination thereof, or for example, an acetate, iii) Polyols, iv) Polysorbate 80, and v) Chelatives may be used depending on the circumstances The ADC includes huMAb2-3-SPDB-DM4.

[0024] In a particular embodiment, the pharmaceutical formulation is at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) For example, a buffer selected from the group consisting of acetates, arginine, histidine, citrates, and any combination thereof, or for example, an acetate, iii) Sorbitol, iv) Polysorbate 80, and v) Chelatives may be used depending on the circumstances The ADC includes huMAb2-3-SPDB-DM4.

[0025] In a particular embodiment, the pharmaceutical formulation is at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Sodium acetate, iii) Sorbitol, iv) Polysorbate 80, and v) Chelatives may be used depending on the circumstances The ADC includes huMAb2-3-SPDB-DM4.

[0026] In a particular embodiment, the pharmaceutical formulation is at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Buffering agent, iii) tonicity agent; iv) Polysorbates, e.g., polysorbate 80, and v) Chelating agents The ADC includes huMAb2-3-SPDB-DM4.

[0027] In a particular embodiment, the pharmaceutical formulation is at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Buffers selected from acetates, arginine, histidine, citrates, and any combination thereof, iii) tonicity agent; iv) Polysorbate 80, and v) Ethylenediaminetetraacetic acid (EDTA) The ADC includes huMAb2-3-SPDB-DM4.

[0028] In a particular embodiment, the pharmaceutical formulation is at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Buffers selected from acetates, histidines, citrates, and any combination thereof, iii) Isotonic agents selected from erythritol, glycerin, lactitol, maltitol, mannitol, sorbitol, sucrose, trethitol, xylitol, and any combination thereof. iv) Polysorbate 80, and v) EDTA The ADC includes huMAb2-3-SPDB-DM4.

[0029] In a particular embodiment, the pharmaceutical formulation is at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Buffers selected from acetates, histidines, citrates, and any combination thereof, iii) Sorbitol, iv) Polysorbate 80, and v) EDTA The ADC includes huMAb2-3-SPDB-DM4.

[0030] In a particular embodiment, the pharmaceutical formulation is at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Sodium acetate, iii) Sorbitol, iv) Polysorbate 80, and v) EDTA The ADC includes huMAb2-3-SPDB-DM4.

[0031] In a particular embodiment, the pharmaceutical formulation is at a pH of approximately 4.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) A buffer selected from acetate, histidine, citrate, and any combination thereof in concentrations of approximately 5 mM to 100 mM. iii) Polyols, iv) Surfactants selected from the group consisting of polysorbate 80, polysorbate 20, poloxamer 188, and any combination thereof, and v) EDTA The ADC includes huMAb2-3-SPDB-DM4.

[0032] In a particular embodiment, the pharmaceutical formulation is at a pH of approximately 4.5 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) A buffer selected from acetate, histidine, citrate, and any combination thereof in concentrations of approximately 5 mM to 100 mM. iii) Polyols, iv) Surfactants selected from the group consisting of polysorbate 80, polysorbate 20, poloxamer 188, and any combination thereof, and v) EDTA The ADC includes huMAb2-3-SPDB-DM4.

[0033] In a particular embodiment, the pharmaceutical formulation is at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) A buffer selected from acetate, histidine, citrate, and any combination thereof in concentrations of approximately 5 mM to 100 mM. iii) Polyols, iv) Surfactants selected from the group consisting of polysorbate 80, polysorbate 20, poloxamer 188, and any combination thereof, and v) EDTA The ADC includes huMAb2-3-SPDB-DM4.

[0034] In a particular embodiment, the pharmaceutical formulation is at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) A buffer selected from acetate, histidine, citrate, and any combination thereof in concentrations of approximately 5 mM to 100 mM. iii) Isotonic agents selected from erythritol, glycerin, lactitol, maltitol, mannitol, sorbitol, sucrose, trethitol, xylitol, and any combination thereof. iv) Polysorbate 80, and v) EDTA The ADC includes huMAb2-3-SPDB-DM4.

[0035] In a particular embodiment, the pharmaceutical formulation is at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Buffers selected from acetates, histidines, citrates, and any combination thereof, iii) Isotonic agents selected from erythritol, glycerin, lactitol, maltitol, mannitol, sorbitol, sucrose, trethitol, xylitol, and any combination thereof in concentrations of approximately 1% (w / v) to approximately 50% (w / v), iv) Polysorbate 80, and v) EDTA, The ADC includes huMAb2-3-SPDB-DM4.

[0036] In a particular embodiment, the pharmaceutical formulation is at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) A buffer selected from acetate, histidine, citrate, and any combination thereof in concentrations of approximately 5 mM to 100 mM. iii) Isotonic agents selected from erythritol, glycerin, lactitol, maltitol, mannitol, sorbitol, sucrose, trethitol, xylitol, and any combination thereof in concentrations of approximately 1% (w / v) to approximately 50% (w / v), iv) Polysorbate 80, and v) EDTA The ADC includes huMAb2-3-SPDB-DM4.

[0037] The aspects of this disclosure are as follows: at pH 5.5, i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Sodium acetate, iii) Sorbitol, iv) Polysorbate 80, and v) Disodium EDTA A pharmaceutical formulation containing [a specific compound], wherein the ADC consists of huMAb2-3-SPDB-DM4.

[0038] In a particular embodiment, the pharmaceutical formulation is at pH 5.5, i) 5 mg / mL anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) 10 mM sodium acetate, iii) 5% (w / v) sorbitol, iv) 0.04% (w / v) polysorbate 80, and v) 1-50 μM disodium EDTA The ADC includes huMAb2-3-SPDB-DM4.

[0039] In a particular embodiment, the pharmaceutical formulation is at pH 5.5, i) 5 mg / mL anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) 10 mM sodium acetate, iii) 5% (w / v) sorbitol, iv) 0.04% (w / v) polysorbate 80, and v) 1 μM EDTA disodium, The ADC includes huMAb2-3-SPDB-DM4.

[0040] In a particular embodiment, the pharmaceutical formulation is at pH 5.5, i) 5 mg / mL anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) 10 mM sodium acetate, iii) 5% (w / v) sorbitol, iv) 0.04% (w / v) polysorbate 80, and v) 10 μM disodium EDTA The ADC includes huMAb2-3-SPDB-DM4.

[0041] In a particular embodiment, the pharmaceutical formulation is at pH 5.5, i) 5 mg / mL anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) 10 mM sodium acetate, iii) 5% (w / v) sorbitol, iv) 0.04% (w / v) polysorbate 80, and v) 50 μM disodium EDTA The ADC includes huMAb2-3-SPDB-DM4.

[0042] A part of the present disclosure is a method for treating cancer, comprising the step of administering an effective amount of one of the above-mentioned pharmaceutical formulations to a subject in need thereof.

[0043] In a particular embodiment, the pharmaceutical formulation is at pH 5.5, i) 5 mg / mL anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) 10 mM sodium acetate, iii) 5% (w / v) sorbitol, iv) 0.04% (w / v) polysorbate 80, and v) 10 μM disodium EDTA The ADC includes huMAb2-3-SPDB-DM4.

[0044] In certain embodiments, the cancer is a highly carcinoembryonic antigen-associated cell adhesion molecule carcinoma.

[0045] In certain embodiments, cancer is selected from the group consisting of colorectal, gastric, lung, breast, prostate, ovarian, cervical, and bladder cancers.

[0046] In certain embodiments, cancer is selected from the group consisting of lung, breast, prostate, ovarian, cervical, and bladder cancers.

[0047] In a particular embodiment, the cancer is lung cancer.

[0048] In certain embodiments, the lung cancer is non-squamous non-small cell lung cancer.

[0049] In certain embodiments, the subject is human. [Brief explanation of the drawing]

[0050] [Figure 1] The image on the left shows the overall structure of huMAb2-3-SPDB-DM4, and the image on the right shows the structure of its chemical part. [Figure 2] This graph shows the SEC-HPLC T0 analytical chromatograms of prototype formulations A-H. It also shows an overlay of the SEC-HPLC graphs of prototypes A-H at T0. See Example 3. [Figure 3] This graph shows t0 analytical chromatograms of free mytansinoids for prototype A (acetate), prototype C (histidine), and prototype E (citrate). It shows the free mytansinoids for prototype A (medium gray), prototype C (light gray), and prototype E (dark gray) at T0. X-axis: min; Y-axis: AU. [Figure 4] Figure 4A shows SDS-PAGE images at t0 for prototype formulations A-H under non-reducing conditions. Figure 4B shows SDS-PAGE images at t0 for prototype formulations A-H under reducing conditions. [Figure 5]This graph shows the evolution of OD350nm after the specified stress. The Y-axis represents OD350nm. For each stress condition, prototypes appear from A to H. [Figure 6] This graph shows the evolution of the drug-to-antibody ratio (DAR) after thermal stress. The X-axis represents the prototype formulation. [Figure 7] This graph shows the evolution of aggregates under thermal stress. For each prototype, the stress conditions appear in the following order: T0, T2w40°C, T4W40°C, T1M5°C. X-axis: Prototype formulation. [Figure 8] This graph shows the evolution of the fragments under thermal stress. For each prototype, the stress conditions appear in the following order: T0, T2w40°C, T4W40°C, T1M5°C. X-axis: Prototype formulation. [Figure 9] This graph shows the total amount of free mytansinoids after heat stress. Except for prototype D, the T0 content is invisible. For all prototypes, T2W (40°C) is visible. The T4W time point was analyzed only for prototypes A, B, C, G, and H. X-axis: Prototype formulation; Y-axis: Free mytansinoid content. [Figure 10] This graph shows the superimposed free mytansinoid profiles of formulation A (acetate, lower trace) and formulation C (histidine, upper trace) after 4 weeks at 40°C. [Figure 11] This graph shows the evolution of acidic isoforms after the indicated thermal stress. For each prototype, the time points are shown in the following order: T0, T2W 40°C, T4W ​​40°C, T4W ​​5°C, T4W ​​25°C. Prototypes E and F were not analyzed for the T4W time point. X-axis: Prototype formulation; Y-axis: Acidic isoform %. [Figure 12] This graph shows the visualized capillary isoelectric focusing (iCIEF) chromatograms of prototype formulation A (acetate) at t0 (upper panel) and 4 weeks after 40°C (lower panel). X-axis: Time (minutes). [Figure 13] This graph shows the superimposed iCIEF chromatograms after shaking stress for formulations A (acetate), C (histidine), and E (citrate). [Figure 14] This graph shows the evolution of DAR under thermal stress (initial formulation development; Example 2). Prototypes A through F are shown for each time point. Y-axis: DAR UV. [Figure 15] This graph shows the icIEF evolution of the major charge isoforms under thermal stress. For each time point, prototypes are shown from A to F. [Figure 16] This graph shows the evolution of total free mytansinoids under heat stress. For each time point, prototypes are shown from A to F. [Figure 17] This graph shows the evolution of LMW under thermal stress. For each time point, prototypes are shown from A to F. [Figure 18] This graph shows the results of a high-precision in-liquid particle counter (HIAC) after simulation during use, with particles ≥ 1.5 μm. For each prototype, the stress is expressed in the following order: no stress after shaking stress (T0), P0 (after dilution in an IV bag of 0.9% NaCl at 0.9 g / L), P1 (24 hours after dilution in an IV bag of 0.9% NaCl at 0.9 g / L), and P2 (24 hours after dilution in an IV bag of 0.9% NaCl at 0.9 g / L and perfusion through an IV infusion with an in-line filter). [Figure 19A] This graph shows the percentage variation in concentration after simulation during use. [Figure 19B] This graph shows the concentration variations after simulation during use. [Figure 20A] This graph shows the DAR variation (%) after simulation during use. [Figure 20B] This graph shows the DAR variation (%) after simulation during use. [Figure 21]This graph shows the evolution of the HMW during simulation. For both time points, the prototypes are shown from A to F. [Figure 22] This graph shows the evolution of the LMW during simulation. For each time point, prototypes A through F are shown. [Figure 23] This graph shows the DAR evolution in a packed vial for samples of the indicated EDTA concentrations after the indicated thermal stress. T0, time zero; T2W 40°C, 2 weeks at 40°C; T1M T2W 40°C, 1 month at 40°C; T3M 25°C, 3 months at 25°C (Example 3). The T3M 25°C time point was analyzed only for samples without EDTA and samples with 10 μM EDTA. [Figure 24] This graph shows the monomer percentage variation in packed vials for samples with the indicated EDTA concentrations after the indicated thermal stress. The T3M25°C time point was analyzed only for samples without EDTA and for the 10 μM EDTA sample. [Figure 25] This graph shows the high molecular weight (HMW)% variation in packed vials for samples with the indicated EDTA concentrations after thermal stress. The T3M25°C time point was analyzed only for samples without EDTA and for the 10 μM EDTA sample. [Figure 26] This graph shows the sum of the evolution of other fragments of the sample at the indicated EDTA concentration after the indicated thermal stress. [Figure 27] This graph shows the H2L 2% variation (non-reducing capillary gel electrophoresis) of samples with the indicated EDTA concentrations after thermal stress. [Figure 28] This graph shows the total percentage of light and heavy chains (reduced capillary gel electrophoresis) of the sample at the indicated EDTA concentration after thermal stress. [Figure 29] This graph shows the PS80 content of samples with the indicated EDTA concentrations after thermal stress. The T3M25°C timepoint was analyzed for 1 μM and 50 μM EDTA samples. [Figure 30] This graph shows the total percentage of major isoforms in a filled vial for the sample with the indicated EDTA concentration after thermal stress. [Figure 31] This graph shows the total percentage of low pI in the filled vial for the samples with the indicated EDTA concentrations after thermal stress. [Modes for carrying out the invention]

[0051] This invention discloses a pharmaceutical formulation of an antibody-drug conjugate (ADC) that specifically binds to the human and cynomolgus monkey (Macaca Fascicularis) CEACAM5 protein. This ADC is known as huMAb2-3-SPDB-DM4, and the formulation disclosed herein is useful for treating any of the various types of CEACAM5-expressing cancers, including non-squamous non-small cell lung cancer. Unexpectedly, it has been found in accordance with this disclosure that the inclusion of a moderate amount of ethylenediaminetetraacetic acid (EDTA) in the formulation of huMAb2-3-SPDB-DM4 significantly increases the stability of the ADC and its formulation.

[0052] Carcinoembryonic antigens (CEAs) are glycoproteins involved in cell adhesion. First identified in 1965 as proteins normally expressed by the fetal intestine during the first six months of pregnancy (Gold and Freedman, J Exp Med, 121, 439, 1965), CEAs have also been found in pancreatic, liver, and colon cancers. CEAs belong to a family within the immunoglobulin superfamily. The CEA family, consisting of 18 genes, is subdivided into two protein subgroups: the carcinoembryonic antigen-associated cell adhesion molecule (CEACAM) subgroup and the pregnancy-specific glycoprotein subgroup (Kammerer & Zimmermann, BMC Biology 2010, 8:12).

[0053] In humans, the CEACAM subgroup consists of seven members: CEACAM1, CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM7, and CEACAM8. Numerous studies have revealed that CEACAM5, which is identical to the initially identified CEA, is highly expressed on the surface of colorectal, gastric, lung, breast, prostate, ovarian, cervical, and bladder tumor cells, while it is expressed only weakly in some normal epithelial tissues, such as columnar epithelium and goblet cells in the colon, gastric glandular mucinous cells in the stomach, and squamous epithelial cells in the esophagus and cervix (Hammarstrom et al., 2002, in “Tumor Markers, Physiology, Pathobiology, Technology and Clinical Applications” Eds. Diamandis EP et al., AACC Press, Washington pp375). Therefore, CEACAM5 constitutes a suitable therapeutic target for tumor-specific targeting approaches, such as immunoconjugates.

[0054] As used herein, “CEACAM5” refers to “carcinoembryonic antigen-associated cell adhesion molecule 5,” also known as CD66e (surface antigen classification 66e) or CEA. CEACAM5 is a glycoprotein involved in cell adhesion. CEACAM5 is highly expressed, particularly on the surface of colorectal, gastric, lung, and uterine tumor cells.

[0055] As used herein, “high-grade CEACAM5 cancer” refers to any of several types of cancer, including colorectal, gastric, lung, breast, prostate, ovarian, cervical, and bladder cancers. In certain embodiments, “high-grade CEACAM5 cancer” refers to any of several types of cancer, including colorectal, lung, gastric, cervical, and pancreatic cancers.

[0056] In some embodiments, the lung cancer is non-squamous non-small cell lung cancer. In certain embodiments, highly CEACAM5 expressioners have an intensity of 2+ or higher in at least 50% of the expressing tumor cell population. In certain embodiments, highly CEACAM5 expressioners have an intensity greater than 2+ in at least 50% of the expressing tumor cell population. Highly CEACAM5 expressioners account for approximately 20% of lung cancers.

[0057] Immunoconjugate ADC huMAb2-3-SPDB-DM4 is an immunoconjugate combining the huMAb2-3 (anti-CEACAM5) antibody with the meitansinoid derivative 4 (DM4), a potent antimitotic agent that inhibits microtubule assembly. DM4 is stable in plasma and covalently binds to huMAb2-3 via an optimized linker SPDB [N-succinimidyl-4-(2-pyridyldithio)butyrate] that is cleavable inside cells. After binding and internalizing into targeted cancer cells, huMAb2-3-SPDB-DM4 is degraded, releasing cytotoxic DM4 metabolites.

[0058] The antibody moiety of huMAb2-3-SPDB-DM4 is a human IgG1κ antibody having a pair of heavy chain (HC) domains (each containing a variable heavy chain (VH) domain) and a pair of light chain (LC) domains (each containing a variable light chain (VL) domain). Each VH domain contains three complementarity-determining regions (CDRs), namely HCDR1, HCDR2, and HCDR3. Each VL domain contains three complementarity-determining regions (CDRs), namely LCDR1, LCDR2, and LCDR3. The amino acid sequences for these polypeptides are as follows: HCDR1 Sequence ID 1 GFVFSSYD HCDR2 Sequence ID 2 ISSGGGIT HCDR3 Sequence ID 3 AAHYFGSSGPFAY LCDR1 Sequence ID 4 ENIFSY LCDR2 NTR LCDR3 Sequence ID 5 QHHYGTPFT VH Sequence ID 6 EVQLQESGPGLVKPGGSLSLSCAASGFVFSSYDMSWVRQTPERGLEWVAYISSGGGITYAPSTVKGRFTVSRDNAKNTLYLQMNSLTSEDTAVYYCAAHYFGSSGPFAYWGQGTLVTVSS VL Sequence ID 7 DIQMTQSPASLSASVGDRVTITCRASENIFSYLAWYQQKPGKSPKLLVYNTRTLAEGVPSRFSGSGSGTDFSLTISSLQPEDFATYYCQHHYGTPFTFGSGTKLEIK HC Sequence ID 8 EVQLQESGPGLVKPGGSLSLSCAASGFVFSSYDMSWVRQTPERGLEWVAYISSGGGITYAPSTVKGRFTVSRDNAKNTLYLQMNSLTSEDTAVYYCAAHYFGSSGPFAYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG LC Sequence ID 9 DIQMTQSPASLSASVGDRVTITCRASENIFSYLAWYQQKPGKSPKLLVYNTRTLAEGVPSRFSGSGSGTDFSLTISSLQPEDFATYYCQHHYGTPFTFGSGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0059] As used herein, “maytansinoid” refers to maytansinoids and maytansinoid analogs. Maytansinoids are drugs that inhibit microtubule formation and are highly toxic to mammalian cells.

[0060] Examples of suitable mayancinoids include mayancinol and mayancinol analogs.

[0061] The cytotoxic conjugate of the present invention, as a cytotoxic agent, is formally N 2’ -deacetyl-N- 2’ We utilize a thiol-containing maytansinoid called (4-methyl-4-mercapto-1-oxopentyl)-maytansine, DM4. DM4 is represented by the following structural formula (I): [ka]

[0062] In some embodiments, the antibody of the present invention is covalently linked to at least one growth inhibitor, either directly or via a cleavable or non-cleavable linker.

[0063] As used herein, "linker" means a chemical moiety that contains a chain of atoms covalently linking a polypeptide to a drug moiety.

[0064] Conjugates can be prepared by in vitro methods. Binding groups are used to link a drug or prodrug to an antibody. Suitable binding groups are well known in the art and include disulfide groups, thioether groups, acid-unstable groups, photo-dissociable groups, peptidase-unstable groups, and esterase-unstable groups. The conjugation of the antibody of the present invention with a cytotoxic agent or proliferation inhibitor involves N-succinimidyl-4-(2-pyridyldithio)butanoic acid (SPDB), 4-[(5-nitro-2-pyridinyl)dithio]-2,5-dioxo-1-pyrrolidinyl ester of butanoic acid (nitro-SPDB), 4-(pyridine-2-yldisulfanyl)-2-sulfobutyric acid (sulfo-SPDB), N-succinimidyl(2-pyridyldithio)propionate (SPDP), succinimidyl(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), and difunctional derivatives of imide esters (for example) This can be achieved using a variety of bifunctional protein coupling agents, including, but not limited to, dimethyl adipimidate HCl, active esters (e.g., disuccinimidylsberate), aldehydes (e.g., glutaraldehyde), bis-azide compounds (e.g., bis(p-azidobenzoyl)-hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene-2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene).

[0065] The linker may be a “cleavable linker” that facilitates the release of cytotoxic agents or growth inhibitors within cells. For example, acid-unstable linkers, peptidase-sensitive linkers, esterase-unstable linkers, photodissociative linkers, or disulfide-containing linkers (see, for example, U.S. Patent No. 5,208,020) may be used. The linker may also be a “non-cleavable linker” (e.g., an SMCC linker), which may provide better tolerability in some cases.

[0066] According to one embodiment, in the conjugate of the present invention, the growth inhibitor is the maytansinoid DM4.

[0067] In the conjugate, the antibody is conjugated to at least one growth inhibitor by a binding group. In one embodiment, the binding group is a cleavable or non-cleavable linker, such as N-succinimidyl-4-(2-pyridyldithio)butanoic acid (SPDB), 4-(pyridine-2-yldisulfanyl)-2-sulfobutyric acid (sulfo-SPDB), or succinimidyl (N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC).

[0068] In one embodiment, the binding group is SPDB, and the conjugate is of formula (II) [ka] The formula consists of an antibody-SPDB-DM4 conjugate, where n is an integer greater than or equal to 1. The average value of n is generally about 3.8. See also Figure 1.

[0069] Generally, conjugates are: (i) A step of contacting a buffered aqueous solution of a cell binding agent (for example, an antibody according to the present invention) with a solution of a linker and a cytotoxic compound; (ii) Next, the conjugate formed in (i) is optionally separated from the unreacted cell binder. It can be obtained through a process that includes this.

[0070] Aqueous solutions of cell binders can be buffered using buffers such as potassium phosphate, acetate, citrate, or N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES buffer). The buffer depends on the properties of the cell binder. Cytotoxic compounds are in solution in polar organic solvents such as dimethyl sulfoxide (DMSO) or dimethylacetamide (DMA).

[0071] The reaction temperature is typically between 20 and 40°C. The reaction time can vary from 1 to 24 hours. The reaction between the cell conjugate and the cytotoxic agent can be monitored by size exclusion chromatography (SEC) equipped with a refractive index detector and / or a UV detector. If the conjugate yield is excessively low, the reaction time can be extended.

[0072] Several different chromatographic methods are available to those skilled in the art to carry out the separation in step (ii): the conjugate can be purified by, for example, SEC, adsorption chromatography (e.g., ion exchange chromatography, IEC, etc.), hydrophobic interaction chromatography (HIC), affinity chromatography, mixed support chromatography (e.g., hydroxyapatite chromatography, etc.), or high-performance liquid chromatography (HPLC). Purification by dialysis or diafiltration is also available.

[0073] According to one embodiment, the conjugate according to the present invention is characterized by a “drug-to-antibody ratio” (or “DAR”) in the range of 1 to 10, for example 2 to 5, and particularly 3 to 4. This generally applies to conjugates containing maytansinoid molecules.

[0074] This DAR number can vary depending on the properties of the antibody and drug used (i.e., the growth inhibitor), along with the experimental conditions used in the conjugation (e.g., the ratio of growth inhibitor to antibody, reaction time, solvent, and the properties of any co-solvent). Therefore, when contact occurs between the antibody and the growth inhibitor, a mixture containing several different conjugates due to different drug-to-antibody ratios may result; sometimes a naked antibody; sometimes aggregates. The DAR determined is therefore an average value.

[0075] The methods available for determining DAR are: λ DAnd at 280 nm, spectrophotometrically measuring the ratio of the absorption of a substantially purified conjugate solution. 280 nm is a wavelength generally used to measure protein concentration, such as antibody concentration, etc. Wavelength λ D is selected so as to be able to distinguish the drug from the antibody, that is, as will be readily appreciated by those skilled in the art, λ D is the high absorption wavelength of the drug, and also λ D is sufficiently separated from 280 nm to avoid substantial overlap of the absorption peaks of the drug and the antibody. In the case of maytansinoid molecules, λ D can be selected as 252 nm. The method for DAR calculation may be derived from Antony S. Dimitrov (ed), LLC, 2009, Therapeutic Antibodies and Protocols, vol 525, 445, Springer Science.

[0076] The absorption of λ D (A λD ) and the absorption at 280 nm (A 280 ) are measured with respect to the monomer peak of size exclusion chromatography (SEC) analysis (enabling the calculation of the "DAR(SEC)" parameter), or using a classical spectrophotometer device (enabling the calculation of the "DAR(UV)" parameter). The absorption is expressed as follows: A λD =(c D ×e DλD )+(c A ×e AλD ) A 280 =(c D ×e D280 )+(c A ×e A280 ) Where: c D and c A are the concentrations of the drug solution and the antibody solution, respectively; e DλD and e D280 are the absorptivities at λD and the molar extinction coefficient of the drug at 280 nm; and e AλD and e A280 These are λ, respectively. D And the molar extinction coefficient of the antibody at 280 nm.

[0077] Solving these two equations, which contain two unknowns, yields the following equation: c D =[(e A280 ×A λD )-(e AλD ×A 280 )] / [(e DλD ×e A280 )-(e AλD ×e D280 )] c A =[A 280 -(c D ×e D280 )] / e A280

[0078] The average DAR is the ratio of drug concentration to antibody concentration: DAR = c D / c A It is calculated from.

[0079] A Phase I / II initial human study evaluating the safety, pharmacokinetics, and antitumor activity of huMAb2-3-SPDB-DM4 in patients with advanced solid tumors is currently nearing completion (NCT02187848).

[0080] A randomized, open-label, phase III trial of huMAb2-3-SPDB-DM4 is currently underway (NCT04154956) comparing it to docetaxel in patients with metastatic non-squamous non-small cell lung cancer who have previously been treated and have CEACAM5-positive tumors.

[0081] As used herein, “Subject” refers to mammals, including mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, sheep, goats, pigs, cattle, horses, non-human primates, and humans. In certain embodiments, the subject is human.

[0082] formulation According to this disclosure, it has been found that formulations of huMAb2-3-SPDB-DM4 are advantageous to contain ADC, buffers, isotonic agents, surfactants, and optionally chelating agents. Furthermore, according to this disclosure, it has been found that formulations of huMAb2-3-SPDB-DM4 are advantageous to contain ADC, acetate, sorbitol, and polysorbate 80 (PS80). Furthermore, according to this disclosure, it has been found that formulations of huMAb2-3-SPDB-DM4 are advantageous to contain ADC, acetate, sorbitol, polysorbate 80 (PS80), and EDTA. The formulations are suitable in both liquid and lyophilized forms.

[0083] The pharmaceutical compositions disclosed herein are understood to be aqueous solutions containing each of the listed components in specified amounts and concentrations.

[0084] In the case of lyophilized products prepared from aqueous solutions, the specified amounts and concentrations of the listed components correspond to the amounts and concentrations in the reconstituted formulation. The liquid used in the reconstitution is generally sterile water for injection in an amount suitable for achieving the specified amounts and concentrations of the listed components.

[0085] The aspects of this disclosure are as follows: i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Buffering agent, iii) Isotonic agents, and iv) Surfactants A pharmaceutical formulation containing [a specific compound], wherein the ADC consists of huMAb2-3-SPDB-DM4.

[0086] In a particular embodiment, the pharmaceutical formulation is a lyophilized formulation.

[0087] In a particular embodiment, the pharmaceutical formulation is a liquid formulation.

[0088] In a particular embodiment, the pharmaceutical formulation is at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Buffering agent, iii) Isotonic agents, such as polyols, iv) Surfactants, such as polysorbates, and v) Depending on the case, a chelating agent may be used. The ADC includes huMAb2-3-SPDB-DM4.

[0089] In a particular embodiment, the pharmaceutical formulation is at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Buffering agent, iii) Polyols, iv) Polysorbates, and v) Depending on the case, a chelating agent may be used. The ADC includes huMAb2-3-SPDB-DM4.

[0090] The aspects of this disclosure are as follows: i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Buffering agent, iii) tonicity agent; iv) Surfactants, and v) Chelating agents A pharmaceutical formulation containing [a specific compound], wherein the ADC consists of huMAb2-3-SPDB-DM4.

[0091] In a particular embodiment, the pharmaceutical formulation is a lyophilized formulation.

[0092] In a particular embodiment, the pharmaceutical formulation is a liquid formulation.

[0093] In a particular embodiment, the pharmaceutical formulation is at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) For example, a buffer selected from the group consisting of acetates, arginine, histidine, citrates, and any combination thereof, or for example, an acetate, iii) Polyols, for example, sorbitol, iv) Polysorbates, for example, polysorbate 80, and v) Chelatives may be used depending on the circumstances The ADC includes huMAb2-3-SPDB-DM4.

[0094] In a particular embodiment, the pharmaceutical formulation is at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) For example, a buffer selected from the group consisting of acetates, arginine, histidine, citrates, and any combination thereof, or for example, an acetate, iii) Sorbitol, iv) Polysorbates, and v) Chelatives may be used depending on the circumstances The ADC includes huMAb2-3-SPDB-DM4.

[0095] In a particular embodiment, the pharmaceutical formulation is at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) For example, a buffer selected from the group consisting of acetates, arginine, histidine, citrates, and any combination thereof, or for example, an acetate, iii) Polyols, iv) Polysorbate 80, and v) Chelatives may be used depending on the circumstances The ADC includes huMAb2-3-SPDB-DM4.

[0096] In a particular embodiment, the pharmaceutical formulation is at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) For example, a buffer selected from the group consisting of acetates, arginine, histidine, citrates, and any combination thereof, or for example, an acetate, iii) Sorbitol, iv) Polysorbate 80, and v) Chelatives may be used depending on the circumstances The ADC includes huMAb2-3-SPDB-DM4.

[0097] In a particular embodiment, the pharmaceutical formulation is at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Sodium acetate, iii) Sorbitol, iv) Polysorbate 80, and v) Chelatives may be used depending on the circumstances The ADC includes huMAb2-3-SPDB-DM4.

[0098] In a particular embodiment, the pharmaceutical formulation is at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Buffering agent, iii) tonicity agent; iv) Polysorbates, and v) Chelating agents The ADC includes huMAb2-3-SPDB-DM4.

[0099] In a particular embodiment, the pharmaceutical formulation is a lyophilized formulation.

[0100] In a particular embodiment, the pharmaceutical formulation is a liquid formulation.

[0101] In a particular embodiment, the pharmaceutical formulation is at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Buffering agent, iii) Polyols, iv) Polysorbates, and v) Chelating agents The ADC includes huMAb2-3-SPDB-DM4.

[0102] Suitable buffers for use in formulation include, but are not limited to, organic acid salts such as citric acid, ascorbic acid, gluconic acid, carbonate, tartaric acid, succinic acid, acetic acid, or phthalic acid salts; tris, tromethamine (tris(hydroxymethyl)-aminomethane) hydrochloride, or phosphate buffers. In addition, amino acid components can also be used as buffers. Non-limited examples of such amino acid components include arginine, glycine, glycylglycine, and histidine. Examples of arginine buffers include arginine acetate, arginine chloride, arginine phosphate, arginine sulfate, and arginine succinate. In one embodiment, the arginine buffer is arginine acetate. Examples of histidine buffers include histidine chloride-arginine chloride, histidine acetate-arginine acetate, histidine phosphate-arginine phosphate, histidine sulfate-arginine sulfate, and histidine succinate-arginine succinate. These are available from several commercial suppliers.

[0103] In some embodiments, the buffer may be acetic acid, arginine, histidine, a salt of citrate, such as an alkali metal salt thereof. The salt of acetic acid may be sodium acetate.

[0104] Isotonic agents considered suitable for use in formulation include, but are not limited to, sugars (reducing and non-reducing sugars), sugar alcohols, and polyols containing sugar acids. “Reducing sugars” are sugars containing hemiacetal groups that can reduce metal ions or react covalently with lysine and other amino groups in proteins, while “non-reducing sugars” are sugars that do not possess these properties of reducing sugars. Examples of reducing sugars include fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose, and glucose. Examples of non-reducing sugars include sucrose, trehalose, sorbose, meletitose, and raffinose. Sugar alcohols are selected from mannitol, xylitol, erythritol, maltitol, lactitol, erythritol, treitol, sorbitol, and glycerol. Sugar acids include L-gluconates and their metal salts. These are available from several commercial suppliers.

[0105] In some embodiments, the polyol may be sorbitol.

[0106] Suitable surfactants for formulation include, but are not limited to, polysorbates and poloxamers. Poloxamers include, for example, poloxamer 188. These are available from several commercial suppliers.

[0107] Polysorbates considered suitable for use in formulation include, but are not limited to, polysorbate 20, polysorbate 40, polysorbate 65, polysorbate 80, polysorbate 81, and polysorbate 85. These are available from several commercial suppliers.

[0108] In some embodiments, the polysorbate may be polysorbate 80.

[0109] Suitable chelating agents for use in formulation include, but are not limited to, diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), hydroxyethylenediaminetriacetic acid (HEDTA), nitrilotriacetic acid, ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and their salts, such as disodium EDTA, calcium disodium EDTA, and tetrasodium EDTA. These are available from several commercial suppliers.

[0110] In some embodiments, the chelating agent may be ethylenediaminetetraacetic acid (EDTA) or a salt thereof, such as disodium EDTA, calcium disodium EDTA, or tetrasodium EDTA.

[0111] In a particular embodiment, the pharmaceutical formulation is at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Buffers selected from acetates, arginine, histidine, citrates, and any combination thereof, iii) tonicity agent; iv) Polysorbate 80, and v) Ethylenediaminetetraacetic acid (EDTA) The ADC includes huMAb2-3-SPDB-DM4.

[0112] In a particular embodiment, the pharmaceutical formulation is at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Buffers selected from acetates, histidines, citrates, and any combination thereof, iii) Isotonic agents selected from erythritol, glycerin, lactitol, maltitol, mannitol, sorbitol, sucrose, trethitol, xylitol, and any combination thereof. iv) Polysorbate 80, and v) EDTA The ADC includes huMAb2-3-SPDB-DM4.

[0113] In a particular embodiment, the pharmaceutical formulation is at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Buffers selected from acetates, histidines, citrates, and any combination thereof, iii) Sorbitol, iv) Polysorbate 80, and v) EDTA The ADC includes huMAb2-3-SPDB-DM4.

[0114] In a particular embodiment, the pharmaceutical formulation is at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Sodium acetate, iii) Sorbitol, iv) Polysorbate 80, and v) EDTA The ADC includes huMAb2-3-SPDB-DM4.

[0115] In a particular embodiment, the pharmaceutical formulation is at a pH of approximately 4.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) A buffer selected from acetate, histidine, citrate, and any combination thereof in concentrations of approximately 5 mM to 100 mM. iii) Polyols, iv) Surfactants selected from the group consisting of polysorbate 80, polysorbate 20, poloxamer 188, and any combination thereof, and v) EDTA The ADC includes huMAb2-3-SPDB-DM4.

[0116] In a particular embodiment, the pharmaceutical formulation is at a pH of approximately 4.5 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) A buffer selected from acetate, histidine, citrate, and any combination thereof in concentrations of approximately 5 mM to 100 mM. iii) Polyols, iv) Surfactants selected from the group consisting of polysorbate 80, polysorbate 20, poloxamer 188, and any combination thereof, and v) EDTA The ADC includes huMAb2-3-SPDB-DM4.

[0117] In a particular embodiment, the pharmaceutical formulation is at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) A buffer selected from acetate, histidine, citrate, and any combination thereof in concentrations of approximately 5 mM to 100 mM. iii) Polyols, iv) A surfactant selected from the group consisting of polysorbate 80, polysorbate 20, poloxamer 188, and any combination thereof, and v) EDTA The ADC includes huMAb2-3-SPDB-DM4.

[0118] In a particular embodiment, the pharmaceutical formulation is at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) A buffer selected from acetate, histidine, citrate, and any combination thereof in concentrations of approximately 5 mM to 100 mM. iii) Isotonic agents selected from erythritol, glycerin, lactitol, maltitol, mannitol, sorbitol, sucrose, trethitol, xylitol, and any combination thereof. iv) Polysorbate 80, and v) EDTA The ADC includes huMAb2-3-SPDB-DM4.

[0119] In a particular embodiment, the pharmaceutical formulation is at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Buffers selected from acetates, histidines, citrates, and any combination thereof, iii) Isotonic agents selected from erythritol, glycerin, lactitol, maltitol, mannitol, sorbitol, sucrose, trethitol, xylitol, and any combination thereof in concentrations of approximately 1% (w / v) to approximately 50% (w / v), iv) Polysorbate 80, and v) EDTA The ADC includes huMAb2-3-SPDB-DM4.

[0120] In a particular embodiment, the pharmaceutical formulation is at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) A buffer selected from acetate, histidine, citrate, and any combination thereof in concentrations of approximately 5 mM to 100 mM. iii) Isotonic agents selected from erythritol, glycerin, lactitol, maltitol, mannitol, sorbitol, sucrose, trethitol, xylitol, and any combination thereof in concentrations of approximately 1% (w / v) to approximately 50% (w / v), iv) Polysorbate 80, and v) EDTA The ADC includes huMAb2-3-SPDB-DM4.

[0121] According to each of the above embodiments, in various specific embodiments, the pH may be about 5.0 to about 6.0. In various specific embodiments, the pH may be about 5.0 to about 5.5. In various specific embodiments, the pH may be about 5.6 to about 6.5. In various specific embodiments, the pH may be about 5.6 to about 6.0. In various specific embodiments, the pH may be about 6.1 to about 6.5.

[0122] According to each of the above embodiments, in various specific embodiments, the pH may be 5.0 to 6.0. In various specific embodiments, the pH may be 5.0 to 5.5. In various specific embodiments, the pH may be 5.6 to 6.5. In various specific embodiments, the pH may be 5.6 to 6.0. In various specific embodiments, the pH may be 6.1 to 6.5.

[0123] According to each of the above embodiments, in various specific embodiments, the pH may be about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, or about 6.5.

[0124] According to each of the above embodiments, in various specific embodiments, the pH may be 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5.

[0125] According to each of the above embodiments, the buffer can be selected from acetate, histidine, citrate, and any combination thereof in concentrations of about 5 mM to about 100 mM. In various embodiments, the buffer is present at a concentration of about 10 mM to about 100 mM. In various embodiments, the buffer is present at a concentration of about 20 mM to about 100 mM. In various embodiments, the buffer is present at a concentration of about 30 mM to about 100 mM. In various embodiments, the buffer is present at a concentration of about 40 mM to about 100 mM. In various embodiments, the buffer is present at a concentration of about 50 mM to about 100 mM. In various embodiments, the buffer is present at a concentration of about 60 mM to about 100 mM. In various embodiments, the buffer is present at a concentration of about 70 mM to about 100 mM. In various embodiments, the buffer is present at a concentration of about 80 mM to about 100 mM. In various embodiments, the buffer is present at a concentration of approximately 90 mM to approximately 100 mM. In various embodiments, the buffer is present at a concentration of approximately 5 mM to approximately 90 mM. In various embodiments, the buffer is present at a concentration of approximately 5 mM to approximately 80 mM. In various embodiments, the buffer is present at a concentration of approximately 5 mM to approximately 70 mM. In various embodiments, the buffer is present at a concentration of approximately 5 mM to approximately 60 mM. In various embodiments, the buffer is present at a concentration of approximately 5 mM to approximately 50 mM. In various embodiments, the buffer is present at a concentration of approximately 5 mM to approximately 40 mM. In various embodiments, the buffer is present at a concentration of approximately 5 mM to approximately 30 mM. In various embodiments, the buffer is present at a concentration of approximately 5 mM to approximately 20 mM. In various embodiments, the buffer is present at a concentration of approximately 5 mM to approximately 10 mM.

[0126] According to each of the above embodiments, the buffer can be selected from acetate, histidine, citrate, and any combination thereof in concentrations of 5 mM to 100 mM. In various embodiments, the buffer is present at a concentration of 10 mM to 100 mM. In various embodiments, the buffer is present at a concentration of 20 mM to 100 mM. In various embodiments, the buffer is present at a concentration of 30 mM to 100 mM. In various embodiments, the buffer is present at a concentration of 40 mM to 100 mM. In various embodiments, the buffer is present at a concentration of 50 mM to 100 mM. In various embodiments, the buffer is present at a concentration of 60 mM to 100 mM. In various embodiments, the buffer is present at a concentration of 70 mM to 100 mM. In various embodiments, the buffer is present at a concentration of 80 mM to 100 mM. In various embodiments, the buffer is present at a concentration of 90 mM to 100 mM. In various embodiments, the buffer is present at a concentration of 5 mM to 90 mM. In various embodiments, the buffer is present at a concentration of 5 mM to 80 mM. In various embodiments, the buffer is present at a concentration of 5 mM to 70 mM. In various embodiments, the buffer is present at a concentration of 5 mM to 60 mM. In various embodiments, the buffer is present at a concentration of 5 mM to 50 mM. In various embodiments, the buffer is present at a concentration of 5 mM to 40 mM. In various embodiments, the buffer is present at a concentration of 5 mM to 30 mM. In various embodiments, the buffer is present at a concentration of 5 mM to 20 mM. In various embodiments, the buffer is present at a concentration of 5 mM to 10 mM.

[0127] According to each of the above embodiments, in various embodiments, the buffer is present at a concentration of about 5 mM. In various embodiments, the buffer is present at a concentration of about 10 mM. In various embodiments, the buffer is present at a concentration of about 15 mM. In various embodiments, the buffer is present at a concentration of about 20 mM. In various embodiments, the buffer is present at a concentration of about 25 mM. In various embodiments, the buffer is present at a concentration of about 30 mM. In various embodiments, the buffer is present at a concentration of about 40 mM. In various embodiments, the buffer is present at a concentration of about 50 mM. In various embodiments, the buffer is present at a concentration of about 60 mM. In various embodiments, the buffer is present at a concentration of about 70 mM. In various embodiments, the buffer is present at a concentration of about 80 mM. In various embodiments, the buffer is present at a concentration of about 90 mM. In various embodiments, the buffer is present at a concentration of about 100 mM.

[0128] According to each of the above embodiments, in various embodiments, the buffer is present at a concentration of 5 mM. In various embodiments, the buffer is present at a concentration of 10 mM. In various embodiments, the buffer is present at a concentration of 15 mM. In various embodiments, the buffer is present at a concentration of 20 mM. In various embodiments, the buffer is present at a concentration of 25 mM. In various embodiments, the buffer is present at a concentration of 30 mM. In various embodiments, the buffer is present at a concentration of 40 mM. In various embodiments, the buffer is present at a concentration of 50 mM. In various embodiments, the buffer is present at a concentration of 60 mM. In various embodiments, the buffer is present at a concentration of 70 mM. In various embodiments, the buffer is present at a concentration of 80 mM. In various embodiments, the buffer is present at a concentration of 90 mM. In various embodiments, the buffer is present at a concentration of 100 mM.

[0129] In some embodiments, the buffer may be a salt of acetate present in an amount ranging from about 2 mM to about 25 mM, or 5 mM to about 20 mM, or about 8 mM to about 15 mM, or about 10 mM, such as a metal alkali salt of acetate, such as sodium acetate.

[0130] According to each of the above embodiments, in a particular embodiment, the isotonic agent is selected from erythritol, glycerin, lactitol, maltitol, mannitol, sorbitol, sucrose, treitol, xylitol, and any combination thereof in concentrations of about 1% (w / v) to about 50% (w / v). In a particular embodiment, the isotonic agent is present at a concentration of about 5% (w / v) to about 50% (w / v). In a particular embodiment, the isotonic agent is present at a concentration of about 10% (w / v) to about 50% (w / v). In a particular embodiment, the isotonic agent is present at a concentration of about 15% (w / v) to about 50% (w / v). In a particular embodiment, the isotonic agent is present at a concentration of about 20% (w / v) to about 50% (w / v). In a particular embodiment, the isotonic agent is present at a concentration of about 25% (w / v) to about 50% (w / v). In certain embodiments, the isotonic agent is present at a concentration of approximately 30% (w / v) to approximately 50% (w / v). In certain embodiments, the isotonic agent is present at a concentration of approximately 40% (w / v) to approximately 50% (w / v). In certain embodiments, the isotonic agent is present at a concentration of approximately 1% (w / v) to approximately 40% (w / v). In certain embodiments, the isotonic agent is present at a concentration of approximately 1% (w / v) to approximately 30% (w / v). In certain embodiments, the isotonic agent is present at a concentration of approximately 1% (w / v) to approximately 25% (w / v). In certain embodiments, the isotonic agent is present at a concentration of approximately 1% (w / v) to approximately 20% (w / v). In certain embodiments, the isotonic agent is present at a concentration of approximately 1% (w / v) to approximately 15% (w / v). In certain embodiments, the isotonic agent is present at a concentration of approximately 1% (w / v) to approximately 10% (w / v). In certain embodiments, the isotonic agent is present at a concentration of approximately 1% (w / v) to approximately 5% (w / v).

[0131] According to each of the above embodiments, in a particular embodiment, the isotonic agent is selected from erythritol, glycerin, lactitol, maltitol, mannitol, sorbitol, sucrose, treitol, xylitol, and any combination thereof in concentrations of 1% (w / v) to 50% (w / v). In a particular embodiment, the isotonic agent is present at a concentration of 5% (w / v) to 50% (w / v). In a particular embodiment, the isotonic agent is present at a concentration of 10% (w / v) to 50% (w / v). In a particular embodiment, the isotonic agent is present at a concentration of 15% (w / v) to 50% (w / v). In a particular embodiment, the isotonic agent is present at a concentration of 20% (w / v) to 50% (w / v). In a particular embodiment, the isotonic agent is present at a concentration of 25% (w / v) to 50% (w / v). In certain embodiments, the isotonic agent is present at a concentration of 30%(w / v) to 50%(w / v). In certain embodiments, the isotonic agent is present at a concentration of 40%(w / v) to 50%(w / v). In certain embodiments, the isotonic agent is present at a concentration of 1%(w / v) to 40%(w / v). In certain embodiments, the isotonic agent is present at a concentration of 1%(w / v) to 30%(w / v). In certain embodiments, the isotonic agent is present at a concentration of 1%(w / v) to 25%(w / v). In certain embodiments, the isotonic agent is present at a concentration of 1%(w / v) to 20%(w / v). In certain embodiments, the isotonic agent is present at a concentration of 1%(w / v) to 15%(w / v). In certain embodiments, the isotonic agent is present at a concentration of 1%(w / v) to 10%(w / v). In certain embodiments, the isotonic agent is present at a concentration of 1% (w / v) to 5% (w / v).

[0132] According to each of the above embodiments, in a particular embodiment, the isotonic agent is selected from erythritol, glycerin, lactitol, maltitol, mannitol, sorbitol, sucrose, treitol, xylitol, and any combination thereof in a concentration of about 1% (w / v). In a particular embodiment, the isotonic agent is present at a concentration of about 2% (w / v). In a particular embodiment, the isotonic agent is present at a concentration of about 3% (w / v). In a particular embodiment, the isotonic agent is present at a concentration of about 4% (w / v). In a particular embodiment, the isotonic agent is present at a concentration of about 5% (w / v). In a particular embodiment, the isotonic agent is present at a concentration of about 10% (w / v). In a particular embodiment, the isotonic agent is present at a concentration of about 15% (w / v). In a particular embodiment, the isotonic agent is present at a concentration of about 20% (w / v). In certain embodiments, the isotonic agent is present at a concentration of approximately 25% (w / v). In certain embodiments, the isotonic agent is present at a concentration of approximately 30% (w / v). In certain embodiments, the isotonic agent is present at a concentration of approximately 40% (w / v). In certain embodiments, the isotonic agent is present at a concentration of approximately 50% (w / v).

[0133] According to each of the above embodiments, in a particular embodiment, the isotonic agent is selected from erythritol, glycerin, lactitol, maltitol, mannitol, sorbitol, sucrose, treitol, xylitol, and any combination thereof at a concentration of 1% (w / v). In a particular embodiment, the isotonic agent is present at a concentration of 2% (w / v). In a particular embodiment, the isotonic agent is present at a concentration of 3% (w / v). In a particular embodiment, the isotonic agent is present at a concentration of 4% (w / v). In a particular embodiment, the isotonic agent is present at a concentration of 5% (w / v). In a particular embodiment, the isotonic agent is present at a concentration of 10% (w / v). In a particular embodiment, the isotonic agent is present at a concentration of 15% (w / v). In a particular embodiment, the isotonic agent is present at a concentration of 20% (w / v). In a particular embodiment, the isotonic agent is present at a concentration of 25% (w / v). In certain embodiments, the isotonic agent is present at a concentration of 30% (w / v). In certain embodiments, the isotonic agent is present at a concentration of 40% (w / v). In certain embodiments, the isotonic agent is present at a concentration of 50% (w / v).

[0134] In some embodiments, the polyol may be sorbitol present in amounts ranging from about 1 to about 10% (w / v), about 2 to about 8% (w / v), or about 4 to about 6% (w / v), or present in about 5% (w / v).

[0135] According to each of the above aspects and embodiments, in a particular embodiment, the surfactant may be selected from polysorbates, for example, polysorbate 20, polysorbate 40, polysorbate 65, polysorbate 80, polysorbate 81, and polysorbate 85, poloxamer, and any combination thereof, in concentrations of about 0.0001% (w / v) to about 10% (w / v). In a particular embodiment, the surfactant is present at a concentration of about 0.0001% (w / v) to about 10% (w / v). In a particular embodiment, the surfactant is present at a concentration of about 0.0005% (w / v) to about 5% (w / v). In a particular embodiment, the surfactant is present at a concentration of about 0.001% (w / v) to about 3% (w / v). In a particular embodiment, the surfactant is present at a concentration of about 0.005% (w / v) to about 2% (w / v). In certain embodiments, the surfactant is present at a concentration of approximately 0.01% (w / v) to approximately 1% (w / v). In certain embodiments, the surfactant is present at a concentration of approximately 0.02% (w / v) to approximately 0.5% (w / v). In certain embodiments, the surfactant is present at a concentration of approximately 0.03% (w / v) to approximately 0.1% (w / v). In certain embodiments, the surfactant is present at a concentration of approximately 0.04% (w / v) to approximately 0.08% (w / v). In certain embodiments, the surfactant is present at a concentration of approximately 0.04% (w / v) to approximately 0.06% (w / v). In certain embodiments, the surfactant is present at a concentration of approximately 0.04% (w / v).

[0136] According to each of the above embodiments, in a particular embodiment, the surfactant is selected from polysorbates, for example, polysorbate 20, polysorbate 40, polysorbate 65, polysorbate 80, polysorbate 81, and polysorbate 85, poloxamer, and any combination thereof, in concentrations of 0.0001% (w / v) to 10% (w / v). In a particular embodiment, the surfactant is present at a concentration of 0.0005% (w / v) to 10% (w / v). In a particular embodiment, the surfactant is present at a concentration of 0.001% (w / v) to 10% (w / v). In a particular embodiment, the surfactant is present at a concentration of 0.005% (w / v) to 10% (w / v). In a particular embodiment, the surfactant is present at a concentration of 0.01% (w / v) to 10% (w / v). In certain embodiments, the surfactant is present at a concentration of 0.02% (w / v) to 10% (w / v). In certain embodiments, the surfactant is present at a concentration of 0.03% (w / v) to 10% (w / v). In certain embodiments, the surfactant is present at a concentration of 0.04% (w / v) to 10% (w / v). In certain embodiments, the surfactant is present at a concentration of 0.001% (w / v) to 5% (w / v). In certain embodiments, the surfactant is present at a concentration of 0.001% (w / v) to 3% (w / v). In certain embodiments, the surfactant is present at a concentration of 0.001% (w / v) to 2% (w / v). In certain embodiments, the surfactant is present at a concentration of 0.001% (w / v) to 1% (w / v). In certain embodiments, the surfactant is present at a concentration of 0.001% (w / v) to 0.5% (w / v). In certain embodiments, the surfactant is present at a concentration of 0.001% (w / v) to 0.1% (w / v). In certain embodiments, the surfactant is present at a concentration of 0.001% (w / v) to 0.08% (w / v).

[0137] According to each of the above aspects and embodiments, in a particular embodiment, the surfactant is selected from polysorbates, for example, polysorbate 20, polysorbate 40, polysorbate 65, polysorbate 80, polysorbate 81, and polysorbate 85, poloxamer, and any combination thereof, in a concentration of about 0.0001% (w / v). In a particular embodiment, the surfactant is present at a concentration of about 0.0005% (w / v). In a particular embodiment, the surfactant is present at a concentration of about 0.001% (w / v). In a particular embodiment, the surfactant is present at a concentration of about 0.005% (w / v). In a particular embodiment, the surfactant is present at a concentration of about 0.01% (w / v). In a particular embodiment, the surfactant is present at a concentration of about 0.02% (w / v). In a particular embodiment, the surfactant is present at a concentration of about 0.03% (w / v). In certain embodiments, the surfactant is present at a concentration of about 0.04% (w / v). In certain embodiments, the surfactant is present at a concentration of about 0.06% (w / v). In certain embodiments, the surfactant is present at a concentration of about 0.08% (w / v). In certain embodiments, the surfactant is present at a concentration of about 0.1% (w / v). In certain embodiments, the surfactant is present at a concentration of about 0.5% (w / v). In certain embodiments, the surfactant is present at a concentration of about 1% (w / v). In certain embodiments, the surfactant is present at a concentration of about 2% (w / v). In certain embodiments, the surfactant is present at a concentration of about 5% (w / v). In certain embodiments, the surfactant is present at a concentration of about 10% (w / v).

[0138] In some embodiments, the surfactant may be a polysorbate, such as polysorbate 80 present in amounts ranging from about 0.005 to about 5% (w / v), or about 0.01 to about 2% (w / v), or about 0.02 to about 1% (w / v), or about 0.03 to about 0.08% (w / v), or present in about 0.04% (w / v).

[0139] The aspects of this disclosure are as follows: at pH 5.5, i) Anti-human carcinoembryonic antigen-related cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Sodium acetate, iii) Sorbitol, iv) Polysorbate 80, and v) Disodium EDTA, A pharmaceutical preparation comprising, wherein the ADC consists of huMAb2-3-SPDB-DM4.

[0140] In certain embodiments, the pharmaceutical preparation comprises from about 1 to about 50 mg / mL of huMAb2-3-SPDB-DM4. In certain embodiments, the pharmaceutical preparation comprises from about 2 to about 40 mg / mL of huMAb2-3-SPDB-DM4. In certain embodiments, the pharmaceutical preparation comprises from about 3 to about 30 mg / mL of huMAb2-3-SPDB-DM4. In certain embodiments, the pharmaceutical preparation comprises from about 4 to about 20 mg / mL of huMAb2-3-SPDB-DM4. In certain embodiments, the pharmaceutical preparation comprises from about 5 to about 10 mg / mL of huMAb2-3-SPDB-DM4. In certain embodiments, the pharmaceutical preparation comprises about 5 mg / mL of huMAb2-3-SPDB-DM4.

[0141] In certain embodiments, the pharmaceutical preparation comprises 1 to 50 mg / mL of huMAb2-3-SPDB-DM4. In certain embodiments, the pharmaceutical preparation comprises 2 to 40 mg / mL of huMAb2-3-SPDB-DM4. In certain embodiments, the pharmaceutical preparation comprises 3 to 30 mg / mL of huMAb2-3-SPDB-DM4. In certain embodiments, the pharmaceutical preparation comprises 4 to 20 mg / mL of huMAb2-3-SPDB-DM4. In certain embodiments, the pharmaceutical preparation comprises 5 to 10 mg / mL of huMAb2-3-SPDB-DM4. In certain embodiments, the pharmaceutical preparation comprises about 5 mg / mL of huMAb2-3-SPDB-DM4. In certain embodiments, the pharmaceutical preparation comprises about 10 mg / mL of huMAb2-3-SPDB-DM4.

[0142] In some embodiments, the chelating agent may be selected from diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), hydroxyethylenediaminetriacetic acid (HEDTA), nitrilotriacetic acid, ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and their salts, such as disodium EDTA, calcium disodium EDTA, and tetrasodium EDTA, present in amounts ranging from about 1 to about 50 μM, or about 1 to about 40 μM, or about 1 to about 30 μM, or about 1 to about 20 μM. In some embodiments, the chelating agent may be disodium EDTA, present in amounts of about 10 to about 50 μM, or about 10 to about 40 μM, or about 10 to about 20 μM. In some embodiments, the chelating agent may be present in amounts of about 10 μM, or about 20 μM, or about 30 μM, or about 40 μM, or about 50 μM.

[0143] In certain embodiments, the pharmaceutical composition contains about 1 to about 50 μM of disodium EDTA. In certain embodiments, the pharmaceutical composition contains about 1 to about 40 μM of disodium EDTA. In certain embodiments, the pharmaceutical composition contains about 1 to about 30 μM of disodium EDTA. In certain embodiments, the pharmaceutical composition contains about 1 to about 20 μM of disodium EDTA. In certain embodiments, the pharmaceutical composition contains about 1 to about 10 μM of disodium EDTA. In certain embodiments, the pharmaceutical composition contains about 10 to about 50 μM of disodium EDTA. In certain embodiments, the pharmaceutical composition contains about 10 to about 40 μM of disodium EDTA. In certain embodiments, the pharmaceutical composition contains about 10 to about 20 μM of disodium EDTA. In certain embodiments, the pharmaceutical composition contains about 10 μM of disodium EDTA. In certain embodiments, the pharmaceutical composition contains about 20 μM of disodium EDTA. In certain embodiments, the pharmaceutical composition contains about 30 μM of disodium EDTA. In certain embodiments, the pharmaceutical composition contains about 40 μM of disodium EDTA. In certain embodiments, the pharmaceutical composition contains about 50 μM of disodium EDTA.

[0144] In certain embodiments, the pharmaceutical composition comprises 1 to 50 μM of disodium EDTA. In certain embodiments, the pharmaceutical composition comprises 1 to 40 μM of disodium EDTA. In certain embodiments, the pharmaceutical composition comprises 1 to 30 μM of disodium EDTA. In certain embodiments, the pharmaceutical composition comprises 1 to 20 μM of disodium EDTA. In certain embodiments, the pharmaceutical composition comprises 1 to 10 μM of disodium EDTA. In certain embodiments, the pharmaceutical composition comprises 10 to 50 μM of disodium EDTA. In certain embodiments, the pharmaceutical composition comprises 10 to 40 μM of disodium EDTA. In certain embodiments, the pharmaceutical composition comprises 10 to 20 μM of disodium EDTA. In certain embodiments, the pharmaceutical composition comprises 10 μM of disodium EDTA. In certain embodiments, the pharmaceutical composition comprises 20 μM of disodium EDTA. In certain embodiments, the pharmaceutical composition comprises 30 μM of disodium EDTA. In certain embodiments, the pharmaceutical composition comprises 40 μM of disodium EDTA. In certain embodiments, the pharmaceutical composition comprises 50 μM of disodium EDTA.

[0145] In some embodiments, the chelating agent can be EDTA or a salt thereof present in an amount in the range of about 1 to about 30 μM or about 2 to about 25 μM or about 5 to about 20 μM or about 8 to about 15 μM, or can be present at about 10 μM.

[0146] In certain embodiments, the pharmaceutical formulation, at pH 5.5, i) 5 mg / mL of anti-human carcinoembryonic antigen-related cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) 10 mM of sodium acetate, iii) 5% (w / v) sorbitol, iv) 0.04% (w / v) polysorbate 80, and v) 1 to 50 μM of disodium EDTA and the ADC consists of huMAb2-3-SPDB-DM4.

[0147] In a particular embodiment, the pharmaceutical formulation is at pH 5.5, i) 5 mg / mL anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) 10 mM sodium acetate, iii) 5% (w / v) sorbitol, iv) 0.04% (w / v) polysorbate 80, and v) 1 μM disodium EDTA The ADC includes huMAb2-3-SPDB-DM4.

[0148] In a particular embodiment, the pharmaceutical formulation is at pH 5.5, i) 5 mg / mL anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) 10 mM sodium acetate, iii) 5% (w / v) sorbitol, iv) 0.04% (w / v) polysorbate 80, and v) 10 μM disodium EDTA The ADC includes huMAb2-3-SPDB-DM4.

[0149] In a particular embodiment, the pharmaceutical formulation is at pH 5.5, i) 5 mg / mL anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) 10 mM sodium acetate, iii) 5% (w / v) sorbitol, iv) 0.04% (w / v) polysorbate 80, and v) 50 μM disodium EDTA The ADC includes huMAb2-3-SPDB-DM4.

[0150] The formulations disclosed herein are generally suitable for administration to a subject by injection or infusion, such as by intravenous injection or infusion. The formulations disclosed herein are generally suitable for administration to a human subject by injection or infusion, such as by intravenous injection or infusion.

[0151] Method Aspects of the disclosure are methods of treating cancer, comprising administering to a subject in need thereof an effective amount of any one of the above pharmaceutical formulations.

[0152] According to such methods, in certain embodiments, the pharmaceutical formulation is at a pH of about 5.0 to about 6.5, i) an anti-human carcinoembryonic antigen-related cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) a buffer, iii) an isotonicity agent, such as a polyol, iv) a surfactant, such as a polysorbate, and v) optionally a chelating agent and the ADC consists of huMAb2-3-SPDB-DM4.

[0153] In certain embodiments according to the method, the pharmaceutical formulation is at a pH of about 5.0 to about 6.5, i) an anti-human carcinoembryonic antigen-related cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), [[ID=3o]]ii) a buffer, iii) a polyol, iv) a polysorbate, and v) optionally a chelating agent and the ADC consists of huMAb2-3-SPDB-DM4.

[0154] In certain embodiments according to the method, the pharmaceutical formulation is at a pH of about 5.oo to about 6.5, i) an anti-human carcinoembryonic antigen-related cell adhesion molecule s (hCEACAM5) antibody-drug conjugate (ADC), ii) For example, a buffer selected from the group consisting of acetates, arginine, histidine, citrates, and any combination thereof, or for example, an acetate, iii) Polyols, for example, sorbitol, iv) Polysorbates, for example, polysorbate 80, and v) Chelatives may be used depending on the circumstances The ADC includes huMAb2-3-SPDB-DM4.

[0155] In a particular embodiment following that method, the pharmaceutical formulation is prepared at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) For example, a buffer selected from the group consisting of acetates, arginine, histidine, citrates, and any combination thereof, or for example, an acetate, iii) Sorbitol, iv) Polysorbates, and v) Chelatives may be used depending on the circumstances The ADC includes huMAb2-3-SPDB-DM4.

[0156] In a particular embodiment following that method, the pharmaceutical formulation is prepared at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) For example, a buffer selected from the group consisting of acetates, arginine, histidine, citrates, and any combination thereof, or for example, an acetate, iii) Polyols, iv) Polysorbate 80, and v) Depending on the case, a chelating agent may be used. The ADC includes huMAb2-3-SPDB-DM4.

[0157] In a particular embodiment following that method, the pharmaceutical formulation is prepared at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) For example, a buffer selected from the group consisting of acetates, arginine, histidine, citrates, and any combination thereof, or for example, an acetate, iii) Sorbitol, iv) Polysorbate 80, and v) Chelatives may be used depending on the circumstances The ADC includes huMAb2-3-SPDB-DM4.

[0158] In a particular embodiment following that method, the pharmaceutical formulation is prepared at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Sodium acetate, iii) Sorbitol, iv) Polysorbate 80, and v) Chelatives may be used depending on the circumstances The ADC includes huMAb2-3-SPDB-DM4.

[0159] In a particular embodiment following that method, the pharmaceutical formulation is prepared at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Buffering agent, iii) tonicity agent; iv) Polysorbates, and v) Chelating agents The ADC includes huMAb2-3-SPDB-DM4.

[0160] In a particular embodiment following that method, the pharmaceutical formulation is prepared at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Buffering agent, iii) Polyols, iv) Polysorbates, and v) Chelating agents The ADC includes huMAb2-3-SPDB-DM4.

[0161] In a particular embodiment following that method, the pharmaceutical formulation is prepared at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Buffers selected from acetates, arginine, histidine, citrates, and any combination thereof, iii) tonicity agent; iv) Polysorbate 80, and v) Ethylenediaminetetraacetic acid (EDTA) The ADC includes huMAb2-3-SPDB-DM4.

[0162] In a particular embodiment following that method, the pharmaceutical formulation is prepared at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Buffers selected from acetates, histidines, citrates, and any combination thereof, iii) Isotonic agents selected from erythritol, glycerin, lactitol, maltitol, mannitol, sorbitol, sucrose, trethitol, xylitol, and any combination thereof. iv) Polysorbate 80, and v) EDTA The ADC includes huMAb2-3-SPDB-DM4.

[0163] In a particular embodiment following that method, the pharmaceutical formulation is prepared at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Buffers selected from acetates, histidines, citrates, and any combination thereof, iii) Sorbitol, iv) Polysorbate 80, and v) EDTA The ADC includes huMAb2-3-SPDB-DM4.

[0164] In a particular embodiment following that method, the pharmaceutical formulation is prepared at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Sodium acetate, iii) Sorbitol, iv) Polysorbate 80, and v) EDTA The ADC includes huMAb2-3-SPDB-DM4.

[0165] In a particular embodiment following that method, the pharmaceutical formulation is prepared at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) A buffer selected from acetate, histidine, citrate, and any combination thereof in concentrations of approximately 5 mM to 100 mM. iii) Isotonic agents selected from erythritol, glycerin, lactitol, maltitol, mannitol, sorbitol, sucrose, trethitol, xylitol, and any combination thereof. iv) Polysorbate 80, and v) EDTA The ADC includes huMAb2-3-SPDB-DM4.

[0166] In a particular embodiment following that method, the pharmaceutical formulation is prepared at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Buffers selected from acetates, histidines, citrates, and any combination thereof, iii) Isotonic agents selected from erythritol, glycerin, lactitol, maltitol, mannitol, sorbitol, sucrose, trethitol, xylitol, and any combination thereof in concentrations of approximately 1% (w / v) to approximately 50% (w / v), iv) Polysorbate 80, and v) EDTA The ADC includes huMAb2-3-SPDB-DM4.

[0167] In a particular embodiment following that method, the pharmaceutical formulation is prepared at a pH of approximately 5.0 to approximately 6.5. i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) A buffer selected from acetate, histidine, citrate, and any combination thereof in concentrations of approximately 5 mM to 100 mM. iii) Isotonic agents selected from erythritol, glycerin, lactitol, maltitol, mannitol, sorbitol, sucrose, trethitol, xylitol, and any combination thereof in concentrations of approximately 1% (w / v) to approximately 50% (w / v), iv) Polysorbate 80, and v) EDTA The ADC includes huMAb2-3-SPDB-DM4.

[0168] In a particular embodiment following that method, the pharmaceutical formulation is prepared at pH 5.5, i) Anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Sodium acetate, iii) Sorbitol, iv) Polysorbate 80, and v) Disodium EDTA The ADC includes huMAb2-3-SPDB-DM4.

[0169] In a particular embodiment following that method, the pharmaceutical formulation is prepared at pH 5.5, i) 5 mg / mL anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) 10 mM sodium acetate, iii) 5% (w / v) sorbitol, iv) 0.04% (w / v) polysorbate 80, and v) 1-50 μM disodium EDTA The ADC includes huMAb2-3-SPDB-DM4.

[0170] In a particular embodiment following that method, the pharmaceutical formulation is prepared at pH 5.5, i) 5 mg / mL anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) 10 mM sodium acetate, iii) 5% (w / v) sorbitol, iv) 0.04% (w / v) polysorbate 80, and v) 1 μM disodium EDTA The ADC includes huMAb2-3-SPDB-DM4.

[0171] In a particular embodiment following that method, the pharmaceutical formulation is prepared at pH 5.5, i) 5 mg / mL anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) 10 mM sodium acetate, iii) 5% (w / v) sorbitol, iv) 0.04% (w / v) polysorbate 80, and v) 10 μM disodium EDTA The ADC includes huMAb2-3-SPDB-DM4.

[0172] In a particular embodiment following that method, the pharmaceutical formulation is prepared at pH 5.5, i) 5 mg / mL anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) 10 mM sodium acetate, iii) 5% (w / v) sorbitol, iv) 0.04% (w / v) polysorbate 80, and v) 50 μM disodium EDTA The ADC includes huMAb2-3-SPDB-DM4.

[0173] In certain embodiments, the cancer is a high-grade carcinoembryonic antigen-associated cell adhesion molecule carcinoma.

[0174] In certain embodiments, cancer is selected from the group consisting of colorectal, gastric, lung, breast, prostate, ovarian, cervical, and bladder cancers.

[0175] In certain embodiments, cancer is selected from the group consisting of lung, breast, prostate, ovarian, cervical, and bladder cancers.

[0176] In a particular embodiment, the cancer is lung cancer.

[0177] In certain embodiments, the lung cancer is non-squamous non-small cell lung cancer.

[0178] In certain embodiments, the subject is human.

[0179] In certain embodiments, cancer is selected from the group consisting of colorectal, gastric, lung, breast, prostate, ovarian, cervical, and bladder cancers.

[0180] In certain embodiments, cancer is selected from the group consisting of lung, breast, prostate, ovarian, cervical, and bladder cancers.

[0181] In a particular embodiment, the cancer is lung cancer.

[0182] In certain embodiments, the lung cancer is non-squamous non-small cell lung cancer.

[0183] Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. NSCLC usually grows and spreads more slowly than the less common small cell lung cancer. There are three main subtypes of NSCLC: squamous cell carcinoma (25% of lung cancers), adenocarcinoma (40% of lung cancers), and large cell carcinoma (10% of lung cancers). Therefore, non-squamous non-small cell lung cancer includes adenocarcinoma (40% of lung cancers) and large cell carcinoma (10% of lung cancers).

[0184] In certain embodiments, the subject is human. [Examples]

[0185] Initial formulation of huMAb2-3-SPDB-DM4 Initially, huMAb2-3-SPDB-DM4 (see Figure 1) was developed as a concentrate for intravenous administration. The dosage form was a single-use 5 mg / mL solution in a glass vial stored at 5°C. The active pharmaceutical ingredient (API) was developed as a -20°C frozen solution in a polycarbonate bottle, representing other containers. The objective of this initial study was to identify a set of pH buffering systems and stabilizing excipients. A total of eight formulations with various excipients were tested under thermal stress at 40°C, 25°C, and 5°C, as well as under freeze / thaw stress and mechanical stress (shaking).

[0186] The prototype is shown in Table 1.

[0187] [Table 1]

[0188] method UF / DF Procedure The starting material was prepared in histidine buffer. To produce acetate-based prototypes (prototypes A, B, F, G, H), diafiltration (88 cm) was performed against 10 mM acetate at pH 5.5 in a Cogent μScale system with a Pellicon 3® cassette. 2 Three membranes, 68.7g protein / m² 2 The starting material was treated with a membrane. To produce a citrate-based prototype (Prototype E), diafiltration (88cm) was performed against 10mM citrate at pH 6.0 in a Cogent μScale system with a Pellicon3® cassette. 2 One membrane contains 51.4 g of protein / m². 2 The starting material was treated with a membrane.

[0189] In both cases, diafiltration was stopped after passing through 10 volumes of diafiltration buffer. After diafiltration, the concentration was targeted to 5 mg / mL by adding a suitable 2x concentrated solution, as shown in Table 3.

[0190] [Table 2]

[0191] Filtration 0.2μm All formulations were prepared with a 2x concentrated solution, filtered through a 0.2 μm filter, collected in Nalgene® (HDPE) bottles, and then filled.

[0192] Stress Heat stress Formulation assays A-H were stored at 40°C for 2 weeks, and at 5°C, 25°C, and 40°C for 4 weeks prior to analysis.

[0193] Freeze / thaw cycle Formulation assays A-H were frozen at -20°C or -80°C and thawed at room temperature. Three cycles of the freeze / thaw procedure were performed, after which the formulations were analyzed.

[0194] Shaking stress Formulation assays A-H were shaken in vials for 15 hours using orbital shaking at a speed of 350 rpm.

[0195] Analysis method During the study, the following analytical methods were used:

[0196] Visual inspection of appearance (transparency, color, and particles): Vials were inspected for 5 seconds on the white surface of the visual inspection table, and then on the black surface.

[0197] Protein concentration was measured by UV measurement, and OD at 280 nm and 254 nm was measured using DAR: lower absorption. Dilution factor of the test solution: F = 10. Absorbance of the test solution at 254 nm: A254D Absorbance of the test solution at 280 nm: A280D Molecular weight of the naked antibody h4D4 = 144522 g / mol Molecular weight of DM4 = 780 g / mol The molecular extinction coefficient of DM4 at 280 nm is 4927 L·mol. -1 ·cm -1 The molecular extinction coefficient of the naked antibody h4D4 at 280 nm = 201400 L·mol -1 ·cm -1 A254 / A280 = 0.43 for h4D4 DM4's A254 / A280 = 4.83 [Drug](M)=[A254-(0.43×A280)] / [(4.83×4927)-(0.43×4927)] [Drug](mg / mL) = [Drug](M) × 780 × F [Protein](M) = [A280 - 4927 × [Drug](M) / 201400 [Protein](mg / mL) = [Protein](M) × 144522 × F

[0198] For opalescence characterization, the absorbance of an undiluted solution with 0.8 mL of turbidity (OD at 350 nm) was measured at 350 nm.

[0199] Protein purity by SEC-UPLC: After T0, 2 weeks and 4 weeks at 40°C, and 4 weeks at 5°C, samples A, B, G, and H were subjected to size exclusion ultrahigh performance liquid chromatography (SEC-UPLC).

[0200] SEC-UPLC conditions: Column: BEH200, 1.7μm, 300mm x 4.6mm Flow rate: 0.30mL / min Detection: 230nm Column temperature: 30℃±2℃ Syringe temperature: 5℃ ± 0.3℃ Injection volume: 4μl Analysis time: 17 minutes

[0201] Mobile phase D-PBS (2×): Prepared with D-PBS (10×) (Dulbeccio phosphate buffered saline (D-PBS) (10×)), Invitrogen reference number: 14200083: This solution was diluted to 1 / 5 with water for injection (WFI) and filtered through 0.22 μm. The DS batch was used as a reference in each sequence (5 μl injected at 5 g / L).

[0202] Protein purity by SDS-PAGE: Measurements were taken only after 2 weeks at T0 and 40°C. Samples were prepared under reducing and non-reducing conditions (with the addition of NEM) and moved within 4–12% Bistris gels. A transfer buffer was prepared by diluting 50 mL of 20× Nu-Page MOPS SDS buffer in 1000 mL of sqf purified water. The transfer parameter was 135 V for 90 minutes. The gels were colored with Bleu rotiphorese solution.

[0203] Charge heterogeneity due to iCIEF: The 4-week analysis was performed only on the acetate preparation with a pH of 5.5.

[0204] IcIEF was performed using a Convergent Biosciences iCE280 capillary electrophoresis system with FC-coated cIEF cartridges. Samples were diluted with a master mixture containing Pharmalyte 3-10, Pharmalyte 8-10.5, 0.35% 4M urea, 1% methylcellulose, and a pI marker.

[0205] Each sample was injected into a capillary using an autosampler. After focusing, detection was performed using a CCD camera that enables direct measurement of 280nm UV absorbance. The iCE280 software calculated the apparent pI of each separated peak, and Empower enabled quantification of isoforms by normalization per region.

[0206] Free mytansinoids: Only the pH 5.5 formulation was analyzed over a 4-week period.

[0207] Free mytansinoids were measured by RP-HPLC using two columns: the first column separated proteins from free mytansinoids, and the second column separated free mytansinoid species, which were then detected by a PDA detector. Mobile phase A was a purified aqueous solution of 0.1% trifluoracetic acid. Mobile phase B was a solution of 0.08% trifluoracetic acid in acetonitrile. DM4 solution was used as a reference.

[0208] Differential scanning calorimetry (DSC) for thermal stability was performed only at T0.

[0209] Dynamic light scattering (DLS) for colloidal aggregation: average particle size and distribution. The 4-week analysis was performed only on the pH 5.5 formulation.

[0210] FCM (flow cytometry) for subvisible aggregation: morphological analysis and counting of particles. The 4-week analysis was performed only on the pH 5.5 formulation.

[0211] Results and Discussion Observation of DS after TFF in histidine buffer and in acetate and citrate buffers, and SEC analysis. At reception, the batch of DS in histidine buffer after 0.2 μm filtration was slightly opalescent in particles. The results after UFDF in acetate and citrate buffers are shown in Table 4.

[0212] [Table 3]

[0213] The acetate buffer appeared to be better than the others in terms of opalescence and yield.

[0214] Stress research Analysis at T0 The prototype was equivalent in T0 for visual inspection, FCM, DLS, SEC-UPLC (Figure 2), and icIEF analytical assays: - Visual inspection revealed that all formulations were only slightly opalescent and did not contain any visible particles. - Regarding DLS, all formulations were polydispersible, and the PDI exceeded 30%.

[0215] We were able to observe some differences between the prototypes at T0: - Regarding the DSC results, Tm was higher in acetate buffer (pH 5.5 or 6) than in the others. - For free mytansinoids, the total content was lower in acetate or citrate buffer than in other buffers, and higher in histidine buffer than in other buffers (<0.1% in acetate and citrate buffers, and approximately 0.6% in histidine buffer). See Figure 3. - Regarding the SDS-PAGE results, formulations G and H had different profiles, but aside from those, all formulations were similar. Formulation H in the non-reducing state showed a supplemental band of 127 kDa, while formulation G in the reduced state showed a supplemental band of 89 kDa (Figures 4A and 4B).

[0216] Heat stress Except for prototype C (regarding pH, concentration, DLS, and FCM) after 4 weeks at 5°C and 25°C, which was suspected of contamination, all prototypes were similar in terms of analytical parameters (visual inspection, SEC-HPLC, FCM, DLS, free mytansinoids, and ic IEF) after 4 weeks at 5°C and 25°C. See Figure 5 for turbidity results.

[0217] After 2 and 4 weeks at 40°C, the prototypes were comparable in analytical assays for protein concentration, SDS-PAGE, and visual inspection: - After 2 weeks at 40°C, a new 145 kDa band was detected in all formulations under reduced conditions on SDS-PAGE. - After two weeks at 40°C, all formulations were only slightly opalescent upon visual inspection. Nevertheless, no evolution against T0 was observed after 40°C stress.

[0218] After 40°C for 2W and 4W, we were able to observe several differences between the prototypes: - After 4 weeks at 40°C, the decrease in DAR was greatest with formulation D (histidine buffer at pH 6.5) and was higher with formulations at pH 6.5 or in histidine (Figure 6). - After 2 weeks at 40°C, the largest increase in HMW was observed with citrate pH 6.0 (prototype E), and was also high in the other two formulations (D & F) with pH above 5.5. For the prototypes (A, B, G, H) at pH 5.5 in acetate buffer, an increase in aggregates (Figure 7) and fragments (Figure 8) was observed after 4W at 40°C, with formulations G and H showing higher increases than the others (prototypes C, D, E, F were not analyzed at 4W at 40°C). - After 2 weeks at 40°C, free mytansinoid analysis showed that formulations D, E, and F exhibited higher levels of free mytansinoids than the others. After 4 weeks at 40°C, only formulations at pH 5.5 (prototypes A, B, C, G, and H) were analyzed, and prototype A showed lower levels of free mytansinoids than the others (Figure 9). The type of buffer (acetate vs. histidine) affected the detected impurity of mytansinoids (Figure 10). - After 4 weeks at 40°C, the greatest increase in OD at 350 nm was observed with prototype D (histidine, pH 6.5), and was higher under high pH conditions or with formulations in histidine. - Regarding the results of charge isoform analysis using iCIEF, the greatest increase in acidic species was observed in formulation D (pH 6.5) after 4W 40°C (Figure 12). The other prototypes tested (prototypes A, B, G, and H) behaved similarly after 2W 40°C and 4W 40°C (Figure 11).

[0219] Shaking stress After shaking stress, the prototype was similar in pH, concentration, and DAR, DLS, turbidity, SEC-UPLC, and free mytansinoids.

[0220] After shaking stress, some differences were observed between the prototypes: - In visual inspection, prototypes A and B showed no particles, but prototypes D, G, and H contained a large number of visible particles. - Regarding FCM, a slight increase in particles ≥10 and 25 μm was observed in prototype H, which may indicate the initiation of aggregation with PS20 (Table 5). - Regarding the icIEF results, prototype C (histidine, pH 5.5) showed the greatest charge variant evolution (acidic +5%, basic -3%, PI shift, Figure 13). The other prototypes did not show charge variant evolution after shaking stress.

[0221] [Table 4]

[0222] Freeze-thaw (F / T) stress After F / T stress, the prototypes were similar in terms of visual inspection, pH, concentration and DAR, turbidity, SEC-UPLC, free mytansinoids, FCM, and DLS. For icIEF, prototype C was the most susceptible to F / T stress (acidic +6% at -20°C, +5% at -80°C, basic -6% at both temperatures, Tables 6 and 7).

[0223] [Table 5]

[0224] [Table 6]

[0225] conclusion The yield after UF / DF, concentrated to approximately 12 mg / mL, was better in acetate buffer (approximately 92%) than in other buffers, and the process time was shorter than in citrate buffer. huMAb2-3-SPDB-DM4 in 10 mM citrate buffer at pH 6.0 was slightly opalescent after UF / DF.

[0226] Regarding the behavior of pharmaceutical formulations under stress: In all formulations, slight opalescence was observed by visual inspection, but it did not evolve. After 40°C stress at OD350nm, turbidity increased in all formulations, but mainly in histidine pH 6.5 (formulation D).

[0227] For all formulations, some visible particles were observed only after shaking stress, but in fewer cases for formulations A and B than for the others (acetate with sucrose and mannitol, pH 5.5).

[0228] Histidine pH 6.5 showed a stronger tendency towards DAR instability after 4 weeks at 40°C compared to other samples.

[0229] Acetate with pH 5.5 showed a stronger tendency to stabilize than the others in terms of HMW (measured by SEC-UPLC) after 2 weeks at 40°C.

[0230] After 4 weeks at 40°C, LMW (measured by SEC-UPLC) increased threefold for all acetate pH 5.5 formulations (other formulations were not analyzed).

[0231] For free mytansinoids and iCIEF, pH 5.5 and acetate buffer showed a stronger tendency to stabilize after stress (mainly heat stress) than other conditions.

[0232] No aggregation of subvisible particles with PS80 was observed by FCM.

[0233] Higher Tm (measured by DSC) was obtained in acetate buffer (pH 5.5 and 6.0).

[0234] In conclusion, acetate pH 5.5 appeared to be the buffer solution with the strongest tendency to stabilize (mainly for DAR, HMW, free mytansinoids, and charge variants), and the presence of PS80 enabled particle reduction. [Examples]

[0235] Selection of sorbitol as an excipient method The formulation development study described in this example was designed based on the results of Example 1 and on the following rationale: • A 10 mM sodium acetate buffer at pH 5.5 was selected to improve several physicochemical properties (primarily DAR, HMW%, free mytansinoids, and charge variants). • The mannitol tested in Example 1 was retained in one formulation, and other polyols, sucrose, and sorbitol at isotonic concentrations were tested as substitutes: • PS80 was selected from Example 1 as a substance that improves aggregation characteristics (mainly subvisible particles produced by FCM after shaking stress). The concentration of PS80 was varied between 0.01% w / v and 0.04% w / v.

[0236] Table 8 shows the six prototype compositions selected for this study.

[0237] [Table 7]

[0238] Six prototype formulations were stored at 40°C for up to one month, and at -20°C, 5°C, and 25°C for up to three months. They were evaluated under stress conditions induced by shaking, and then subjected to in-use simulations. Table 9 details the time points, stress conditions, and analytical methods used in the formulation studies.

[0239] [Table 8]

[0240] The active pharmaceutical ingredient (API) used in this example originated from a single batch. This API was characterized by formulation in 10 mM acetate, pH 5.5, 12.4 g / L. The sample was diluted in acetate buffer, and excipients were added to obtain the six formulations shown in Table 8.

[0241] Visual inspection of the appearance (transparency and particle size) was performed on a visual inspection table. The vials were inspected for 5 seconds on a white surface, and then on a black surface, to assess the presence of visible particles. The vials were further inspected using MOTIC MLC-150C optical fiber (color temperature: 2000-3500K) with the scoring detailed in Tables 10 and 11.

[0242] [Table 9]

[0243] [Table 10]

[0244] Protein concentration and DAR(UV) Protein concentration and DAR were measured by UV measurement using the following method: The sample was diluted 10-fold, and then the OD at 280 nm and 254 nm was measured. DAR and protein concentration were calculated using the following formula. [Drug](M)=A254-(0.43×A280) / (4.83×4927)-(0.43×4927) [Drug] mg / mL = [Drug] (M) × 780 × F [Protein](M) = A280 - 4927 × [Drug](M) / 201400 [Protein](mg / mL) = [Protein](M) × 144522 × F DAR = [Drugs](M) / [Proteins](M)

[0245] Turbidity Turbidity was assessed by measuring the absorbance of a 0.8 mL undiluted solution at 350 nm using a Cary UV100 spectrometer.

[0246] light shielding Subvisible particles were counted using a light-shielding technique with a high-precision liquid particle counter (HIAC). Each result was the average of four measurements taken from a 0.9 mL sample. Approximately 4 mL of sample was required to perform the analysis.

[0247] Protein purity measured by SDS-PAGE Protein purity was determined by SDS-PAGE using the following procedure. Sample preparation is described in Table 12.

[0248] [Table 11]

[0249] The sample was diluted to a concentration of 1 g / L. A reference was run through the sample. A 1x transfer buffer was prepared by diluting 50 mL of 20x Nu-Page MOPS SDS buffer (Invitrogen NP0001) with enough purified water to make 1 L. The gel was 4–12% bistris. Electrophoretic transfer parameters were 135 volts for 90 minutes. The gel was then stained with "Blue Rotiphorese" solution. Data processing was performed using Quantity One.

[0250] result Thermal stress studies (1 month at 5°C, 25°C, and 40°C, and 3 months at 5°C, 25°C, and -20°C).

[0251] DSC The DSC results for T0 are shown in Table 13.

[0252] [Table 12]

[0253] Thermal stability was comparable among the six formulations at T0.

[0254] Visual inspection The results of the visual inspection are shown in Table 14 (particles) and Table 15 (transparency).

[0255] [Table 13]

[0256] [Table 14]

[0257] Overall, only very small amounts of particles were observed in all formulations at all time points. All formulations were clear or slightly opalescent, and there was no significant evolution over time.

[0258] Turbidity (OD at 350nm) Table 16 shows the turbidity (OD at 350 nm) results after thermal stress.

[0259] [Table 15]

[0260] Significant evolution was observed for all formulations after T1M40°C. Slight differences were observed between the sorbitol-based formulations E and F and the other formulations at T1M40°C and T3M25°C, with slightly lower diffusion in the sorbitol-based prototypes.

[0261] pH The pH results are reported in Table 17.

[0262] [Table 16]

[0263] No pH evolution was observed in any of the formulations under stress.

[0264] DAR and concentration The protein concentration and DAR results are reported in Table 18.

[0265] [Table 17]

[0266] No evolution of concentration was observed during stress for any of the formulations. For DAR, a decrease was observed during thermal stress after T1M 40°C (approximately -1 unit) and T3M 25°C (approximately -0.5 units), but it was not possible to highlight differences between prototypes (Figure 14).

[0267] icIEF The results for major isoform %, acidic form %, and basic form % are shown in Table 19. The results for major isoform content according to icIEF are shown in Figure 15.

[0268] [Table 18]

[0269] No significant differences were observed in the percentages of major isoforms, acidic isoforms, and basic isoforms for all formulations and all stress conditions except T1M40°C. After T1M40°C, there was a significant decrease in the percentages of major isoforms and basic isoforms, along with an increase in the percentage of acidic isoforms. No significant differences were observed between prototypes.

[0270] Free mytansinoids The results for the total free mytansinoid content during thermal stress are shown in Figure 16 and Table 20.

[0271] [Table 19]

[0272] After T1M25℃ (slight increase), T3M25℃, and T1M40℃, the free mytansinoid content increased in all formulations. For all prototypes, comparable levels were observed under each condition. Formulations E and F had slightly lower free mytansinoid content after T3M25℃ and T1M40℃. Formulation D had a slightly higher free mytansinoid content after T1M40℃.

[0273] Overall, the sorbitol-containing prototypes (formulations E and F) showed better total free mytansinoid content than the others, as observed at T1M 40°C and T3M 25°C.

[0274] SEC-UPLC The results of SEC-UPLC are shown in Table 21.

[0275] [Table 20]

[0276] For all formulations and all thermal stress conditions, monomer % and HMWS% evolved similarly.

[0277] Regarding LMWS%, similar levels of fragmentation were observed for all formulations at T0, T1M, and T3M 5°C, T3M -20°C, and T1M 25°C. As shown in Figure 17, formulations E and F appeared to have lower LMWS% after T3M 25°C and T1M 40°C compared to the other formulations (-0.3% to -0.6% after T1M 40°C compared to the other formulations).

[0278] SDS PAGE Observations under non-reducing conditions were as follows: After T3M5°C and T3M25°C, the same pattern as T0 was observed, and no differences were observed between formulations. After T1M40°C, an additional band appeared at 46kDa for all formulations. Its relative amount was 0.1% to 0.6%.

[0279] Observations under reducing conditions were as follows: The same pattern was observed after T3m5℃ and T3m25℃, and no differences were observed between formulations. After T1m40℃, up to 9 additional bands appeared at 33, 42, 44, 83, 87, 90, 130, 150, and 160 kDa, depending on the formulation. No significant differences were observed between formulations.

[0280] Overall, no differences between formulations were highlighted by SDS-PAGE across all tested thermal stress conditions.

[0281] FCM The FCM results are shown in Table 22.

[0282] [Table 21]

[0283] For all prototypes and all thermal stress conditions, there was no significant evolution in the subvisible particle evolution by FCM.

[0284] ELISA-mediated binding to CEACAM5 Table 23 shows the binding to CEACAM5 based on ELISA results. The formulations were tested against T0 samples after T3M-20°C, T3M5°C, and T3M25°C (relative potency EC2). 50 T0 / EC 50 T3m).

[0285] [Table 22]

[0286] After T3M-20°C, T3M5°C, and T3M25°C, the relative titer of binding to CEACAM5 by ELISA remained stable. Under these experimental conditions, no differences were observed between formulation prototypes.

[0287] Shaking stress Table 24 shows the results of visual observations after shaking stress.

[0288] [Table 23]

[0289] After shaking stress, only a small number of particles were observed in all formulations. These particles were often identified as exogenous fibers.

[0290] LO(HIAC) and FCM Tables 25 and 26 show the results for subvisible particles obtained by HIAC and FCM after shaking stress.

[0291] [Table 24]

[0292]

Table 25

[0293] For formulation C, a slight increase in particles ≥ 10 μm was observed by FCM rather than HIAC. Overall, after agitation stress, the stability of all formulations was satisfied for sub-visible particles.

[0294] In-use simulation Visual observation Three sampling points were evaluated: · P0: T0 after dilution to 0.8 g / L with 0.9% NaCl · P1: T24h at RT without injection through the injection line and filter after dilution to 0.8 g / L with 0.9% NaCl · P2: T24h at RT after injection through the injection line and filter after dilution to 0.8 g / L with 0.9% NaCl.

[0295] All formulations showed visible particles after dilution with 0.9% NaCl: At the P0 sampling point, the following ranking was proposed: F < (better than) D < C < B < E < A, where a is the worst case and f is better than other formulations.

[0296] At the P1 sampling point, the following ranking was proposed: F < D < C < E < B < A (the differences between samples are smaller than when at P0).

[0297] At the P2 sampling point, all formulations except prototype E had far fewer particles than others. Prototype E showed a large number of particles: the same was true for other prototypes, which had fewer particles than prototype E.

[0298] In conclusion, dilution with 0.9% NaCl increased the level of visible particles in all formulations. After injection (P2 sample), the particle levels returned to levels comparable to the T0 level, except for formulation E where ps80 was 100 ppm.

[0299] Subvisible particles Tables 27 and 28 show the HIAC results for subvisible particles after in-use simulations, for particles / ml and particles / container, respectively.

[0300] [Table 26]

[0301] [Table 27]

[0302] As can be seen from Figure 18, osmotic stress due to dilution with saline (0.9% NaCl) generated subvisible particles of ≥1.5 μm at the P0 and P1 sampling points in all formulations. Formulation A had a higher number of ≥1.5 μm particles than the others. Formulations A and E had a higher number of ≥10 μm particles at the P0 sampling point than the others. Both formulations A and E had a lower tested PS80 concentration of 100 ppm than the others.

[0303] After injection through the infusion line and 0.2 μm filter (P2 sample collection point), subvisible particles decreased for all formulations. All formulations passed the pharmacopoeia standard in a 50 mL infusion bag. Formulation E would not pass the pharmacopoeia standard for volumes exceeding 100 mL (less than 25 particles / mL of ≥10 μm and less than 3 particles / mL of ≥25 μm). The risk of non-compliance was highlighted as higher at a PS80 concentration of 100 ppm than other concentrations.

[0304] Concentration and DAR Figures 19 and 20 show the protein concentration and DAR results after in-use simulation. For all formulations, a decrease in concentration (-15 to -20%) and DAR (-10 to -15%) was observed after approximately 10 ml of in-use simulation using a standard infusion kit (containing a 0.2 μm PES in-line filter). Differences between prototypes could not be highlighted.

[0305] SEC-UPLC Figures 21 and 22 show the HMW% and LMW% results after the in-use simulation. During the in-use simulation, no significant evolution by SEC was observed for any prototype after dilution and 24 hours of bag storage at room temperature.

[0306] conclusion Based on thermal stress, the sorbitol-based formulation (E&F) performed better than the others in terms of free mytansinoids (T1M40°C), fragmentation (T3M25°C and T1M40°C), and turbidity (T1M40°C). For other parameters tested, all formulations were comparable.

[0307] In addition, vibration stress and subsequent in-use simulations demonstrated the following:

[0308] Under shaking stress, all formulations showed satisfactory results, and no significant differences were highlighted among the prototypes.

[0309] Dilution with 0.9% NaCl at 0.8 mg / mL caused subvisible particle formation, particularly in formulations containing 100 ppm PS80. Formulation E (5% sorbitol, 100 ppm PS80) failed to meet the most stringent pharmacopoeia standards set by LO(HIAC).

[0310] Infusion of 0.8 mg / mL diluted huMAb2-3-SPDB-DM4 through a PE line + PES filter resulted in a significant decrease in protein concentration and DAR in the first milliliter.

[0311] Based on these results, the initial huMAb2-3-SPDB-DM4 formulation was selected: 10 mM sodium acetate at pH 5.5, 5% (w / v) sorbitol, and 400 ppm (0.04%) PS80. [Examples]

[0312] The effect of adding EDTA to huMAb2-3-SPDB-DM4 formulations The presence of metallic residues may accelerate the degradation of polysorbate. Even at very low concentrations (ppb), iron, along with other metals such as copper, is known to be involved in the degradation of polysorbate 80 (PS80). (Kranz et al., J Pharm Sci, 2019 108(6):2022~2032). To evaluate its ability to limit PS80 oxidation, the addition of the chelating agent disodium EDTA was investigated.

[0313] Research design The objective of this study was to assess the protective role of EDTA at three different concentrations (1, 10, and 50 μM) in huMAb2-3-SPDB-DM4 material containing metal residue (representing achievable levels). The formulation (DP) filling step was simulated at a laboratory scale using a filling platform.

[0314] The stability of the samples was evaluated for up to one month at 5°C, 25°C, and 40°C. After formulation locking, additional analyses were performed for up to six months on the selected EDTA concentration and the control sample without EDTA.

[0315] [Table 28]

[0316] Four test formulations were stored at 5°C, 25°C, and 40°C for up to one month prior to formulation lock. A limited number of prototypes and analytical methods underwent complementary analyses at 25°C (T6M25°C) for up to six months. Table 30 details the time points, stress conditions, and analytical methods used in the formulation studies.

[0317] [Table 29]

[0318] The active pharmaceutical ingredient (DS) used in this study contained 24 ng / mL of iron and 7 ng / mL of nickel. The prepared DS was formulated with 10 mM acetate, 5% sorbitol, 0.04% PS80, pH 5.5, and 5 g / L, and stored at -20°C.

[0319] method Preparation of major packaging materials The glass vials were manually washed with water for injection (WFI), depyrinated in an oven, and then filled. The stoppers were sterilized in an autoclave.

[0320] Formulation preparation Three different concentrations of EDTA stock solution (50 μM, 500 μM, and 2500 μM) were prepared by dissolving disodium EDTA in WFI in a graduated glass flask. The resulting solutions were filtered using a 0.22 μm sterile syringe filter.

[0321] ADC DS was melted and pooled in a 10L polycarbonate (PC) bottle, where it was homogenized by manually inverting the bottle. The ADC DS was then divided into six 2L PC bottles, each containing 1.5L (1.527kg), using a peristaltic pump.

[0322] Subsequently, the EDTA stock solution was added to ADC DS and homogenized by manually inverting the bottle. Table 31 shows the calculated volume of EDTA stock solution to be added to ADC DS to obtain the following target concentrations: no EDTA, 1 μM, 10 μM, and 50 μM.

[0323] [Table 30]

[0324] Once prepared, the solution was stored at 5°C until DP filling began. After EDTA spiking, the dilution factor of the initial formulation was estimated to be approximately 2%.

[0325] vial filling After formulation, the product was filtered into a 10L intermediate bag and then filled into 10R vials using a dedicated filling platform that mimicked the industrial formulation (DP) process. The filled vials were then manually capped and crimped.

[0326] Analysis method Visual observation The samples were visually inspected in a pharmacopoeia chamber and under a MOTIC MLC-150C optical fiber to analyze the presence of visible particles (scoring is detailed in Table 32).

[0327] [Table 31]

[0328] MicroFlow Imaging (MFI) MFI analysis required 1 mL of undiluted sample. The procedure used was as follows: the sample was flowed into the cells at 0.1 mL / min, and recording began after a 0.2 mL purge. Measurements were performed at room temperature. 3% Hellmanex and MilliQ filtration water flushing was performed before and after each measurement.

[0329] Dynamic light scattering An 80 μl solution was analyzed using a nanosizer (Zetasizer nano-S, Malvern) with the following parameters: 3 measurements of 11 runs for 10 seconds at an incidence angle of 173 degrees. No dilution was required. The results are shown as the average of the three measurements.

[0330] pH The pH was measured at room temperature by dropping a pH probe into a 2 mL sample.

[0331] Osmotic pressure Osmotic pressure was measured using a freezing point osmometer. The measurement was repeated three times, and the results are shown as the average. A volume of 20 μl was required for each measurement.

[0332] PS80 content The PS80 content was measured by gas chromatography (GC).

[0333] Results and Discussion Since no differences or evolution were observed between samples under storage conditions of 5°C, the results discussed below are limited to stress conditions of 40°C and 25°C. Formulation locking was performed after the T1M results, and it was decided to analyze only the 10 μM selected formulation and the comparison sample without EDTA at subsequent time points.

[0334] Visual observation The results of the visual observation are shown in Table 33.

[0335] According to the European Pharmacopoeia method, all formulations were considered to be essentially free of visible particles at all stress points except T2W40℃. At T1M40℃, the presence of visible particles was not confirmed, and therefore this was thought to be due to preparation in a non-GMP environment.

[0336] When using the fiber optic method (which has higher sensitivity than the European Pharmacopoeia method), some particles were observed in all formulations and at all time points, and no trend was observed.

[0337] No significant differences or evolutionary changes were observed in the presence or absence of EDTA.

[0338] pH and osmotic pressure The pH and osmotic pressure are shown in Table 34.

[0339] No evolution was highlighted for pH. The values ​​remained constant and conformed to the approval standards (5.2-5.8). Osmotic pressure values ​​did not show any evolution over time for any of the samples, regardless of EDTA concentration.

[0340] Protein concentration and DAR(UV) Protein concentrations and DAR results are shown in Table 35.

[0341] Protein concentration values ​​did not show significant evolution over time for any of the samples, regardless of EDTA concentration.

[0342] The DAR values ​​decreased over time for all samples after T2M40°C and T1M40°C (Figure 23).

[0343] A greater decrease over time was observed in the sample without EDTA compared to the others (for the three EDTA concentrations, the decrease at T1M 40°C was -1.2 compared to -0.9 at T1M 40°C for the sample without EDTA). This trend was confirmed at the 3M 25°C time point, where the decrease was greater in the sample without EDTA than in the sample with 10 μM EDTA.

[0344] [Table 32]

[0345] [Table 33]

[0346] [Table 34]

[0347] [Table 35]

[0348] [Table 36]

[0349] Submicron particles (DLS) Table 36 shows the PdI% values ​​and the Z-mean values ​​over time.

[0350] Regarding Zav, no trends were observed for any time point or EDTA concentration. For PDI%, a slight increase was observed for most samples at the 25°C and 40°C time points. However, no clear trends could be highlighted for EDTA content.

[0351] Subvisible particles (MFI) The results for subvisible particles are shown in Table 37.

[0352] No differences were observed between prototypes at any given time point. At the T3M25°C time point, a very slight increase in subvisible particles was observed: this increase was similar for samples without EDTA and with 10 μM EDTA.

[0353] Purity and HMW% by size exclusion chromatography (SEC) Table 38 shows the results for SEC (monomer %) and HMWs %).

[0354] The monomer percentage decreased after T1M40°C for all samples, and the rate of decrease was faster in the absence of EDTA than in the others (-3.4% at T1M40°C for samples without EDTA, and -1.3 / -1.8% for samples with EDTA; see Figure 24).

[0355] This result was confirmed by an increase in HMW%, which was greater in the sample without EDTA (at T1M40℃, +2.0% for the sample without EDTA, and +1.0 / +1.3% for the sample with EDTA; see Figure 25).

[0356] No significant differentiation was observed among the three EDTA concentrations tested for both monomer % and HMW %.

[0357] Purification and fragmentation by CGE Tables 39 and 40 show the CGE results under reducing conditions (total %) of the light and heavy chains and under non-reducing conditions (total %) of purity, major fragments, and other fragments.

[0358] In non-reducing cGE (Table 40, Figure 26, and Figure 27), a significant decrease in purity was observed after 1M 40°C in the absence of EDTA, confirmed by an increase in fragmentation (total of all fragments). Significant evolution was observed at three different EDTA concentrations: T2W 40°C, T1M 25°C, and T1M 40°C.

[0359] In reduced cGE (see Table 39 and Figure 28), a significant decrease in the total light and heavy chains was observed after 1M 40°C in the absence of EDTA. No significant variations were observed at the three EDTA concentrations: T2W 40°C, T1M 25°C, and T1M 40°C.

[0360] PS80 content determined by gas chromatography (GC) Table 41 shows the results for PS80 content.

[0361] For all EDTA concentrations, no significant decrease in PS80 content was recorded up to T6M25°C. In the absence of EDTA, a significant decrease in PS80 was observed after T1M40°C and T6M25°C (see also Figure 29).

[0362] Charge variant analysis by imaged capillary isoelectric focusing (iCIEF). Table 42 shows the iCIEF results (total %) for major isoforms, total %) for high pI isoforms, and total %) for low PI isoforms.

[0363] Under 40°C stress conditions, samples containing EDTA showed improved stability for charge isoforms. The increase in the total number of low pI isoforms and the decrease in the major isoforms were more pronounced in the absence of EDTA than in the others (low pI: +22% without EDTA at T1M 40°C, +12-13% with EDTA; major isoforms: -17% without EDTA, -9 / -10% with EDTA; see also Figures 30 and 31). No significant differences were highlighted for EDTA concentration.

[0364] Free mytansinoid content Table 43 shows the results of the free drug analysis.

[0365] Regarding the total amount of free drug, no significant difference could be highlighted between the samples with and without EDTA for all samples. However, a difference was observed in the percentage of impurity A: after 2 weeks and 1 month at 40°C, the percentage of impurity A was higher in the absence of EDTA than in the EDTA sample. The three EDTA concentrations could not be differentiated. In addition, oxidized species appeared after 1M at 40°C in the absence of EDTA, whereas no oxidized species were detected in the presence of EDTA.

[0366] Post-translation modification Since no differences were observed between formulations of other PTMs, this section will discuss only Met255 and Met431 oxidation.

[0367] PTM analysis showed that the degree of oxidation at positions M255 and M431 was lower in the presence of EDTA compared to samples without EDTA. No significant difference was observed among the three EDTA concentrations (see Table 44).

[0368] conclusion For all conditions tested, three different EDTA concentrations resulted in efficient stabilization of PS80 content and other qualitative attributes that may be affected by the oxidation process (particularly PTM, free mytansinoids, cGE, iCIEF, and SEC).

Claims

1. A liquid pharmaceutical preparation, The present invention comprises an antibody-drug conjugate (ADC) containing an antibody against human cancer fetal antigen-associated cell adhesion molecule 5 (hCEACAM5) conjugated to a meitansinoid derivative 4 (DM4) by an N-succinimidyl-4-(2-pyridyldithio)butanoic acid (SPDB) linker, wherein the antibody comprises a variable heavy chain (VH) domain of the amino acid sequence of SEQ ID NO: 6 and a variable light chain (VL) domain of the amino acid sequence of SEQ ID NO:

7. The formulation contains the following excipients: i) Sodium acetate at a concentration of approximately 10 mM; ii) Sorbitol at a concentration of approximately 5% w / v; iii) Polysorbate 80 at a concentration of approximately 0.04% w / v; and iv) EDTA disodium at a concentration of approximately 1 μM to approximately 50 μM Includes, The formulation is a liquid pharmaceutical preparation with a pH of approximately 5.

5.

2. The pharmaceutical formulation according to claim 1, wherein the antibody-drug conjugate has a concentration of approximately 5 mg / ml.

3. The pharmaceutical preparation according to claim 1, wherein the EDTA disodium is present in a concentration of about 1 μml, about 10 μml, or about 50 μml.

4. The pharmaceutical formulation according to claim 1, wherein the antibody-drug conjugate has a drug-to-antibody ratio in the range of about 1 to about 10, about 2 to about 5, or about 3 to about 4.

5. A liquid pharmaceutical preparation, i) 5 mg / mL anti-human cancer fetal antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC); ii) Sodium acetate at a concentration of 10 mM; iii) Sorbitol at a concentration of 5% w / v; iv) Polysorbate 80 at a concentration of 0.04% w / v; and v) EDTA disodium at a concentration of 10 μM; It consists of, The formulation has a pH of 5.

5. ADC is a liquid pharmaceutical formulation consisting of huMAb2-3-SPDB-DM4.

6. Use of an effective amount of the pharmaceutical preparation according to claim 1 or 5 in the manufacture of a drug for treating cancer.

7. The use according to claim 6, wherein the cancer expresses carcinoembryonic antigen-associated cell adhesion molecule 5 (CEACAM5).

8. The use according to claim 6, wherein the cancer is a highly carcinoembryonic antigen-associated cell adhesion molecule 5 (CEACAM5) expressing cancer.

9. The use according to claim 6, wherein the cancer is selected from the group consisting of colorectal, gastric, lung, breast, prostate, ovarian, cervical, pancreatic, and bladder cancer.

10. The use according to claim 6, wherein the cancer is selected from the group consisting of prostate, pancreatic, lung, and colorectal cancer.

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