Ceacam5 antibody-drug conjugate formulation and uses thereof

Abstract

This invention provides a stable formulation of the human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5)-target antibody-drug conjugate huMAb2-3-SPDB-DM4, as well as its preparation and usage methods.

Description

Technical Field

[0001] sequence list

[0002] This application contains a sequence list, which was submitted electronically in ASCII format and is incorporated herein by reference in its entirety. This ASCII copy was created on July 31, 2020, named 707628_SA9-297-1_ST25.txt, and is 9,175 bytes in size.

[0003] This invention relates to the field of therapeutic treatment of cancers (such as non-squamous non-small cell lung cancer expressing CEACAM5). Certain aspects of this invention relate to formulations and uses of CEACAM5 antagonists (such as anti-CEACAM5 immune conjugates) for the treatment of cancer. Prior Technology

[0004] Carcinoembryonic antigen (CEA) is a glycoprotein involved in cell adhesion. First discovered in 1965 (Gold and Freedman, J Exp Med, 121, 439, 1965), CEA is a protein typically expressed by the fetal gut during the first six months of pregnancy and has been found in pancreatic, liver, and colon cancers. CEA is part of the immunoglobulin superfamily. The CEA family consists of 18 genes, 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).

[0005] Numerous studies have demonstrated that CEACAM5, a subgroup of CEACAM, is highly expressed on the surface of tumor cells in the colorectal, stomach, lung, breast, prostate, ovary, cervix, and bladder, similar to the initially discovered CEA. However, it is less expressed in a few normal epithelial tissues (such as columnar epithelium and goblet cells in the colon, mucus neck cells in the stomach, and squamous epithelium in the esophagus and cervix) (Hammarstrom et al., 2002, "Tumor Markers, Physiology, Pathobiology, Technology and Clinical Applications," Eds. Diamandis EP et al., AACC Press, Washington, pp. 375 ff). Therefore, CEACAM5 is considered a potential therapeutic target for tumor-specific targeting methods (such as immune conjugates).

[0006] huMAb2-3-SPDB-DM4 is an immunoconjugate (antibody-drug conjugate, ADC) containing a humanized anti-CEACAM5 antibody linked to maytansin derivative 4 (DM4), a potent antimitotic agent that inhibits microtubule assembly. DM4 is covalently bound to the antibody via an optimized linker SPDB [N-succinimidyl-4-(2-pyridyldithio)butyric acid], which is stable in plasma and cleavable intracellularly. Upon binding and internalization in targeted cancer cells, huMAb2-3-SPDB-DM4 is degraded, releasing cytotoxic DM4 metabolites.

[0007] Currently, huMAb2-3-SPDB-DM4 is undergoing multiple clinical trials and is expected to be used to treat any type of CEACAM5-expressing cancer. For example, a recent clinical study has demonstrated the efficacy of huMAb2-3-SPDB-DM4 in treating non-squamous non-small cell lung cancer (NSQ NSCLC), which accounts for approximately 60% of lung cancers (see WO 2020 / 161214). However, ADCs such as huMAb2-3-SPDB-DM4 are difficult to formulate in terms of long-term stability and shelf life. Therefore, novel formulations of huMAb2-3-SPDB-DM4 have attracted considerable interest. Summary of the Invention

[0008] This invention provides, in particular, a modified formulation of huMAb2-3-SPDB-DM4, an antibody-drug conjugate (ADC) consisting of a monoclonal antibody specifically binding to human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5), which is linked to N2'-acetyl-N-2'-(4-methyl-4-mercapto-1-sideoxypentyl)-matansin (DM4). The formulation disclosed herein differs from and is optimized relative to the huMAb2-3-SPDB-DM4 formulations used to date in clinical trials.

[0009] One embodiment of the present invention is a pharmaceutical preparation comprising... i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) Buffer, iii) Tensioning agents, and iv) Surfactants, At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0010] In some specific cases, pharmaceutical preparations are lyophilized preparations.

[0011] In some specific cases, pharmaceutical preparations are liquid preparations.

[0012] One embodiment of the present invention is a pharmaceutical preparation comprising... i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) Buffer, iii) Tensioner, iv) Surfactants, and v) Chelating agents, At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0013] In some specific cases, pharmaceutical preparations are lyophilized preparations.

[0014] In some specific cases, pharmaceutical preparations are liquid preparations.

[0015] In some specific examples, the buffer is selected from the group consisting of acetate, arginine, histidine, citrate, and any combination thereof. In some specific examples, the buffer is an acetate, such as sodium acetate.

[0016] In some specific examples, the tensioning agent is a polyol. In some specific examples, the polyol is selected from the group consisting of erythritol, glycerol, lactitol, maltitol, mannitol, sorbitol, sucrose, threitol, xylitol, and any combination thereof. In some specific examples, the tensioning agent is sorbitol.

[0017] In some specific examples, the surfactant is selected from the group consisting of polysorbate 20, polysorbate 80, poloxamer 188, and any combination thereof.

[0018] In some specific examples, the surfactant is polysorbate 80.

[0019] In certain specific examples, 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), nitrotriacetic acid, and any combination thereof. In certain specific examples, the chelating agent is EDTA.

[0020] In certain specific cases, pharmaceutical preparations contain i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) Buffers, such as those selected from the group consisting of acetate, arginine, histidine, citrate, and any combination thereof, or for example, acetate. iii) Polyols, such as sorbitol, iv) Polysorbates, such as polysorbate 80, and v) Chelating agent to be selected as appropriate. At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0021] In certain specific cases, pharmaceutical preparations contain i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) Buffers, such as those selected from the group consisting of acetate, arginine, histidine, citrate, and any combination thereof, or for example, acetate. iii) Sorbitol, iv) Polysorbate 80, and v) Chelating agent to be selected as appropriate. At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0022] In some forms, pharmaceutical preparations contain i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) Buffers, such as those selected from the group consisting of acetate, arginine, histidine, citrate, and any combination thereof, or for example, acetate. iii) Polyols, iv) Polysorbate 80, and v) Chelating agent to be selected as appropriate. At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0023] In certain specific cases, pharmaceutical preparations contain i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) Buffers, such as those selected from the group consisting of acetate, arginine, histidine, citrate, and any combination thereof, or for example, acetate. iii) Sorbitol, iv) Polysorbate 80, and v) Chelating agent to be selected as appropriate. At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0024] In certain specific cases, pharmaceutical preparations contain i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) Sodium acetate, iii) Sorbitol, iv) Polysorbate 80, and v) Chelating agent to be selected as appropriate. At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0025] In certain specific cases, pharmaceutical preparations contain i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) Buffer, iii) Tensioner, iv) Polysorbates, such as polysorbate 80, and v) Chelating agents, At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0026] In certain specific cases, pharmaceutical preparations contain i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) A buffer selected from acetate, arginine, histidine, citrate, and any combination thereof. iii) Tensioner, iv) Polysorbate 80, and v) Ethylenediaminetetraacetic acid (EDTA), At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0027] In certain specific cases, pharmaceutical preparations contain i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) A buffer selected from acetates, histidines, citrates, and any combination thereof. iii) Tendon-releasing agents, selected from erythritol, glycerol, lactitol, maltitol, mannitol, sorbitol, sucrose, threitol, xylitol, and any combination thereof. iv) Polysorbate 80, and v) EDTA, At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0028] In certain specific cases, pharmaceutical preparations contain i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) A buffer selected from acetates, histidines, citrates, and any combination thereof. iii) Sorbitol, iv) Polysorbate 80, and v) EDTA, At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0029] In some forms, pharmaceutical preparations contain i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) Sodium acetate, iii) Sorbitol, iv) Polysorbate 80, and v) EDTA, At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0030] In certain specific cases, pharmaceutical preparations contain i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) A buffer selected from acetate, histidine, citrate, and any combination thereof, at a concentration of about 5 mM to about 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, At a pH of approximately 4.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0031] In certain specific cases, pharmaceutical preparations contain i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) A buffer selected from acetate, histidine, citrate, and any combination thereof, at a concentration of about 5 mM to about 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, At a pH of approximately 4.5 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0032] In certain specific cases, pharmaceutical preparations contain i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) A buffer selected from acetate, histidine, citrate, and any combination thereof, at a concentration of about 5 mM to about 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, At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0033] In certain specific cases, pharmaceutical preparations contain i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) A buffer selected from acetate, histidine, citrate, and any combination thereof, at a concentration of about 5 mM to about 100 mM. iii) Tendon-releasing agents, selected from erythritol, glycerol, lactitol, maltitol, mannitol, sorbitol, sucrose, threitol, xylitol, and any combination thereof. iv) Polysorbate 80, and v) EDTA, At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0034] In certain specific cases, pharmaceutical preparations contain i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) A buffer selected from acetates, histidines, citrates, and any combination thereof. iii) A tensioning agent selected from erythritol, glycerol, lactitol, maltitol, mannitol, sorbitol, sucrose, threitol, xylitol, and any combination thereof, at a concentration of about 1% (w / v) to about 50% (w / v). iv) Polysorbate 80, and v) EDTA, At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0035] In certain specific cases, pharmaceutical preparations contain i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) A buffer selected from acetate, histidine, citrate, and any combination thereof, at a concentration of about 5 mM to about 100 mM. iii) A tensioning agent selected from erythritol, glycerol, lactitol, maltitol, mannitol, sorbitol, sucrose, threitol, xylitol, and any combination thereof, at a concentration of about 1% (w / v) to about 50% (w / v). iv) Polysorbate 80, and v) EDTA, At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0036] One embodiment of the present invention is a pharmaceutical preparation comprising... i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) Sodium acetate, iii) Sorbitol, iv) Polysorbate 80, and v) Disodium EDTA At pH 5.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0037] In certain specific cases, pharmaceutical preparations contain i) 5 mg / mL anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Sodium acetate 10 mM, iii) Sorbitol 5% (w / v), iv) 0.04% (w / v) polysorbate 80, and v) 1-50 μM disodium EDTA At pH 5.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0038] In certain specific cases, pharmaceutical preparations contain i) 5 mg / mL anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Sodium acetate 10 mM, iii) Sorbitol 5% (w / v), iv) 0.04% (w / v) polysorbate 80, and v) 1 μM disodium EDTA, At pH 5.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0039] In certain specific cases, pharmaceutical preparations contain i) 5 mg / mL anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Sodium acetate 10 mM, iii) Sorbitol 5% (w / v), iv) 0.04% (w / v) polysorbate 80, and v) 10 μM disodium EDTA, At pH 5.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0040] In certain specific cases, pharmaceutical preparations contain i) 5 mg / mL anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Sodium acetate 10 mM, iii) Sorbitol 5% (w / v), iv) 0.04% (w / v) polysorbate 80, and v) 50 μM disodium EDTA, At pH 5.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0041] One aspect of the present invention is a method for treating cancer, the method comprising administering an effective amount of any of the aforementioned pharmaceutical formulations to an individual in need.

[0042] In certain specific cases, pharmaceutical preparations contain i) 5 mg / mL anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Sodium acetate 10 mM, iii) Sorbitol 5% (w / v), iv) 0.04% (w / v) polysorbate 80, and v) 10 μM disodium EDTA, At pH 5.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0043] In some specific cases, the cancer is a high-carcinoembryonic antigen-associated cell adhesion molecule cancer.

[0044] In some specific cases, cancer is selected from the group consisting of colorectal cancer, stomach cancer, lung cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, and bladder cancer.

[0045] In some specific cases, cancer is selected from the group consisting of lung cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, and bladder cancer.

[0046] In some specific cases, the cancer is lung cancer.

[0047] In some specific cases, the lung cancer is non-squamous non-small cell lung cancer.

[0048] In some specific cases, the individual is a human being. Simple Explanation of the Diagram

[0049] Figure 1 is a general representation of the structure of huMAb2-3-SPDB-DM4 (left) and its chemical fraction (right). Figure 2 is a SEC-UPLC T0 chromatogram depicting the prototype formulation AH. It shows the overlay of the prototype AH SEC-HPLC chromatograms at T0. See Example 3. Figure 3 is a graph depicting the t0 analytical chromatograms of free maytansine derivatives A (acetate), C (histidine), and E (citrate). It shows the free maytansine derivatives A (medium gray), C (light gray), and E (dark gray) at T0. X-axis, centimeters; Y-axis, AU. Figure 4A shows the SDS-PAGE image of the prototype formulation AH under non-reducing conditions at time t0. Figure 4B shows the SDS-PAGE image of the prototype formulation AH under reducing conditions at time t0. Figure 5 depicts the evolution of OD 350 nm after a specified pressure. Y-axis, OD 350 nm. For each pressure condition, the prototype is presented as A to H. Figure 6 is a graph depicting the evolution of the drug-to-antibody ratio (DAR) after a specified thermal stress. X-axis: prototype formulation. Figure 7 is a diagram depicting the evolution of aggregates during specified thermal pressures. For each prototype, the pressure conditions are presented in the following order: T0, T2w40℃, T4w40℃, T1M5℃. X-axis: prototype formulation. Figure 8 is a diagram depicting the evolution of fragments during specified thermal pressures. For each prototype, the pressure conditions are presented in the following order: T0, T2w40℃, T4w40℃, T1M5℃. X-axis: prototype formulation. Figure 9 is a graph depicting the total free maytansine-like substances after a specified thermal pressure. Except for prototype D, the T0 content is not visible. All prototypes are visible at T2W 40℃. Only prototypes A, B, C, G, and H are analyzed at the T4W time point. X-axis: prototype formulation; Y-axis: free maytansine-like substance content. Figure 10 is a graph depicting the free maytansine curves of formulations A (acetate, lower outline) and C (histidine, upper outline) after 4 weeks at 40°C. Figure 11 is a graph depicting the evolution of the acid isoform after a specified thermal pressure. For each prototype, the time points are presented in the following order: T0, T2W40℃, T4W40℃, T4W5℃, T4W25℃. Prototypes E and F were not analyzed at the T4W time point. X-axis: prototype formulation; Y-axis: acid isoform. Figure 12 is an imaging capillary isoelectric focusing (iCIEF) tomography diagram depicting the prototype formulation A (acetate) after 4 weeks at t0 (top) and 40°C (bottom). X-axis: time (minutes). Figure 13 is a graph depicting the superimposed iCIEF chromatography of formulations A (acetate), C (histidine), and E (citrate) after shaking pressure. Figure 14 is a graph depicting the evolution of DAR during thermal stress (initial formulation development; Example 2). For each time point, the prototype is presented as A to F. Y-axis: DAR UV. Figure 15 is a diagram depicting the isomorphic evolution of the primary charge in the icIEF during thermal stress. For each time point, the prototype is represented by A through F. Figure 16 is a diagram depicting the evolution of total free maytansine during thermostress. For each time point, the prototype is represented by A to F. Figure 17 is a diagram depicting the evolution of LMW during thermal stress. For each time point, the prototype is presented as A to F. Figure 18 is a graph depicting the results of the high-precision liquid particle counter (HIAC) after simulation in use, with particles ≥1.5 µm. For each prototype, the pressures are presented in the following order: no applied pressure (T0), after shaking pressure, P0 (after dilution in 0.9 g / L NaCl 0.9% IV bag), P1 (24 hours after dilution in 0.9 g / L NaCl 0.9% IV bag), and P2 (24 hours after dilution in 0.9 g / L NaCl 0.9% IV bag and perfusion via IV infusion set with in-line filter). Figure 19A is a graph depicting the % change in concentration after simulation during use. Figure 19B is a graph depicting the concentration changes after simulation during use. Figure 20A is a graph depicting the change (%) of DAR after simulation during use. Figure 20B is a graph depicting the change (%) of DAR after simulation in use. Figure 21 is a diagram depicting the evolution of HMW during the simulation in use. For both time points, the prototype is presented as A to F. Figure 22 is a diagram depicting the evolution of the LMW during the simulation. For each time point, the prototype is presented as A to F. Figure 23 is a graph depicting the DAR evolution of samples with a specified concentration of EDTA in filled vials after a specified thermal pressure. T0, time zero; T2W 40℃, two weeks at 40℃; T1M T2W 40℃, one month at 40℃; T3M 25℃, three months at 25℃ (Example 3). Samples without EDTA and with 10µM EDTA were analyzed only at the T3M 25℃ time point. Figure 24 depicts the change in monomer percentage in vials for samples with a specified concentration of EDTA after a specified thermal pressure. Samples without EDTA and those with 10 µM EDTA were analyzed only at the T3M 25℃ time point. Figure 25 depicts the change in the percentage of high molecular weight substance (HMW) in vials of samples with a specified concentration of EDTA after a specified thermal pressure. Samples without EDTA and those with 10 µM EDTA were analyzed only at the T3M25℃ time point. Figure 26 is a graph depicting the sum of the evolution of other fragments in a sample with a specified concentration of EDTA after a specified thermal pressure. Figure 27 is a graph depicting the change in H2L2% of a sample with a specified concentration of EDTA after a specified thermal pressure (non-reducing capillary gel electrophoresis). Figure 28 is a graph depicting the total percentage of light and heavy chains in a sample with a specified concentration of EDTA after a specified thermal pressure (reducing capillary gel electrophoresis). Figure 29 is a graph depicting the PS80 content of samples with a specified concentration of EDTA after a specified thermal pressure. No analysis was performed at the T3M 25℃ time point for the 1 µM and 50 µM EDTA samples. Figure 30 is a graph depicting the main isotype summation of a sample with a specified concentration of EDTA in a filled vial after being subjected to a specified thermal pressure. Figure 31 is a graph depicting the sum of low pI% of a sample with a specified concentration of EDTA in a filled vial after a specified thermal pressure. Implementation

[0050] This invention discloses pharmaceutical formulations of antibody-drug conjugates (ADCs) that specifically bind to the CEACAM5 protein in humans and cynomolgus monkeys. This ADC, termed huMAb2-3-SPDB-DM4, and the formulations disclosed herein, can be used to treat any type of cancer expressing CEACAM5, including non-squamous non-small cell lung cancer. Unexpectedly, according to the invention, the inclusion of an appropriate amount of ethylenediaminetetraacetic acid (EDTA) in the huMAb2-3-SPDB-DM4 formulation has been found to significantly increase the stability of the ADC and its formulations.

[0051] Carcinoembryonic antigen (CEA) is a glycoprotein involved in cell adhesion. First discovered in 1965 (Gold and Freedman, J Exp Med, 121, 439, 1965), CEA is a protein typically expressed by the fetal gut during the first six months of pregnancy and has been found in pancreatic, liver, and colon cancers. CEA is part of the immunoglobulin superfamily. The CEA family consists of 18 genes, 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).

[0052] In humans, the CEACAM subgroup consists of seven members: CEACAM1, CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM7, and CEACAM8. Numerous studies have demonstrated that CEACAM5, identical to the originally discovered CEA, is highly expressed on the surface of tumor cells in the colorectal, stomach, lung, breast, prostate, ovary, cervix, and bladder, but less so in a few normal epithelial tissues (such as columnar and goblet cells in the colon, mucus neck cells in the stomach, and squamous epithelial cells in the esophagus and cervix) (Hammarstrom et al., 2002, "Tumor Markers, Physiology, Pathobiology, Technology and Clinical Applications," Eds. Diamandis EP et al., AACC Press, Washington, pp. 375). Therefore, CEACAM5 is considered a potential therapeutic target for tumor-specific targeting approaches, such as immune conjugates.

[0053] As used in this article, "CEACAM5" stands for "Carcinoembryonic antigen-associated cell adhesion molecule 5," also known as CD66e (differentiation cluster 66e) or CEA. CEACAM5 is a glycoprotein involved in cell adhesion. CEACAM5 is particularly highly expressed on the surface of tumor cells in the colorectal, stomach, lung, and uterus.

[0054] As used in this article, "high CEACAM5 cancer" refers to any of a variety of cancer types, including colorectal cancer, stomach cancer, lung cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, and bladder cancer. In some specific cases, "high CEACAM5 cancer" refers to any of a variety of cancer types, including colon cancer, lung cancer, stomach cancer, cervical cancer, and pancreatic cancer.

[0055] In some specific cases, the lung cancer is non-squamous non-small cell lung cancer. In some specific cases, high CEACAM5 expression is present in at least 50% of the phenotyped tumor cell populations with an intensity greater than or equal to 2+. High CEACAM5 expression accounts for approximately 20% of lung cancers. immune conjugates

[0056] ADC huMAb2-3-SPDB-DM4 is an immunoconjugate that binds huMAb2-3 (anti-CEACAM5) antibody and maytansin derivative 4 (DM4), a potent antimitotic agent that inhibits microtubule assembly. DM4 is covalently bound to huMAb2-3 via an optimized linker SPDB [N-4-(2-pyridyldithio)-butyrate succinimide], which is stable in plasma and cleavable intracellularly. Upon binding and internalization in targeted cancer cells, huMAb2-3-SPDB-DM4 is degraded, releasing cytotoxic DM4 metabolites.

[0057] The antibody portion of huMAb2-3-SPDB-DM4 is a human IgG1κ antibody, possessing a pair of heavy chains (HC), each containing a variable heavy (VH) domain; and a pair of light chains (LC), each containing a variable light (VL) domain. Each VH domain contains three complementarity-determining regions (CDRs): HCDR1, HCDR2, and HCDR3. Each VL domain contains three complementarity-determining regions (CDRs): LCDR1, LCDR2, and LCDR3. The amino acid sequences of these peptides are as follows: HCDR1 SEQ ID NO: 1 GFVFSSYD HCDR2 SEQ ID NO: 2 ISSGGGIT HCDR3 SEQ ID NO: 3 AAHYFGSSGPFAY LCDR1 SEQ ID NO: 4 ENIFSY LCDR2 NTR LCDR3 SEQ ID NO: 5 QHHYGTPFT VH SEQ ID NO: 6 EVQLQESGPGLVKPGGSLSLSCAASGFVFSSYDMSWVRQTPERGLEWVAYISSGGGITYAPSTVKGRFTVSRDNAKNTLYLQMNSLTSEDTAVYYCAAHYFGSSGPFAYWGQGTLVTVSS VL SEQ ID NO: 7 DIQMTQSPASLSASVGDRVTITCRASENIFSYLAWYQQKPGKSPKLLVYNTRTLAEGVPSRFSGSGSGTDFSLTISSLQPEDFATYYCQHHYGTPFTFGSGTKLEIK HC SEQ ID NO: 8 EVQLQESGPGLVKPGGSLSLSCAASGFVFSSYDMSWVRQTPERGLEWVAYISSGGGITYAPSTVKGRFTVSRDNAKNTLYLQMNSLTSEDTAVYYCAAHYFGSSGPFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG LC SEQ ID NO: 9 DIQMTQSPASSLSASVGDRVTITCRASENIFSYLAWYQQKPGKSPKLLVYNTRTLAEGVPSRFSGSGSGTDFSLTISSLQPEDFATYYCQHHYGTPFTFGSGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0058] As used in this article, "maytansin-like drugs" refers to maytansin-like drugs and their analogues. Maytansin-like drugs are inhibitors of microtubule formation and are highly toxic to mammalian cells.

[0059] Examples of suitable maytansine analogues include maytanol and maytanol analogues.

[0060] The cytotoxic conjugate of this invention utilizes thiol-containing maytansin DM4 as a cytotoxic agent. DM4 is formally named N2-deacetylated-N-2'-(4-methyl-4-mercapto-1-sideoxypentyl)-matansin. DM4 is represented by the following structural formula (I): (I)

[0061] In some specific examples, the antibodies of the present invention are covalently attached to at least one growth inhibitor, either directly or via cleavable or non-cleavable linkers.

[0062] As used in this article, "linker" refers to a chemical part that contains covalent bonds or chains of atoms that covalently attach a polypeptide to a drug moiety.

[0063] The conjugate can be prepared by in vitro methods. A linker group is used to link the drug or prodrug to the antibody. Suitable linker groups are known in the art and include disulfide groups, thioether groups, acid-labile groups, light-labile groups, peptidase-labile groups, and esterase-labile groups. The conjugation of the antibody of the present invention to a cytotoxic agent or growth inhibitor can be achieved using various bifunctional protein conjugates, including but not limited to N-succinimidyl-4-(2-pyridyldithio)butyric acid (SPDB), 4-[(5-nitro-2-pyridyl)dithio]-2,5-disideloxy-1-pyridinedimethyl ester of butyric acid (nitro-SPDB), 4-(pyridin-2-yldihydrothio)-2-sulfo-butyric acid (sulfon-SPDB), N-(2-pyridyldithio)propionate succinimidyl ester (SPDP), and (N-maleimino) Methylcyclohexane-1-carboxylic acid succinimide (SMCC), iminothiones (IT), bifunctional derivatives of succinimide esters (such as dimethyl adipate HCl), active esters (such as disuccinimide octanoate), aldehydes (such as glutaraldehyde), diazid compounds (such as bis(p-azidobenzoyl)-hexanediamine), diazido derivatives (such as bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and difluorinated compounds (such as 1,5-difluoro-2,4-dinitrobenzene).

[0064] Linkers can be "cleavable linkers" that promote the release of cytotoxic agents or growth inhibitors into cells. For example, acid-labile linkers, peptidase-sensitive linkers, esterase-labile linkers, light-labile linkers, or disulfide-containing linkers can be used (see, for example, U.S. Patent No. 5,208,020). Linkers can also be "non-cleavable linkers" (e.g., SMCC linkers), which may have better tolerability in certain situations.

[0065] According to one specific example, in the conjugate of the present invention, the growth inhibitor is a maytansin DM4.

[0066] In this conjugate, the antibody is attached to the at least one growth inhibitor via a linker group. In one specific example, the linker group is a cleavable or non-cleavable linker, such as N-succinimidyl-4-(2-pyridyldithio)butyric acid (SPDB), 4-(pyridin-2-yldihydrothio)-2-sulfonyl-butyric acid (sulfon-SPDB), or (N-maleiminomethyl)cyclohexane-1-carboxylic acid succinimidyl ester (SMCC).

[0067] In one specific example, the linker is SPDB, and the conjugate consists of the antibody-SPDB-DM4 conjugate of formula (II). (II); Where n is an integer greater than or equal to 1. The average value of n is typically about 3.8. See also Figure 1.

[0068] Generally, the joint can be obtained by a method including the following steps: (i) Contacting, as appropriate, a buffered aqueous solution of a cell binder (e.g., an antibody according to the invention) with a solution of a linker and a cytotoxic compound; (ii) Then, depending on the situation, the conjugate formed in (i) will be separated from the unreacted cell binder.

[0069] Aqueous solutions of cell binders can be buffered with buffers such as potassium phosphate, acetate, citrate, or N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES buffer). The buffer depends on the nature of the cell binder. Cytotoxic compounds are solutions in polar organic solvents such as dimethyl sulfoxide (DMSO) or dimethyl acetamide (DMA).

[0070] The reaction temperature is typically between 20 and 40°C. Reaction times can range from 1 hour to 24 hours. The reaction between the cell binder and the cytotoxic agent can be monitored using size exclusion chromatography (SEC) with a refractive and / or UV detector. If the binder yield is too low, the reaction time can be extended.

[0071] Those skilled in the art can use a variety of different chromatographic methods to separate the product in step (ii): the conjugate can be purified, for example, by SEC, adsorption chromatography (such as ion exchange chromatography, IEC), hydrophobic interaction chromatography (HIC), affinity chromatography, mixed support chromatography (such as hydroxyapatite chromatography), or high performance liquid chromatography (HPLC). Purification by dialysis or diafiltration can also be used.

[0072] According to one specific example, the conjugates according to the invention are characterized by a "drug-antibody ratio" (or "DAR") ranging from 1 to 10, for example, from 2 to 5, particularly from 3 to 4. This is typically the case for conjugates that include maytansine-like molecules.

[0073] This DAR number can vary depending on the nature of the antibody and drug (i.e., growth inhibitor) used, as well as the experimental conditions used for conjugation (such as the growth inhibitor / antibody ratio, reaction time, and the nature of the solvent and co-solvent, if any). Therefore, contact between the antibody and growth inhibitor results in a mixture containing several conjugates with varying drug-to-antibody ratios; in some cases, naked antibodies; in others, aggregates. Thus, the measured DAR is an average value.

[0074] A method for determining DAR involves spectrophotometric measurement of the absorbance ratio of a substantially pure conjugate solution at λD and 280 nm. 280 nm is a commonly used wavelength for measuring protein concentrations (such as antibody concentrations). The wavelength λD is chosen to allow differentiation between the drug and antibody, i.e., readily known to those skilled in the art, λD is the wavelength at which the drug exhibits high absorbance, while λD is far enough from 280 nm to avoid significant overlap of absorbance peaks for the drug and antibody. In the case of maytansine molecules, λD can be chosen as 252 nm. The method for calculating DAR is available from Antony S. Dimitrov (ed), LLC, 2009, Therapeutic Antibodies and Protocols, vol 525, 445, Springer Science.

[0075] The absorbance of the conjugate at λD (A λD) and 280 nm (A 280) is measured for monomer peaks by size exclusion chromatography (SEC) analysis (allowing for calculation of the "DAR (SEC)" parameter) or using typical spectrophotometer equipment (allowing for calculation of the "DAR (UV)" parameter). Absorbance can be expressed as follows: A λD= (C Dx ɛ DλD) + (C Ax ɛ AλD) A 280= (C Dx ɛ D280) + (C Ax ɛ A280) in: CD and CA are the concentrations of the drug and antibody solutions, respectively. ɛDλD and ɛD280 are the molar extinction coefficients of the drug at λD and 280 nm, respectively; and ɛAλD and ɛA280 are the molar extinction coefficients of the antibody at λD and 280 nm, respectively.

[0076] Solving these two equations using two unknowns yields the following equation: c D= [(ɛ A280x A λD) - (ɛ AλDx A 280)] / [(ɛ DλDx ɛ A280) - (ɛ AλDx ɛ D280)] c A= [A 280- (c Dx ɛ D280)] / ɛ A280

[0077] Then, the average DAR is calculated based on the ratio of drug concentration to antibody concentration: DAR = cD / cA.

[0078] The Phase 1 / 2 first-in-human study of huMAb2-3-SPDB-DM4 has been completed to evaluate its safety, pharmacokinetics, and antitumor activity in patients with advanced solid tumors (NCT02187848).

[0079] huMAb2-3-SPDB-DM4 is currently undergoing a randomized, open-label phase 3 study comparing it to docetaxel in patients with previously treated metastatic non-squamous non-small cell lung cancer with CEACAM5-positive tumors (NCT04154956).

[0080] As used in this article, "individual" refers to mammals, including mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, sheep, goats, pigs, cattle, horses, non-human primates, and humans. In some specific instances, the individual is a human.

[0081] formulation According to the present invention, it has now been found that formulations of huMAb2-3-SPDB-DM4 advantageously include an ADC, a buffer, a tensioning agent, a surfactant, and, if appropriate, a chelating agent. Furthermore, according to the present invention, formulations of huMAb2-3-SPDB-DM4 advantageously include an ADC, an acetate, sorbitol, and polysorbate 80 (PS80). According to the present invention, it has now been further found that formulations of huMAb2-3-SPDB-DM4 advantageously include an ADC, an acetate, sorbitol, polysorbate 80 (PS80), and EDTA. This formulation is suitable for both liquid and lyophilized forms.

[0082] It should be understood that the pharmaceutical compositions disclosed herein may be aqueous solutions containing the various listed components in specified amounts and concentrations.

[0083] In the case of lyophilized products prepared from aqueous solutions, the amounts and concentrations of the listed components are related to the amounts and concentrations in the formulation during reduction. The liquid used for reduction is typically sterile water for injection, in an amount suitable for achieving the listed amounts and concentrations of the specified components.

[0084] One embodiment of the present invention is a pharmaceutical preparation comprising... i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) Buffer, iii) Tensioning agents, and iv) Surfactants, At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0085] In some specific cases, pharmaceutical preparations are lyophilized preparations.

[0086] In some specific cases, pharmaceutical preparations are liquid preparations.

[0087] In certain specific cases, pharmaceutical preparations contain i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) Buffer, iii) Tendency agents, such as polyols, iv) Surfactants, such as polysorbate, and v) Chelating agent to be selected as appropriate. At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0088] In certain specific cases, pharmaceutical preparations contain i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) Buffer, iii) Polyols, iv) Polysorbate, and v) Chelating agent to be selected as appropriate. At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0089] One embodiment of the present invention is a pharmaceutical preparation comprising... i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) Buffer, iii) Tensioner, iv) Surfactants, and v) Chelating agents, At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0090] In some specific cases, pharmaceutical preparations are lyophilized preparations.

[0091] In some specific cases, pharmaceutical preparations are liquid preparations.

[0092] In some forms, pharmaceutical preparations contain i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) Buffers, such as those selected from the group consisting of acetate, arginine, histidine, citrate, and any combination thereof, or for example, acetate. iii) Polyols, such as sorbitol, iv) Polysorbates, such as polysorbate 80, and v) Chelating agent to be selected as appropriate. At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0093] In certain specific cases, pharmaceutical preparations contain i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) Buffers, such as those selected from the group consisting of acetate, arginine, histidine, citrate, and any combination thereof, or for example, acetate. iii) Sorbitol, iv) Polysorbate, and v) Chelating agent to be selected as appropriate. At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0094] In certain specific cases, pharmaceutical preparations contain i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) Buffers, such as those selected from the group consisting of acetate, arginine, histidine, citrate, and any combination thereof, or for example, acetate. iii) Polyols, iv) Polysorbate 80, and v) Chelating agent to be selected as appropriate. At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0095] In certain specific cases, pharmaceutical preparations contain i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) Buffers, such as those selected from the group consisting of acetate, arginine, histidine, citrate, and any combination thereof, or for example, acetate. iii) Sorbitol, iv) Polysorbate 80, and v) Chelating agent to be selected as appropriate. At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0096] In certain specific cases, pharmaceutical preparations contain i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) Sodium acetate, iii) Sorbitol, iv) Polysorbate 80, and v) Chelating agent to be selected as appropriate. At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0097] In certain specific cases, pharmaceutical preparations contain i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) Buffer, iii) Tensioner, iv) Polysorbate, and v) Chelating agents, At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0098] In some specific cases, pharmaceutical preparations are lyophilized preparations.

[0099] In some specific cases, pharmaceutical preparations are liquid preparations.

[0100] In certain specific cases, pharmaceutical preparations contain i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) Buffer, iii) Polyols, iv) Polysorbate, and v) Chelating agents, At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0101] Suitable buffers for formulations include, but are not limited to, organic acid salts such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, trimethylamine (tris(hydroxymethyl)-aminomethane) hydrochloride, or phosphate buffers. In addition, amino acid components can also be used as buffers. Such amino acid components include, but are not limited to, arginine, glycine, glycylglycine, and histidine. Arginine buffers include arginine acetate, arginine chloride, arginine phosphate, arginine sulfate, arginine succinate, etc. In one specific example, 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, histidine succinate-arginine succinate, etc. These are available from many commercial suppliers.

[0102] In some specific examples, the buffer can be a salt of acetic acid, arginine, histidine, citric acid, or its alkali metal salts. A salt of acetic acid can be sodium acetate.

[0103] Tensors suitable for formulations include, but are not limited to, polyols, including sugars (reducing and non-reducing sugars), sugar alcohols, and sugar acids. A "reducing sugar" is a sugar containing a hemiacetal group, which can reduce metal ions or covalently react with lysine and other amino groups in proteins, while a "non-reducing sugar" is a sugar that does not possess these reducing sugar properties. Examples of reducing sugars are fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose, and glucose. Non-reducing sugars include sucrose, trehalose, sorbitol, metriose, and raffinose. Sugar alcohols are selected from mannitol, xylitol, erythritol, maltitol, lactitol, erythritol, threitol, sorbitol, and glycerol. Sugar acids include L-gluconic acid and its metal salts. These are available from many commercial suppliers.

[0104] In some specific examples, the polyol can be sorbitol.

[0105] Surfactants suitable for formulations include, but are not limited to, polysorbates and poloxamers. Poloxamers include, for example, poloxamer 188. These are available from many commercial suppliers.

[0106] Polysorbate esters suitable for formulations include, but are not limited to, polysorbate 20, polysorbate 40, polysorbate 65, polysorbate 80, polysorbate 81, and polysorbate 85. These are available from many commercial suppliers.

[0107] In some specific examples, polysorbate can be polysorbate 80.

[0108] Chelating agents suitable for formulations include, but are not limited to, diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), hydroxyethylenediaminetriacetic acid (HEDTA), nitrotriacetic acid, ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and their salts, such as disodium EDTA, disodium calcium EDTA, and tetrasodium EDTA. These are available from many commercial suppliers.

[0109] In some specific examples, the chelating agent can be ethylenediaminetetraacetic acid (EDTA) or its salts, such as disodium EDTA, disodium calcium EDTA, and tetrasodium EDTA.

[0110] In certain specific cases, pharmaceutical preparations contain i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) A buffer selected from acetates, arginine, histidine, citrates, and any combination thereof. iii) Tensioner, iv) Polysorbate 80, and v) Ethylenediaminetetraacetic acid (EDTA) At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0111] In certain specific cases, pharmaceutical preparations contain i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) A buffer selected from acetates, histidines, citrates, and any combination thereof. iii) Tendon-releasing agents, selected from erythritol, glycerol, lactitol, maltitol, mannitol, sorbitol, sucrose, threitol, xylitol, and any combination thereof. iv) Polysorbate 80, and v) EDTA, At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0112] In certain specific cases, pharmaceutical preparations contain i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) A buffer selected from acetates, histidines, citrates, and any combination thereof. iii) Sorbitol, iv) Polysorbate 80, and v) EDTA, At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0113] In some forms, pharmaceutical preparations contain i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) Sodium acetate, iii) Sorbitol, iv) Polysorbate 80, and v) EDTA, At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0114] In certain specific cases, pharmaceutical preparations contain i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) A buffer selected from acetate, histidine, citrate, and any combination thereof, at a concentration of about 5 mM to about 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, At a pH of approximately 4.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0115] In certain specific cases, pharmaceutical preparations contain i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) A buffer selected from acetate, histidine, citrate, and any combination thereof, at a concentration of about 5 mM to about 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, At a pH of approximately 4.5 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0116] In certain specific cases, pharmaceutical preparations contain i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) A buffer selected from acetate, histidine, citrate, and any combination thereof, at a concentration of about 5 mM to about 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, At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0117] In certain specific cases, pharmaceutical preparations contain i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) A buffer selected from acetate, histidine, citrate, and any combination thereof, at a concentration of about 5 mM to about 100 mM. iii) Tendon-releasing agents, selected from erythritol, glycerol, lactitol, maltitol, mannitol, sorbitol, sucrose, threitol, xylitol, and any combination thereof. iv) Polysorbate 80, and v) EDTA, At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0118] In certain specific cases, pharmaceutical preparations contain i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) A buffer selected from acetates, histidines, citrates, and any combination thereof. iii) A tensioning agent selected from erythritol, glycerol, lactitol, maltitol, mannitol, sorbitol, sucrose, threitol, xylitol, and any combination thereof, at a concentration of about 1% (w / v) to about 50% (w / v). iv) Polysorbate 80, and v) EDTA, At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0119] In certain specific cases, pharmaceutical preparations contain i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) A buffer selected from acetate, histidine, citrate, and any combination thereof, at a concentration of about 5 mM to about 100 mM. iii) A tensioning agent selected from erythritol, glycerol, lactitol, maltitol, mannitol, sorbitol, sucrose, threitol, xylitol, and any combination thereof, at a concentration of about 1% (w / v) to about 50% (w / v). iv) Polysorbate 80, and v) EDTA, At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0120] According to each of the foregoing states and specific examples, in various specific examples, the pH may be from about 5.0 to about 6.0. In various specific examples, the pH may be from about 5.0 to about 5.5. In various specific examples, the pH may be from about 5.6 to about 6.5. In various specific examples, the pH may be from about 5.6 to about 6.0. In various specific examples, the pH may be from about 6.1 to about 6.5.

[0121] Depending on each of the foregoing states and specific examples, in various specific examples, the pH may be 5.0 to 6.0. In various specific examples, the pH may be 5.0 to 5.5. In various specific examples, the pH may be 5.6 to 6.5. In various specific examples, the pH may be 5.6 to 6.0. In various specific examples, the pH may be 6.1 to 6.5.

[0122] Depending on each of the foregoing states and specific examples, in various specific examples, 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.

[0123] Depending on each of the aforementioned states and specific examples, in various specific examples, 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.

[0124] According to each of the foregoing states and specific examples, the buffer may be selected from acetate, histidine, citrate, and any combination thereof, at a concentration of about 5 mM to about 100 mM. In various specific examples, the buffer is present at a concentration of about 10 mM to about 100 mM. In various specific examples, the buffer is present at a concentration of about 20 mM to about 100 mM. In various specific examples, the buffer is present at a concentration of about 30 mM to about 100 mM. In various specific examples, the buffer is present at a concentration of about 40 mM to about 100 mM. In various specific examples, the buffer is present at a concentration of about 50 mM to about 100 mM. In various specific examples, the buffer is present at a concentration of about 60 mM to about 100 mM. In various specific examples, the buffer is present at a concentration of about 70 mM to about 100 mM. In various specific examples, the buffer is present at a concentration of about 80 mM to about 100 mM. In various specific examples, the buffer is present at a concentration of about 90 mM to about 100 mM. In various specific examples, the buffer is present at a concentration of about 5 mM to about 90 mM. In various specific examples, the buffer is present at a concentration of about 5 mM to about 80 mM. In various specific examples, the buffer is present at a concentration of about 5 mM to about 70 mM. In various specific examples, the buffer is present at a concentration of about 5 mM to about 60 mM. In various specific examples, the buffer is present at a concentration of about 5 mM to about 50 mM. In various specific examples, the buffer is present at a concentration of about 5 mM to about 40 mM. In various specific examples, the buffer is present at a concentration of about 5 mM to about 30 mM. In various specific examples, the buffer is present at a concentration of about 5 mM to about 20 mM. In various specific examples, the buffer is present at a concentration of about 5 mM to about 10 mM.

[0125] According to each of the foregoing states and specific examples, the buffer may be selected from acetate, histidine, citrate, and any combination thereof, at a concentration of 5 mM to 100 mM. In various specific examples, the buffer is present at a concentration of 10 mM to 100 mM. In various specific examples, the buffer is present at a concentration of 20 mM to 100 mM. In various specific examples, the buffer is present at a concentration of 30 mM to 100 mM. In various specific examples, the buffer is present at a concentration of 40 mM to 100 mM. In various specific examples, the buffer is present at a concentration of 50 mM to 100 mM. In various specific examples, the buffer is present at a concentration of 60 mM to 100 mM. In various specific examples, the buffer is present at a concentration of 70 mM to 100 mM. In various specific examples, the buffer is present at a concentration of 80 mM to 100 mM. In various specific examples, the buffer is present at a concentration of 90 mM to 100 mM. In various specific examples, the buffer is present at a concentration of 5 mM to 90 mM. In various specific examples, the buffer is present at a concentration of 5 mM to 80 mM. In various specific examples, the buffer is present at a concentration of 5 mM to 70 mM. In various specific examples, the buffer is present at a concentration of 5 mM to 60 mM. In various specific examples, the buffer is present at a concentration of 5 mM to 50 mM. In various specific examples, the buffer is present at a concentration of 5 mM to 40 mM. In various specific examples, the buffer is present at a concentration of 5 mM to 30 mM. In various specific examples, the buffer is present at a concentration of 5 mM to 20 mM. In various specific examples, the buffer is present at a concentration of 5 mM to 10 mM.

[0126] According to each of the foregoing states and specific examples, in various specific examples, the buffer is present at a concentration of approximately 5 mM. In various specific examples, the buffer is present at a concentration of approximately 10 mM. In various specific examples, the buffer is present at a concentration of approximately 15 mM. In various specific examples, the buffer is present at a concentration of approximately 20 mM. In various specific examples, the buffer is present at a concentration of approximately 25 mM. In various specific examples, the buffer is present at a concentration of approximately 30 mM. In various specific examples, the buffer is present at a concentration of approximately 40 mM. In various specific examples, the buffer is present at a concentration of approximately 50 mM. In various specific examples, the buffer is present at a concentration of approximately 60 mM. In various specific examples, the buffer is present at a concentration of approximately 70 mM. In various specific examples, the buffer is present at a concentration of approximately 80 mM. In various specific examples, the buffer is present at a concentration of approximately 90 mM. In various specific examples, the buffer is present at a concentration of approximately 100 mM.

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

[0128] In some specific examples, the buffer may be a salt of acetic acid, such as an alkali metal salt of acetic acid, for example sodium acetate, 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.

[0129] According to each of the foregoing states and specific examples, in some specific examples, the tensioning agent is selected from erythritol, glycerol, lactitol, maltitol, mannitol, sorbitol, sucrose, threitol, xylitol, and any combination thereof, at a concentration of about 1% (w / v) to about 50% (w / v). In some specific examples, the tensioning agent is present at a concentration of about 5% (w / v) to about 50% (w / v). In some specific examples, the tensioning agent is present at a concentration of about 10% (w / v) to about 50% (w / v). In some specific examples, the tensioning agent is present at a concentration of about 15% (w / v) to about 50% (w / v). In some specific examples, the tensioning agent is present at a concentration of about 20% (w / v) to about 50% (w / v). In some specific examples, the tensioning agent is present at a concentration of about 25% (w / v) to about 50% (w / v). In some specific examples, the tensioning agent is present at a concentration of about 30% (w / v) to about 50% (w / v). In some specific examples, the tensioning agent is present at a concentration of about 40% (w / v) to about 50% (w / v). In some specific examples, the tensioning agent is present at a concentration of about 1% (w / v) to about 40% (w / v). In some specific examples, the tensioning agent is present at a concentration of about 1% (w / v) to about 30% (w / v). In some specific examples, the tensioning agent is present at a concentration of about 1% (w / v) to about 25% (w / v). In some specific examples, the tensioning agent is present at a concentration of about 1% (w / v) to about 20% (w / v). In some specific examples, the tensioning agent is present at a concentration of about 1% (w / v) to about 15% (w / v). In some specific examples, the tensioning agent is present at a concentration of about 1% (w / v) to about 10% (w / v). In some specific examples, the tensioning agent is present at a concentration of about 1% (w / v) to about 5% (w / v).

[0130] According to each of the foregoing states and specific examples, in some specific examples, the tensioning agent is selected from erythritol, glycerol, lactitol, maltitol, mannitol, sorbitol, sucrose, threitol, xylitol, and any combination thereof, at a concentration of 1% (w / v) to 50% (w / v). In some specific examples, the tensioning agent is present at a concentration of 5% (w / v) to 50% (w / v). In some specific examples, the tensioning agent is present at a concentration of 10% (w / v) to 50% (w / v). In some specific examples, the tensioning agent is present at a concentration of 15% (w / v) to 50% (w / v). In some specific examples, the tensioning agent is present at a concentration of 20% (w / v) to 50% (w / v). In some specific examples, the tensioning agent is present at a concentration of 25% (w / v) to 50% (w / v). In some specific examples, the tension agent is present at a concentration of 30% (w / v) to 50% (w / v). In some specific examples, the tension agent is present at a concentration of 40% (w / v) to 50% (w / v). In some specific examples, the tension agent is present at a concentration of 1% (w / v) to 40% (w / v). In some specific examples, the tension agent is present at a concentration of 1% (w / v) to 30% (w / v). In some specific examples, the tension agent is present at a concentration of 1% (w / v) to 25% (w / v). In some specific examples, the tension agent is present at a concentration of 1% (w / v) to 20% (w / v). In some specific examples, the tension agent is present at a concentration of 1% (w / v) to 15% (w / v). In some specific examples, the tension agent is present at a concentration of 1% (w / v) to 10% (w / v). In some specific cases, the tensioning agent is present at a concentration of 1% (w / v) to 5% (w / v).

[0131] According to each of the foregoing states and specific examples, in some specific examples, the tensioning agent is selected from erythritol, glycerol, lactitol, maltitol, mannitol, sorbitol, sucrose, threitol, xylitol, and any combination thereof, at a concentration of about 1% (w / v). In some specific examples, the tensioning agent is present at a concentration of about 2% (w / v). In some specific examples, the tensioning agent is present at a concentration of about 3% (w / v). In some specific examples, the tensioning agent is present at a concentration of about 4% (w / v). In some specific examples, the tensioning agent is present at a concentration of about 5% (w / v). In some specific examples, the tensioning agent is present at a concentration of about 10% (w / v). In some specific examples, the tensioning agent is present at a concentration of about 15% (w / v). In some specific examples, the tensioning agent is present at a concentration of about 20% (w / v). In some specific examples, the tensioning agent is present at a concentration of about 25% (w / v). In some specific examples, the tension agent is present at a concentration of approximately 30% (w / v). In some specific examples, the tension agent is present at a concentration of approximately 40% (w / v). In some specific examples, the tension agent is present at a concentration of approximately 50% (w / v).

[0132] According to each of the foregoing states and specific examples, in some specific examples, the tensioning agent is selected from erythritol, glycerol, lactitol, maltitol, mannitol, sorbitol, sucrose, threitol, xylitol, and any combination thereof, at a concentration of 1% (w / v). In some specific examples, the tensioning agent is present at a concentration of 2% (w / v). In some specific examples, the tensioning agent is present at a concentration of 3% (w / v). In some specific examples, the tensioning agent is present at a concentration of 4% (w / v). In some specific examples, the tensioning agent is present at a concentration of 5% (w / v). In some specific examples, the tensioning agent is present at a concentration of 10% (w / v). In some specific examples, the tensioning agent is present at a concentration of 15% (w / v). In some specific examples, the tensioning agent is present at a concentration of 20% (w / v). In some specific examples, the tensioning agent is present at a concentration of 25% (w / v). In some specific examples, the tension agent is present at a concentration of 30% (w / v). In some specific examples, the tension agent is present at a concentration of 40% (w / v). In some specific examples, the tension agent is present at a concentration of 50% (w / v).

[0133] In some specific examples, the polyol may be sorbitol, present in amounts of about 1 to about 10% (w / v), or about 2 to about 8% (w / v), or about 4 to about 6% (w / v), or about 5% (w / v).

[0134] According to each of the foregoing states and examples, in some examples, the surfactant may be selected from polysorbates, such as polysorbate 20, polysorbate 40, polysorbate 65, polysorbate 80, polysorbate 81 and polysorbate 85, poloxamer, and any combination thereof, at a concentration of about 0.0001% (w / v) to about 10% (w / v). In some examples, the surfactant is present at a concentration of about 0.0001% (w / v) to about 10% (w / v). In some examples, the surfactant is present at a concentration of about 0.0005% (w / v) to about 5% (w / v). In some examples, the surfactant is present at a concentration of about 0.001% (w / v) to about 3% (w / v). In some examples, the surfactant is present at a concentration of about 0.005% (w / v) to about 2% (w / v). In some specific examples, the surfactant is present at a concentration of about 0.01% (w / v) to about 1% (w / v). In some specific examples, the surfactant is present at a concentration of about 0.02% (w / v) to about 0.5% (w / v). In some specific examples, the surfactant is present at a concentration of about 0.03% (w / v) to about 0.1% (w / v). In some specific examples, the surfactant is present at a concentration of about 0.04% (w / v) to about 0.08% (w / v). In some specific examples, the surfactant is present at a concentration of about 0.04% (w / v) to about 0.06% (w / v). In some specific examples, the surfactant is present at a concentration of about 0.04% (w / v).

[0135] According to each of the foregoing states and specific examples, in some specific examples, the surfactant is selected from polysorbates, such as polysorbate 20, polysorbate 40, polysorbate 65, polysorbate 80, polysorbate 81 and polysorbate 85, poloxamer, and any combination thereof, at a concentration of 0.0001% (w / v) to 10% (w / v). In some specific examples, the surfactant is present at a concentration of 0.0005% (w / v) to 10% (w / v). In some specific examples, the surfactant is present at a concentration of 0.001% (w / v) to 10% (w / v). In some specific examples, the surfactant is present at a concentration of 0.005% (w / v) to 10% (w / v). In some specific examples, the surfactant is present at a concentration of 0.005% (w / v) to 10% (w / v). In some specific examples, the surfactant is present at a concentration of 0.01% (w / v) to 10% (w / v). In some specific examples, the surfactant is present at a concentration of 0.02% (w / v) to 10% (w / v). In some specific examples, the surfactant is present at a concentration of 0.03% (w / v) to 10% (w / v). In some specific examples, the surfactant is present at a concentration of 0.04% (w / v) to 10% (w / v). In some specific examples, the surfactant is present at a concentration of 0.001% (w / v) to 5% (w / v). In some specific examples, the surfactant is present at a concentration of 0.001% (w / v) to 3% (w / v). In some specific examples, the surfactant is present at a concentration of 0.001% (w / v) to 2% (w / v). In some specific examples, the surfactant is present at a concentration of 0.001% (w / v) to 1% (w / v). In some specific examples, the surfactant is present at a concentration of 0.001% (w / v) to 0.5% (w / v). In some specific examples, the surfactant is present at a concentration of 0.001% (w / v) to 0.1% (w / v). In some specific examples, the surfactant is present at a concentration of 0.001% (w / v) to 0.08% (w / v).

[0136] According to each of the foregoing states and specific examples, in some specific examples, the surfactant is selected from polysorbates, such as polysorbate 20, polysorbate 40, polysorbate 65, polysorbate 80, polysorbate 81 and polysorbate 85, poloxamer, and any combination thereof, at a concentration of about 0.0001% (w / v). In some specific examples, the surfactant is present at a concentration of about 0.0005% (w / v). In some specific examples, the surfactant is present at a concentration of about 0.001% (w / v). In some specific examples, the surfactant is present at a concentration of about 0.005% (w / v). In some specific examples, the surfactant is present at a concentration of about 0.01% (w / v). In some specific examples, the surfactant is present at a concentration of about 0.02% (w / v). In some specific examples, the surfactant is present at a concentration of about 0.03% (w / v). In some specific examples, the surfactant is present at a concentration of about 0.04% (w / v). In some specific examples, the surfactant is present at a concentration of about 0.06% (w / v). In some specific examples, the surfactant is present at a concentration of about 0.08% (w / v). In some specific examples, the surfactant is present at a concentration of about 0.1% (w / v). In some specific examples, the surfactant is present at a concentration of about 0.5% (w / v). In some specific examples, the surfactant is present at a concentration of about 1% (w / v). In some specific examples, the surfactant is present at a concentration of about 2% (w / v). In some specific examples, the surfactant is present at a concentration of about 5% (w / v). In some specific examples, the surfactant is present at a concentration of about 10% (w / v).

[0137] In some specific examples, 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 about 0.04% (w / v).

[0138] One embodiment of the present invention is a pharmaceutical preparation comprising... i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) Sodium acetate, iii) Sorbitol, iv) Polysorbate 80, and v) Disodium EDTA At pH 5.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0139] In some specific examples, the pharmaceutical formulation contains about 1 to about 50 mg / mL huMAb2-3-SPDB-DM4. In some specific examples, the pharmaceutical formulation contains about 2 to about 40 mg / mL huMAb2-3-SPDB-DM4. In some specific examples, the pharmaceutical formulation contains about 3 to about 30 mg / mL huMAb2-3-SPDB-DM4. In some specific examples, the pharmaceutical formulation contains about 4 to about 20 mg / mL huMAb2-3-SPDB-DM4. In some specific examples, the pharmaceutical formulation contains about 5 to about 10 mg / mL huMAb2-3-SPDB-DM4. In some specific examples, the pharmaceutical formulation contains about 5 mg / mL huMAb2-3-SPDB-DM4.

[0140] In some specific examples, the pharmaceutical formulation contains 1-50 mg / mL huMAb2-3-SPDB-DM4. In some specific examples, the pharmaceutical formulation contains 2-40 mg / mL huMAb2-3-SPDB-DM4. In some specific examples, the pharmaceutical formulation contains 3-30 mg / mL huMAb2-3-SPDB-DM4. In some specific examples, the pharmaceutical formulation contains 4-20 mg / mL huMAb2-3-SPDB-DM4. In some specific examples, the pharmaceutical formulation contains 5-10 mg / mL huMAb2-3-SPDB-DM4. In some specific examples, the pharmaceutical formulation contains 5 mg / mL huMAb2-3-SPDB-DM4. In some specific examples, the pharmaceutical formulation contains 10 mg / mL huMAb2-3-SPDB-DM4.

[0141] In some specific examples, the chelating agent may be selected from diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), hydroxyethylenediaminetriacetic acid (HEDTA), azirmonotriacetic acid, ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and their salts, such as disodium EDTA, disodium calcium EDTA, and tetrasodium EDTA, in an amount of 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, or about 1 to about 10 μM. In some specific examples, the chelating agent may be present in an amount of about 10 to about 50 μM, or about 10 to about 40 μM, or about 10 to about 20 μM disodium EDTA. In some specific examples, the chelating agent is present in an amount of about 10 μM, or about 20 μM, or about 30 μM, or about 40 μM, or about 50 μM.

[0142] In some specific examples, the pharmaceutical composition contains about 1 to about 50 μM disodium EDTA. In some specific examples, the pharmaceutical composition contains about 1 to about 40 μM disodium EDTA. In some specific examples, the pharmaceutical composition contains about 1 to about 30 μM disodium EDTA. In some specific examples, the pharmaceutical composition contains about 1 to about 20 μM disodium EDTA. In some specific examples, the pharmaceutical composition contains about 1 to about 10 μM disodium EDTA. In some specific examples, the pharmaceutical composition contains about 10 to about 50 μM disodium EDTA. In some specific examples, the pharmaceutical composition contains about 10 to about 40 μM disodium EDTA. In some specific examples, the pharmaceutical composition contains about 10 to about 20 μM disodium EDTA. In some specific examples, the pharmaceutical composition contains about 10 μM disodium EDTA. In some specific examples, the pharmaceutical composition contains about 20 μM disodium EDTA. In some specific examples, the pharmaceutical composition contains about 30 μM disodium EDTA. In some specific examples, the pharmaceutical composition contains about 40 μM disodium EDTA. In some specific examples, the pharmaceutical composition contains about 50 μM disodium EDTA.

[0143] In some specific examples, the pharmaceutical composition contains 1 to 50 μM disodium EDTA. In some specific examples, the pharmaceutical composition contains 1 to 40 μM disodium EDTA. In some specific examples, the pharmaceutical composition contains 1 to 30 μM disodium EDTA. In some specific examples, the pharmaceutical composition contains 1 to 20 μM disodium EDTA. In some specific examples, the pharmaceutical composition contains 1 to 10 μM disodium EDTA. In some specific examples, the pharmaceutical composition contains 10 to 50 μM disodium EDTA. In some specific examples, the pharmaceutical composition contains 10 to 40 μM disodium EDTA. In some specific examples, the pharmaceutical composition contains 10 to 20 μM disodium EDTA. In some specific examples, the pharmaceutical composition contains 10 μM disodium EDTA. In some specific examples, the pharmaceutical composition contains 20 μM disodium EDTA. In some specific examples, the pharmaceutical composition contains 30 μM disodium EDTA. In some specific examples, the pharmaceutical composition contains 40 μM disodium EDTA. In some specific examples, the pharmaceutical composition contains 50 μM disodium EDTA.

[0144] In some specific examples, the chelating agent may be EDTA or a salt thereof, which is present in amounts ranging from 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 about 10 μM.

[0145] In certain specific cases, pharmaceutical preparations contain i) 5 mg / mL anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Sodium acetate 10 mM, iii) Sorbitol 5% (w / v), iv) 0.04% (w / v) polysorbate 80, and v) 1-50 μM disodium EDTA At pH 5.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0146] In certain specific cases, pharmaceutical preparations contain i) 5 mg / mL anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Sodium acetate 10 mM, iii) Sorbitol 5% (w / v), iv) 0.04% (w / v) polysorbate 80, and v) 1 μM disodium EDTA, At pH 5.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0147] In certain specific cases, pharmaceutical preparations contain i) 5 mg / mL anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Sodium acetate 10 mM, iii) Sorbitol 5% (w / v), iv) 0.04% (w / v) polysorbate 80, and v) 10 μM disodium EDTA, At pH 5.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0148] In certain specific cases, pharmaceutical preparations contain i) 5 mg / mL anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Sodium acetate 10 mM, iii) Sorbitol 5% (w / v), iv) 0.04% (w / v) polysorbate 80, and v) 50 μM disodium EDTA, At pH 5.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0149] The formulations disclosed herein are generally suitable for administration to an individual by injection or infusion, such as intravenous injection or infusion.

[0150] method One aspect of the present invention is a method for treating cancer, the method comprising administering an effective amount of any of the aforementioned pharmaceutical formulations to an individual in need.

[0151] According to this method, in certain specific cases, pharmaceutical preparations contain i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) Buffer, iii) Tendency agents, such as polyols, iv) Surfactants, such as polysorbate, and v) Chelating agent to be selected as appropriate. At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0152] In certain specific examples according to this method, the pharmaceutical preparation contains i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) Buffer, iii) Polyols, iv) Polysorbate, and v) Chelating agent to be selected as appropriate. At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0153] In certain specific examples according to this method, the pharmaceutical preparation contains i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) Buffers, such as those selected from the group consisting of acetate, arginine, histidine, citrate, and any combination thereof, or for example, acetate. iii) Polyols, such as sorbitol, iv) Polysorbates, such as polysorbate 80, and v) Chelating agent to be selected as appropriate. At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0154] In certain specific examples according to this method, the pharmaceutical preparation contains i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) Buffers, such as those selected from the group consisting of acetate, arginine, histidine, citrate, and any combination thereof, or for example, acetate. iii) Sorbitol, iv) Polysorbate, and v) Chelating agent to be selected as appropriate. At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0155] In certain specific examples according to this method, the pharmaceutical preparation contains i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) Buffers, such as those selected from the group consisting of acetate, arginine, histidine, citrate, and any combination thereof, or for example, acetate. iii) Polyols, iv) Polysorbate 80, and v) Chelating agent to be selected as appropriate. At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0156] In certain specific examples according to this method, the pharmaceutical preparation contains i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) Buffers, such as those selected from the group consisting of acetate, arginine, histidine, citrate, and any combination thereof, or for example, acetate. iii) Sorbitol, iv) Polysorbate 80, and v) Chelating agent to be selected as appropriate. At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0157] In certain specific examples according to this method, the pharmaceutical preparation contains i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) Sodium acetate, iii) Sorbitol, iv) Polysorbate 80, and v) Chelating agent to be selected as appropriate. At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0158] In certain specific examples according to this method, the pharmaceutical preparation contains i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) Buffer, iii) Tensioner, iv) Polysorbate, and v) Chelating agents, At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0159] In certain specific examples according to this method, the pharmaceutical preparation contains i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) Buffer, iii) Polyols, iv) Polysorbate, and v) Chelating agents, At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0160] In certain specific examples according to this method, the pharmaceutical preparation contains i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) A buffer selected from acetate, arginine, histidine, citrate, and any combination thereof. iii) Tensioner, iv) Polysorbate 80, and v) Ethylenediaminetetraacetic acid (EDTA) At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0161] In certain specific examples according to this method, the pharmaceutical preparation contains i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) A buffer selected from acetates, histidines, citrates, and any combination thereof. iii) Tendon-releasing agents, selected from erythritol, glycerol, lactitol, maltitol, mannitol, sorbitol, sucrose, threitol, xylitol, and any combination thereof. iv) Polysorbate 80, and v) EDTA, At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0162] In certain specific examples according to this method, the pharmaceutical preparation contains i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) A buffer selected from acetates, histidines, citrates, and any combination thereof. iii) Sorbitol, iv) Polysorbate 80, and v) EDTA, At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0163] In certain specific examples according to this method, the pharmaceutical preparation contains i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) Sodium acetate, iii) Sorbitol, iv) Polysorbate 80, and v) EDTA, At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0164] In certain specific examples according to this method, the pharmaceutical preparation contains i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) A buffer selected from acetate, histidine, citrate, and any combination thereof, at a concentration of about 5 mM to about 100 mM. iii) Tendon-releasing agents, selected from erythritol, glycerol, lactitol, maltitol, mannitol, sorbitol, sucrose, threitol, xylitol, and any combination thereof. iv) Polysorbate 80, and v) EDTA, At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0165] In certain specific examples according to this method, the pharmaceutical preparation contains i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) A buffer selected from acetates, histidines, citrates, and any combination thereof. iii) A tensioning agent selected from erythritol, glycerol, lactitol, maltitol, mannitol, sorbitol, sucrose, threitol, xylitol, and any combination thereof, at a concentration of about 1% (w / v) to about 50% (w / v). iv) Polysorbate 80, and v) EDTA, At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0166] In certain specific examples according to this method, the pharmaceutical preparation contains i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) A buffer selected from acetate, histidine, citrate, and any combination thereof, at a concentration of about 5 mM to about 100 mM. iii) A tensioning agent selected from erythritol, glycerol, lactitol, maltitol, mannitol, sorbitol, sucrose, threitol, xylitol, and any combination thereof, at a concentration of about 1% (w / v) to about 50% (w / v). iv) Polysorbate 80, and v) EDTA, At a pH of approximately 5.0 to approximately 6.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0167] In certain specific examples according to this method, the pharmaceutical preparation contains i) Antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) ii) Sodium acetate, iii) Sorbitol, iv) Polysorbate 80, and v) Disodium EDTA At pH 5.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0168] In certain specific examples according to this method, the pharmaceutical preparation contains i) 5 mg / mL anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Sodium acetate 10 mM, iii) Sorbitol 5% (w / v), iv) 0.04% (w / v) polysorbate 80, and v) 1-50 μM disodium EDTA At pH 5.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0169] In certain specific examples according to this method, the pharmaceutical preparation contains i) 5 mg / mL anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Sodium acetate 10 mM, iii) Sorbitol 5% (w / v), iv) 0.04% (w / v) polysorbate 80, and v) 1 μM disodium EDTA, At pH 5.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0170] In certain specific examples according to this method, the pharmaceutical preparation contains i) 5 mg / mL anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Sodium acetate 10 mM, iii) Sorbitol 5% (w / v), iv) 0.04% (w / v) polysorbate 80, and v) 10 μM disodium EDTA, At pH 5.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0171] In certain specific examples according to this method, the pharmaceutical preparation contains i) 5 mg / mL anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody-drug conjugate (ADC), ii) Sodium acetate 10 mM, iii) Sorbitol 5% (w / v), iv) 0.04% (w / v) polysorbate 80, and v) 50 μM disodium EDTA, At pH 5.5, The ADC is composed of huMAb2-3-SPDB-DM4.

[0172] In some specific cases, the cancer is a high-carcinoembryonic antigen-associated cell adhesion molecule cancer.

[0173] In some specific cases, cancer is selected from the group consisting of colorectal cancer, stomach cancer, lung cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, and bladder cancer.

[0174] In some specific cases, cancer is selected from the group consisting of lung cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, and bladder cancer.

[0175] In some specific cases, the cancer is lung cancer.

[0176] In some specific cases, the lung cancer is non-squamous non-small cell lung cancer.

[0177] In some specific cases, the individual is a human being.

[0178] In some specific cases, cancer is selected from the group consisting of colorectal cancer, stomach cancer, lung cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, and bladder cancer.

[0179] In some specific cases, cancer is selected from the group consisting of lung cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, and bladder cancer.

[0180] In some specific cases, the cancer is lung cancer.

[0181] In some specific cases, the lung cancer is non-squamous non-small cell lung cancer.

[0182] Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. It typically grows and spreads more slowly than the less common small cell lung cancer. NSCLC is primarily divided into three subtypes: squamous cell carcinoma (accounting for 25% of lung cancers), adenocarcinoma (accounting for 40% of lung cancers), and large cell carcinoma (accounting for 10% of lung cancers). Therefore, non-squamous NSCLC includes both adenocarcinoma (accounting for 40% of lung cancers) and large cell carcinoma (accounting for 10% of lung cancers).

[0183] In some specific cases, the individual is a human being. Example Example 1. Initial formulation of huMAb2-3-SPDB-DM4

[0184] huMAb2-3-SPDB-DM4 (see Figure 1) was initially developed as a concentrate for infusion solution. The dosage form is a 5 mg / mL solution, for single use, stored in glass vials at 5°C. The active pharmaceutical ingredient was developed as a bulk frozen solution in polycarbonate vials at -20°C, which are representative of other containers. The aim of this preliminary study was to identify a set of pH buffering systems and stabilizing excipients. A total of eight formulations with combined excipients were tested under thermal stress at 40°C, 25°C, and 5°C, as well as freezing / thawing stress and mechanical stress (shaking).

[0185] The prototypes are shown in Table 1. Table 1. Excipient combinations evaluated for humab2-3-SPDB-DM4 at approximately pH 6.0 [N°] [Buffer System] [excipient] [pH] A Acetate 10 mM 2.5% sucrose Mannitol 3.75% PS80 0.005% 5.5 B Acetate 10 mM 2.5% sucrose Mannitol 3.75% PS80 0.1% 5.5 C Histidine 10 mM Sucrose 5.0% Glycine 130 mM PS80 0.1% 5.5 D Histidine 10 mM 10% sucrose PS80 0.1% 6.5 E 10 mM citrate 10% sucrose PS80 0.1% 6.0 F Acetate 10 mM 10% sucrose PS80 0.1% 6.0 G Acetate 10 mM 10% sucrose PS80 0.1% 5.5 H Acetate 10 mM 10% sucrose PS20 0.1% 5.5

[0186] method UF / DF program Starting materials were provided in histidine buffer. To prepare acetate-based prototypes (prototypes A, B, F, G, H), the starting materials were permeated with 10 mM acetate at pH 5.5 (three 88 cm² membranes, 68.7 g protein / m² membrane) using a Pellicon 3® cartridge on the Cogent µScale system. To prepare citrate-based prototypes (prototype E), the starting materials were permeated with 10 mM citrate at pH 6.0 (one 88 cm² membrane, 51.4 g protein / m² membrane) using a Pellicon 3® cartridge on the Cogent µScale system.

[0187] In both cases, dialysis was stopped after passing through 10 times the volume of dialysis buffer. After dialysis, an appropriate amount of 2X concentrated solution was added to a concentration of 5 mg / mL, as described in Table 3. Table 3. Formulation of 2X Excipient Concentrate Solution [N°] [Buffer System] [excipient] [Target] [pH] A Acetate 10 mM 5% sucrose Mannitol 7.5% PS80 0.01% 5.5 B Acetate 10 mM 5% sucrose Mannitol 7.5% PS80 0.2% 5.5 C Histidine 10 mM 10% sucrose Glycine 260 mM PS80 0.2% 5.5 D Histidine 10 mM 20% sucrose PS80 0.2% 6.5 E 10 mM citrate 20% sucrose PS80 0.2% 6.0 F Acetate 10 mM 20% sucrose PS80 0.2% 6.0 G Acetate 10 mM 20% sucrose PS80 0.2% 5.5 H Acetate 10 mM 20% sucrose PS20 0.2% 5.5

[0188] Filtering 0.2 μm After being prepared with the 2X concentrated solution, all preparations were filtered through a 0.2 μm filter and collected on Nalgene® (HDPE) bottles before filling.

[0189] Pressure conditions

[0190] thermal pressure Preparations A through H were stored at 40°C for 2 weeks before analysis; and then placed at 5°C, 25°C, and 40°C for 4 weeks before analysis.

[0191] Freeze / thaw cycle Preparations A through H were frozen at -20°C or -80°C and thawed at room temperature. Three cycles of the freeze / thaw procedure were performed before analyzing the preparations.

[0192] Shaking pressure During the 15-hour period, the formulations A to H were shaken in vials by a track-shaking motion at 350 rpm.

[0193] Analytical methods The following analytical methods were used during the study:

[0194] Visual inspection of appearance (transparency, color, and particle size): Inspect the vial on a white surface for 5 seconds, then inspect it on a black surface of the visual inspection table.

[0195] Protein concentration and DAR measured by UV: OD measured at 280 nm and 254 nm using the following absorption coefficients. Check the dilution factor of the solution: F=10. Check the absorbance of the solution at 254 nm: A254D The absorbance of the solution was checked at 280 nm: A280D The molecular weight of naked antibody h4D4 is 144522 g / mol. The molecular weight of DM4 is 780 g / mol. The molar extinction coefficient of DM4 at 280 nm is 4927 L*mol⁻¹*cm⁻¹ The molar extinction coefficient of naked antibody h4D4 at 280 nm is 201400 L*mol⁻¹*cm⁻¹ The ratio of A254 to A280 in h4D4 is 0.43. The ratio of A254 to A280 in DM4 is 4.83. [Drug] (M) = [A254 – (0.43xA280)] / [(4.83x4927) – (0.43x4927)] [Drug] (mg / mL) = [Drug] (M) x 780 x F [Protein] (M) = [A280 – 4927 x [Drug] (M)] / 201400 [Protein] (mg / mL) = [Protein] (M) x 144522 x F

[0196] Regarding the turbidity (OD at 350 nm) for identifying opalescence characteristics: the absorbance of the solution was measured at 350 nm for a 0.8 mL undiluted solution.

[0197] Based on protein purity determined by SEC-UPLC: Samples A, B, G, and H were subjected to size exclusion ultra-high performance liquid chromatography (SEC-UPLC) at T0, after 2 and 4 weeks at 40°C, and after 4 weeks at 5°C.

[0198] SEC-UPLC conditions: Column: BEH 200, 1.7 μm, 300 mm * 4.6 mm Flow rate: 0.30 mL / min Detection: 230 nm Column temperature: 30℃±2℃ Syringe temperature: 5℃±0.3℃ Injection volume: 4 μL Analysis time: 17 minutes Phase-flow D-PBS (2X): Prepared with D-PBS (10X) (Dubecklite phosphate-buffered saline (D-PBS)) (10X), reference Invitrogen: 14200083: This solution was diluted 1 / 5 with water for injection (WFI) and filtered at 0.22 μm. DS batches were used as references in each sequence (5 μL injected at 5 g / L).

[0199] Protein purity based on SDS-PAGE: Measurements were taken only at T0 and after 2 weeks at 40°C. Samples were prepared under reducing and non-reducing conditions (by adding NEM) and migrated on 4–12% Bis-Tris gels. The migration buffer was prepared by diluting 50 mL of Nu-Page MOPS SDS buffer 20X in 1000 mL of sqf pure water. The migration parameter was 135 V during 90 minutes. The gels were stained with Bleu rotiphorese solution.

[0200] Based on the charge heterogeneity of iCIEF: The acetate formulation was analyzed over a 4-week period only at pH 5.5.

[0201] IcIEF was performed using an FC-coated cIEF Cartbridge and Convergent Biosciences iCE280 imaging capillary electrophoresis system. Samples were diluted in Master mix containing Pharmalyte 3-10, Pharmalyte 8-10.5, 4M urea, 0.35% methylcellulose 1%, and pI-labeled.

[0202] Each sample was injected into a capillary using an autosampler. After focusing, it was detected by a CCD camera, which directly measured the 280 nm UV absorbance. The iCE280 software calculated the apparent pI of each separated peak, and Empower allowed for quantification of isomorphism by area normalization.

[0203] Free methylphenidate: The formulation was analyzed over a 4-week period only at pH 5.5.

[0204] Free maytansine was measured using RP-HPLC with two columns: the first column separated the protein from the free maytansine, and the second column separated the free maytansine and detected it on a PDA detector. Mobile phase A was a 0.1% trifluoroacetic acid solution in pure water. Mobile phase B was a 0.08% trifluoroacetic acid solution in acetonitrile. DM4 solution was used as a reference.

[0205] Thermal stability was measured only at T0 using DSC (differential scanning calorimetry).

[0206] DLS (Dynamic Light Scattering) of colloidal aggregates: mean particle size and distribution. Preparations were analyzed over 4 weeks only at pH 5.5.

[0207] Subvisible aggregates were analyzed by flow cytometry (FCM): morphological analysis and counting of particles. Preparations were analyzed over a 4-week period only at pH 5.5.

[0208] Results and Discussion

[0209] Observation and SEC analysis of DS after TFF in histidine buffer and acetate and citrate buffer.

[0210] Upon receipt, a batch of DS in histidine buffer after filtration to 0.2 μm was slightly milky white and contained particles. Results after UFDF in acetate and citrate buffers are provided in Table 4. Table 4. UFDF Results [batch] [TFF] [Yield] [(%) [Processing time and volume] [UFDF] [Concentration after] [(mg / mL)] [Single purity] [(%) [HMW] [(%) [LMW] [(%) After TFF, in acetate buffer pH 5.5 92.0 46 points 160 mL 11.3 94.2 3.0 2.75 After TFF, in citrate buffer pH 6.0 81.5 68 points 40 mL 12.7 94.4 3.1 2.5

[0211] Acetate buffer appears to be better in terms of opacity and yield.

[0212] Stress research

[0213] Analysis in T0

[0214] The visual inspection, FCM, DLS, SEC-UPLC (Figure 2), and icIEF analysis of the prototype at T0 are the same: - Regarding visual inspection, all formulations are very slightly milky white and contain no visible particles. - Regarding DLS, all formulations are multidisperse with a PDI greater than 30%.

[0215] Some differences between the prototypes can be observed at T0: - Regarding the results of DSC, the Tm is higher in acetate buffer (pH 5.5 or 6). - Regarding free maytansine, the total concentration is lower in acetate or citrate buffers and higher in histidine buffers (<0.1% in acetate and citrate buffers, approximately 0.6% in histidine buffers). See Figure 3. - Regarding the SDS-PAGE results, the formulations were similar, except that formulations G and H had different profiles. Under non-reducing conditions, formulation H showed an additional band at 127 kDa, while under reducing conditions, formulation G showed an additional band at 89 kDa (Figures 4A and 4B).

[0216] thermal pressure

[0217] After 4 weeks at 5°C and 25°C, the analytical parameters (visual inspection, SEC-HPLC, FCM, DLS, free maytansin, icIEF) of all prototypes were similar, except for prototype C, which was suspected of being contaminated after 4 weeks at 25°C (correlated with pH, ​​concentration, DLS, and FCM). See Figure 5 for turbidity results.

[0218] After 2 and 4 weeks at 40°C, the prototype was identical in terms of protein concentration, SDS-PAGE, and visual inspection. - After 2 weeks at 40°C, under SDS-PAGE reduction conditions, a new band was detected at 145 kDa in all formulations. - After 2 weeks at 40°C, all formulations showed a very slight milky white color upon visual inspection. However, after pressure at 40°C, no actual evolution was observed compared to T0.

[0219] Some differences between the prototypes can be observed after 2W and 4W at 40℃: - After 4 weeks at 40°C, the DAR of formulation D (histidine buffer at pH 6.5) decreased the most, while the DAR of formulations at pH 6.5 or histidine decreased the most (Figure 6). - After 2 weeks at 40°C, the highest increase in HMW was observed in citrate at pH 6.0 (prototype E), while the two other formulations (D and F) at pH 5.5 showed higher increases. Regarding the prototypes in acetate buffers (A, B, G, H) at pH 5.5, an increase in aggregates (Fig. 7) and fragments (Fig. 8) was observed after 4W at 40°C, with formulations G and H showing higher increases. (Prototypes C, D, E, and F were not analyzed at 4W at 40°C). - After 2 weeks at 40°C, formulations D, E, and F showed higher levels of free maytansine-like substances. After 4 weeks at 40°C, only formulations at pH 5.5 (prototypes A, B, C, G, and H) were analyzed; prototype A showed a lower level of free maytansine-like substances (Figure 9). The type of buffer (acetate versus histidine) affected the impurities detected by maytansine-like substances (Figure 10). - After 4 weeks at 40°C, the highest increase in OD was observed in the original D (histidine, pH 6.5) at 350 nm, while the increase was higher in formulations at high pH or in histidine. - Regarding the charge isotype results for iCIEF, the highest increase in acidity was observed in formulation D (pH 6.5) after 4W 40℃ (Figure 12). Other test prototypes (prototypes A, B, G, and H) showed similar results after 2W 40℃ and 4W 40℃ (Figure 11).

[0220] Shaking pressure

[0221] After shaking and pressure, the prototype was similar in terms of pH, concentration & DAR, DLS, turbidity, SEC-UPLC, and free maytansine.

[0222] After applying pressure, some differences were observed between the prototypes: - Regarding visual inspection, prototypes A and B did not exhibit particles, while prototypes D, G, and H contained many visible particles. - Regarding FCM, prototype H observed a slight increase in particles ≥10 and 25 μm, which may indicate that aggregation begins from PS20 (Table 5). Regarding the icIEF results, prototype C (histidine, pH 5.5) exhibited the highest charge variant generation (+5% acid, -3% base, PI shift, Figure 13). Other prototypes depicted no charge variant evolution after shaking pressure. Table 5. FCM results after shaking: particle count / mL T0 shake formulation ≥2 μm ≥10 μm ≥25 μm ≥2 μm ≥10 μm ≥25 μm A 138 13 6 263 35 15 B 72 6 1 165 29 9 C 128 11 7 178 34 10 D 194 twenty three 5 653 18 4 E 236 17 7 229 18 2 F 100 5 2 390 18 6 G 83 2 1 268 32 6 H 133 10 1 413 149 50

[0223] Freezing and thawing / (F / T) pressure

[0224] After F / T pressure, the prototypes were similar in terms of visual inspection, pH, concentration & DAR, turbidity, SEC-UPLC, free maytansine, FCM, and DLS. Regarding icIEF, prototype C was the most sensitive to F / T pressure (+6% for acid at -20°C and +5% for DAR at -80°C, and -6% for alkali at both temperatures, Tables 6 and 7). Table 6. iCIEF results after F / T -20℃ / RT formulation Main pI Main peak% Σacid% Σalkali% Observed number of homotypes A 7.62 20.5 34.1 45.4 11 B 7.61 20.6 33.2 46.3 11 C 7.62 20.2 40.1 39.1 11 D 7.60 20.2 35.8 44.1 11 E 7.56 20.3 34.6 45.1 11 F 7.58 20.6 34.5 44.9 11 G 7.57* 20.0 34.8 45.3 11 H 7.58 20.4 34.3 45.4 11 *Second major isoform, but maintaining the consistency percentage between conditions Table 7. iCIEF results after F / T -80 °C / RT Formulation Major pI Major peak % Σ acid % Σ base % Observed number of isoforms A 7.63 20.8 33.8 45.4 11 B 7.62 20.5 34.6 44.6 11 C 7.62 20.6 38.8 40.6 11 D 7.62 19.6 35.6 44.8 11 E 7.56 20.1 35.2 44.7 11 F 7.57 21.2 34.3 44.5 11 G 7.56 20.3 34.7 45.0 11 H 7.56* 20.3 35.2 44.6 11 *The second major isotype, but the percentage of conditions maintaining consistency.

[0225] Conclusion After UF / DF concentration to up to approximately 12 mg / mL, the yield in acetate buffer was better (~92%), and the processing time was shorter than in citrate buffer. huMAb2-3-SPDB-DM4 in 10 mM citrate buffer pH 6.0 turned slightly milky white after UF / DF.

[0226] Regarding the behavior of formulations after stress:

[0227] Visual inspection revealed very slight opalescence in all formulations, but no further evolution was observed. At OD 350 nm, turbidity increased in all formulations after pressure at 40°C, but primarily due to histidine at pH 6.5 (Formulation D).

[0228] For all formulations, only some visible particles were observed after shaking pressure, but fewer were observed in formulations A and B (acetate, pH 5.5, containing sucrose and mannitol).

[0229] After 4 weeks at 40°C, the DAR instability of histidine at pH 6.5 was greater.

[0230] After 2 weeks at 40°C, the HMW of acetate at pH 5.5 was more stable (according to SEC-UPLC).

[0231] After 4 weeks at 40°C, the LMW of all acetate pH 5.5 formulations increased by up to 3 (based on SEC-UPLC) (other formulations were not analyzed).

[0232] For free maytansine and iCIEF, pH 5.5 and acetate buffer are more stable after pressure (mainly thermal pressure).

[0233] According to FCM, no subvisible particle aggregation was observed in PS80.

[0234] Higher Tm was obtained in acetate buffer (pH 5.5 and 6.0) (according to DSC).

[0235] In summary, acetate at pH 5.5 appears to be the best stabilizing buffer (primarily against DAR, HMW, free maytansine, and charge variants), and the presence of PS80 allows for particle reduction.

[0236] Example 2. Sorbitol was selected as the excipient.

[0237] method

[0238] The formulation development study described in this example is designed based on the results of Example 1 for the following reasons: • 10 mM sodium acetate buffer at pH 5.5 was chosen to improve various physicochemical properties (primarily DAR, HMW%, free maytansine, and charge variants); • The mannitol tested in Example 1 was retained for one formulation, and other polyols were tested as substitutes: sucrose and sorbitol, at concentrations compatible with isotonicity; and • PS80 was selected from Example 1 to improve aggregation tendency (primarily subvisible particles based on FCM after shaking pressure). PS80 concentrations ranged between 0.01% w / v and 0.04% w / v.

[0239] The composition of the six prototypes selected in this study is provided in Table 8. Table 8. Prototype Formulation [Preparation] [ID] [Buffer System] [Target] [pH] [excipient] [concentration] [(mg / mL)] A Acetate 10 mM 5.5 Sucrose 2.5% + Mannitol 3.75% + PS80 0.01% 5.0 B Acetate 10 mM 5.5 10% sucrose + 80% PS 0.01% 5.0 C Acetate 10 mM 5.5 10% sucrose + 80% PS 0.02% 5.0 D Acetate 10 mM 5.5 10% sucrose + 80% PS (0.04%) 5.0 E Acetate 10 mM 5.5 Sorbitol 5% + PS 80 0.01% 5.0 F Acetate 10 mM 5.5 Sorbitol 5% + PS 80 0.04% 5.0

[0240] The 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 also evaluated under pressure conditions by shaking, followed by in-use simulations. Table 9 describes the time points, pressure conditions, and analytical methods used in the formulation studies. Table 9. Time Points and Analysis Methods [Time point] [Purpose] [Analysis Methods] T0 Initial time point DSC, visual observation, turbidity, pH, DAR and protein concentration, icIEF, free maytansin, SEC-UPLC (monomer %, HMW%, LMW%), DLS, FCM, binding based on ELISA, SDS-PAGE under reducing and non-reducing conditions. Shaking pressure (SS) The track oscillates at 350 rpm for 15 hours. Visual inspection, LO (HIAC) and FCM Simulation in use (executed after SS) The sample was diluted at a concentration of 0.8 mg / mL in a 0.9% NaCl polyolefin bag (P0 analysis). The diluted bags were kept at room temperature for 24 hours (P1 analysis). The diluted bag was then perfused via a Hospira infusion kit (polyethylene tubing and a 0.2 μm polyether filter) and a pump at a rate of 1 mL / min. The collected sample (approximately 10 mL) was analyzed (P2 analysis). Visual observation, LO (HIAC), DAR and protein concentration, SEC-UPLC (monomer%, HMW%, LMW%)

[0241] The active pharmaceutical ingredient used in this example was from a single batch and was characterized as a formulation in acetate at 10 mM, pH 5.5, and 12.4 g / L. Samples were diluted in acetate buffer and excipients were added to obtain the six formulations shown in Table 8.

[0242] Perform a visual inspection of the vials (transparency and particle size) on a visual inspection table. Inspect the vials for 5 seconds on a white surface and then on a black surface, and assess the presence of visible particles. Additionally, inspect the vials using a fiber optic MLC-150C (color temperature: 2000-3500K) from MOTIC; the scoring is detailed in Tables 10 and 11. Table 10. Visual examination scores for opacity assessment Opal L clear SO Slightly milky white O milky Table 11. Visual inspection scores for particle assessment [Particle Detection] [Number of particles] 0 Undetectable by cold light (fiber optics) + Countable (1≦n≦5) 1 Detected using only cold light (fiber optics) ++ Several (n>5) 2 Visible particles on the visual inspection table +++ a lot of

[0243] Protein concentration and DAR (UV)

[0244] Protein concentration and DAR measurements were performed using UV measurements, employing the following method: the sample was diluted 10-fold, and then the OD was measured at 280 nm and 254 nm. The formulas below are used to calculate DAR and protein concentration. [Drug] (M) = A254 – (0.43xA280) / (4.83x4927) – (0.43x4927) [Drug] mg / mL = [Drug] (M) x 780 x F [Protein] (M) = A280 – 4927 x [Drug] (M) / 201400 [Protein] mg / mL = [Protein] (M) x 144522 x F DAR = [Drugs] (M) / [Proteins] (M)

[0245] Turbidity Turbidity was assessed by measuring the absorbance of 0.8 mL of undiluted solution at 350 nm using a Cary UV 100 spectrophotometer.

[0246] Light shading Subvisible particle counting was performed using light-masking techniques with a high-precision liquid particle counter (HIAC). Each result is the average of four measurements from a 0.9 mL sample. Approximately 4 mL of sample is required for the analysis.

[0247] Protein purity based on SDS-PAGE Protein purity was determined by SDS-PAGE according to the following procedure. Sample preparation is as described in Table 12. Table 12. Sample preparation for SDS-PAGE Restoration conditions Non-reducing conditions Sample (5 mg / mL solution or reference) 5 µL 5 µL Nu page LDS buffer (x4) 12.5 µL + nem 12.5 µL Reducing agent (x10) 5 µL - Prepare buffer solution 20 µL 20 µL water 7.5 µL 12.5 µL heating 10 minutes at 90℃ 10 minutes at 70℃ sediment 20 µL 10 µL

[0248] The sample was diluted to a concentration of 1 g / L. The reference standard was used along with the sample. 1X migration buffer was prepared by diluting 50 mL of Nu-Page MOPS SDS Buffer 20X (Invitrogen NP0001) in 1 L of pure water. The gel was 4-12% Bis-Tris. Electrophoresis was performed at 135 W for 90 minutes. The gel was then stained with Blue Rotiphorese solution. Data processing was performed using Quantity One.

[0249] 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).

[0250] DSC The DSC results for T0 are presented in Table 13. Table 13. DSC results for T0 [Preparation] [Td (°C)] A – Suc 2.5% Man 3.75% PS80 0.01% 86.4 B – Suc 10% PS80 0.01% 86.7 C – Suc 10% PS80 0.02% 86.7 D – Suc 10% PS80 0.04% 86.7 E – Sorb 5% PS80 0.01% 86.4 F – Sorb 5% PS80 0.04% 86.4

[0251] At T0, the thermal stability of the six formulations is equal.

[0252] Visual inspection The results of the visual inspection are provided in Table 14 (Particles) and Table 15 (Clarity). Table 14. Visual Observation Results (Particles) [Visual observation] [5℃] [25℃] [40℃] [Preparation] [T0] [T1M] [T3M] [T1M] [T3M] [T1M] A – Suc 2.5% Man 3.75% PS80 0.01% 2+ 0 0 0 2+ 0 B – Suc 10% PS80 0.01% 2+ 0 0 1+ 2+ 0 C – Suc 10% PS80 0.02% 2+ 0 0 0 0 0 D – Suc 10% PS80 0.04% 0 0 0 0 0 0 E – Sorb 5% PS80 0.01% 2+ 0 0 1+ 0 0 F – Sorb 5% PS80 0.04% 2+ 0 0 0 0 0 Table 15. Visual Observation Results (Clarity) [Visual observation] [5℃] [25℃] [40℃] [Preparation] [T0] [T1M] [T3M] [T1M] [T3M] [T1M] A – Suc 2.5% Man 3.75% PS80 0.01% L SO L SO L SO B – Suc 10% PS80 0.01% L SO L SO L SO C – Suc 10% PS80 0.02% L SO L SO L SO D – Suc 10% PS80 0.04% L SO L SO L SO E – Sorb 5% PS80 0.01% L SO L SO L SO F – Sorb 5% PS80 0.04% L* SO L SO L SO

[0253] Overall, very few particles were observed in all formulations and at all time points. All formulations were transparent or slightly milky white, with no significant changes at other times.

[0254] Turbidity (OD at 350 nm) Table 16 presents the turbidity (OD at 350 nm) results after hot pressing. Table 16. Turbidity [Turbidity] [5℃] [25℃] [40℃] [-20℃] [Preparation] [T0] [T1M] [T3M] [T1M] [T3M] [T1M] [T3M] A – Suc 2.5% Man 3.75% PS80 0.01% 0.0145 0.0153 0.0157 0.0185 0.0294 0.0631 0.0150 B – Suc 10% PS80 0.01% 0.0169 0.0177 0.0159 0.0260 0.0318 0.0698 0.0149 C – Suc 10% PS80 0.02% 0.0152 0.0163 0.0155 0.0214 0.0337 0.0692 0.0157 D – Suc 10% PS80 0.04% 0.0164 0.0167 0.0167 0.0221 0.0332 0.0681 0.0163 E – Sorb 5% PS80 0.01% 0.0140 0.0152 0.0138 0.0193 0.0277 0.0618 0.0138 F – Sorb 5% PS80 0.04% 0.0146 0.0152 0.0143 0.0199 0.0289 0.0599 0.0157

[0255] Significant evolution was observed after T1M40℃ for all formulations. At T1M40℃ and T3M25℃, slight differences were noted between the E and F sorbitol formulations and the other formulations, with slightly less diffusion of the sorbitol prototype.

[0256] pH The pH results are reported in Table 17. Table 17. pH Results [pH] [5℃] [25℃] [40℃] [-20℃] [Preparation] [T0] [T1M] [T3M] [T1M] [T3M] [T1M] [T3M] A – Juice 2.5% Price 3.75% PS80 0.01% 5.49 5.49 5.49 5.48 5.46 5.48 5.50 B – Suc 10% PS80 0.01% 5.50 5.48 5.49 5.49 5.49 5.48 5.49 C – Suc 10% PS80 0.02% 5.50 5.49 5.48 5.48 5.48 5.48 5.50 D – Suc 10% PS80 0.04% 5.51 5.48 5.50 5.49 5.48 5.49 5.49 E – Sorb 5% PS80 0.01% 5.48 5.49 5.49 5.49 5.50 5.49 5.50 F – Sorb 5% PS80 0.04% 5.48 5.49 5.50 5.49 5.52 5.49 5.50

[0257] No pH change was observed in any of the formulations during the pressure period.

[0258] DAR and concentration The results of protein concentration and DAR are reported in Table 18. Table 18. Concentration and DAR Results [5℃] [25℃] [40℃] [-20℃] [Preparation] [T0] [T1M] [T3M] [T1M] [T3M] [T1M] [T3M] [concentration] A – Suc 2.5% Man 3.75% PS80 0.01% 5.2 5.2 5.1 5.1 5.2 5.3 5.2 B – Suc 10% PS80 0.01% 5.1 5.1 5.1 5.1 5.2 5.2 5.1 C – Suc 10% PS80 0.02% 5.1 5.1 5.2 5.1 5.2 5.3 5.2 D – Suc 10% PS80 0.04% 5.1 5.1 5.2 5.1 5.2 5.3 5.2 E – Sorb 5% PS80 0.01% 5.1 5.1 5.1 5.1 5.2 5.2 5.2 F – Sorb 5% PS80 0.04% 5.2 5.2 5.2 5.2 5.2 5.3 5.2 [DAR] A – Suc 2.5% Man 3.75% PS80 0.01% 3.5 3.5 3.4 3.8 3.1 2.5 3.4 B – Suc 10% PS80 0.01% 3.5 3.4 3.4 3.3 3.1 2.4 3.5 C – Suc 10% PS80 0.02% 3.5 3.4 3.5 3.3 3.2 2.5 3.5 D – Suc 10% PS80 0.04% 3.5 3.5 3.6 3.4 3.2 2.4 3.6 E – Sorb 5% PS80 0.01% 3.4 3.4 3.3 3.3 3.0 2.5 3.3 F – Sorb 5% PS80 0.04% 3.5 3.5 3.5 3.4 3.2 2.5 3.5

[0259] No concentration evolution was observed in any formulation during the pressure period. Regarding DAR, a decrease was observed after thermal stress at T1M 40°C (approximately -1 unit) and T3M 25°C (approximately -0.5 units), but no significant difference was observed between the prototypes (Figure 14).

[0260] icIEF Results for major isoform %, acidic form %, and basic form % are provided in Table 19. Results for major isoform content, based on icIEF, are depicted in Figure 15. Table 19. icIEF Results [5] [℃]

[25] [℃]

[40] [℃] [-20] [℃] [Preparation] [T0] [T1M] [T3M] [T1M] [T3M] [T1M] [T3M] [Mainly the same type] [%] A – Suc 2.5% Man 3.75% PS80 0.01% 20 21 20 21 20 15 20 B – Suc 10% PS80 0.01% 20 21 20 21 19 14 19 C – Suc 10% PS80 0.02% 20 21 19 21 19 14 19 D – Suc 10% PS80 0.04% 20 21 20 20 19 14 19 E – Sorb 5% PS80 0.01% 20 21 20 20 19 15 19 F – Sorb 5% PS80 0.04% 20 21 20 19 20 15 20 [Acid homotype] [%] A – Suc 2.5% Man 3.75% PS80 0.01% 53 51 54 52 53 67 53 B – Suc 10% PS80 0.01% 53 50 53 53 54 68 54 C – Suc 10% PS80 0.02% 53 50 54 53 54 69 54 D – Suc 10% PS80 0.04% 53 52 53 54 56 69 56 E – Sorb 5% PS80 0.01% 54 51 54 54 54 67 54 F – Sorb 5% PS80 0.04% 54 52 54 53 54 67 54 [Basic Homoform] [%] A – Suc 2.5% Man 3.75% PS80 0.01% 27 28 27 27 27 18 27 B – Suc 10% PS80 0.01% 27 29 27 27 27 18 27 C – Suc 10% PS80 0.02% 27 29 27 26 27 18 27 D – Suc 10% PS80 0.04% 27 28 28 26 twenty four 17 twenty four E – Sorb 5% PS80 0.01% 26 28 27 26 27 18 27 F – Sorb 5% PS80 0.04% 26 27 27 27 26 18 26

[0261] No significant differences were observed in major isoform % and acidic isoform % for all formulations and at all pressure conditions other than T1M40℃. After T1M40℃, major isoform % and basic isoform % decreased significantly, accompanied by an increase in acidic isoform %. No significant differences were observed between prototypes.

[0262] Free maytansin The results of total free maytansine content during thermo-stressing are presented in Figure 16 and Table 20. Table 20. Results of free maytansine [5] [℃]

[25] [℃]

[40] [℃] [-20] [℃] [Preparation] [T0] [T1M] [T3M] [T1M] [T3M] [T1M] [T3M] [Free sum] [%] A – Suc 2.5% Man 3.75% PS80 0.01% 0.3 0.4 0.6 0.8 2.7 4.5 0.4 B – Suc 10% PS80 0.01% 0.4 0.5 0.5 0.9 2.8 4.8 0.4 C – Suc 10% PS80 0.02% 0.4 0.5 0.5 1.0 2.8 4.7 0.4 D – Suc 10% PS80 0.04% 0.4 0.5 0.6 1.1 2.8 5.2 0.5 E – Sorb 5% PS80 0.01% 0.3 0.4 0.5 1.0 2.5 4.3 0.4 F – Sorb 5% PS80 0.04% 0.4 0.5 0.6 1.2 2.6 4.4 0.5 [DM4-TBA (%)] A – Suc 2.5% Man 3.75% PS80 0.01% 0.2 0.4 0.5 0.7 2.2 2.5 0.3 B – Suc 10% PS80 0.01% 0.3 0.4 0.4 0.8 2.1 2.5 0.3 C – Suc 10% PS80 0.02% 0.3 0.4 0.4 0.9 2.2 2.6 0.3 D – Suc 10% PS80 0.04% 0.3 0.4 0.5 1.0 2.1 2.9 0.4 E – Sorb 5% PS80 0.01% 0.2 0.3 0.4 0.8 2.0 2.4 0.3 F – Sorb 5% PS80 0.04% 0.3 0.4 0.5 1.0 2.1 2.7 0.4 [DM4 – (%)] A – Suc 2.5% Man 3.75% PS80 0.01% 0.1 0.1 0.1 - - - 0.1 B – Suc 10% PS80 0.01% 0.1 0.1 0.1 - - - 0.1 C – Suc 10% PS80 0.02% 0.1 0.1 0.1 - - - 0.1 D – Suc 10% PS80 0.04% 0.1 0.1 0.1 0.1 - - 0.1 E – Sorb 5% PS80 0.01% 0.1 0.1 0.1 0.1 - - 0.1 F – Sorb 5% PS80 0.04% 0.1 0.1 0.1 0.1 - - 0.1

[0263] After T1M25℃ (slightly), T3M25℃, and T1M40℃, the free maytansine content increased in all formulations. Under all conditions, the same content was observed in all prototypes. After T3M25℃ and T1M40℃, formulations E and F exhibited slightly lower free maytansine content. After T1M40℃, formulation D showed a slightly higher free maytansine content.

[0264] Overall, as observed at T1M40℃ and T3M25℃, the sorbitol-containing prototypes (formulations E and F) showed better total free maytansin content.

[0265] SEC-UPLC The results of SEC-UPLC are shown in Table 21. Table 21. Monomer % , HMW % and LMW % under thermal stress, based on SEC-UPLC [5] [℃]

[25] [℃]

[40] [℃] [-20] [℃] [Preparation] [T0] [T1M] [T3M] [T1M] [T3M] [T1M] [T3M] [monomer] [%] A – Suc 2.5% Man 3.75% PS80 0.01% 98.3 98.3 98.2 98.1 96.3 93.7 98.3 B – Suc 10% PS80 0.01% 98.3 98.3 98.2 98.1 96.2 93.7 98.4 C – Suc 10% PS80 0.02% 98.3 98.3 98.2 98.1 96.2 93.5 98.4 D – Suc 10% PS80 0.04% 98.3 98.3 98.2 98.1 96.1 93.5 98.3 E – Sorb 5% PS80 0.01% 98.3 98.3 98.2 98.1 96.5 94.0 98.3 F – Sorb 5% PS80 0.04% 98.3 98.3 98.2 98.1 96.4 93.9 98.3 [HMW%] A – Suc 2.5% Man 3.75% PS80 0.01% 1.7 1.7 1.7 1.8 2.1 3.2 1.7 B – Suc 10% PS80 0.01% 1.7 1.7 1.7 1.8 2.0 3.1 1.6 C – Suc 10% PS80 0.02% 1.7 1.7 1.7 1.8 2.0 3.1 1.6 D – Suc 10% PS80 0.04% 1.7 1.7 1.7 1.8 2.0 3.1 1.6 E – Sorb 5% PS80 0.01% 1.7 1.7 1.7 1.8 2.0 3.3 1.7 F – Sorb 5% PS80 0.04% 1.7 1.7 1.7 1.8 2.0 3.2 1.7 [LMW%] A – Suc 2.5% Man 3.75% PS80 0.01% 0.1 0.1 0.1 0.1 1.7 3.1 <0.1 B – Suc 10% PS80 0.01% 0.1 0.1 0.1 0.1 1.8 3.3 <0.1 C – Suc 10% PS80 0.02% 0.1 0.1 0.1 0.1 1.8 3.4 <0.1 D – Suc 10% PS80 0.04% 0.1 0.1 0.1 0.1 1.8 3.4 <0.1 E – Sorb 5% PS80 0.01% 0.1 0.1 0.1 0.1 1.4 2.8 <0.1 F – Sorb 5% PS80 0.04% 0.1 0.1 <0.1 0.1 1.5 2.8 <0.1

[0266] For all formulations and under all thermo-pressure conditions, monomer % and HMW % evolve in a similar manner.

[0267] Regarding LMW%, similar fragmentation content was observed for all formulations at T0, T1M and T3M 5°C, T3M -20°C and T1M 25°C. After T3M 25°C and T1M 40°C, formulations E and F appeared to have lower LMW% than the other formulations (-0.3% to -0.6% after T1M 40°C compared to the other formulations), as depicted in Figure 17.

[0268] SDS-PAGE

[0269] The observations under non-reducing conditions are as follows: After T3M 5℃ and T3M 25℃, the pattern was the same as at T0, and no differences were observed between formulations. After T1m 40℃, all formulations showed an additional band at 46 kDa. Its relative amount ranged from 0.1% to 0.6%.

[0270] The observations under reducing conditions are as follows: After T3m5℃ and T3m25℃, the patterns were identical and there were no significant differences between the formulations. After T1m40℃, up to nine 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 the formulations.

[0271] Overall, based on the SDS-PAGE, it can be seen that there are no differences between the formulations in terms of the thermal stress conditions of all tests.

[0272] FCM The FCM results are presented in Table 22. Table 22. FCM results during thermal stress [Particles] [ / mL] [Preparation] [T0] [5℃]

[25] [℃]

[40] [℃] [-20] [℃] [T1M] [T3M] [T1M] [T3M] [T1M] [T3M] [≧] [2µm] A – Juice 2.5% Value 3.75% PS80 0.01% 436 192 710 129 362 384 336 B – Suc 10% PS80 0.01% 446 126 642 192 525 321 131 C – Suc 10% PS80 0.02% 211 371 491 144 495 1806 374 D – Suc 10% PS80 0.04% 105 208 287 168 1098 162 137 E – Sorb 5% PS80 0.01% 158 284 422 194 326 210 168 F – Sorb 5% PS80 0.04% 161 180 511 698 346 508 108 [≧] [10µm] A – Suc 2.5% Man 3.75% PS80 0.01% 40 15 15 10 10 20 13 B – Suc 10% PS80 0.01% 35 10 28 9 12 23 12 C – Suc 10% PS80 0.02% 15 16 13 10 13 54 33 D – Suc 10% PS80 0.04% 6 16 11 15 16 11 11 E – Sorb 5% PS80 0.01% 7 21 11 13 14 10 13 F – Sorb 5% PS80 0.04% 8 14 11 63 15 16 13 [≧] [25µm] A – Suc 2.5% Man 3.75% PS80 0.01% 4 3 1 1 1 3 2 B – Suc 10% PS80 0.01% 1 1 5 1 2 4 1 C – Suc 10% PS80 0.02% 2 1 2 2 2 5 2 D – Suc 10% PS80 0.04% 1 1 1 4 1 1 1 E – Sorb 5% PS80 0.01% 1 1 1 2 4 2 1 F – Sorb 5% PS80 0.04% 1 3 0 4 6 0 1

[0273] For all prototypes and under all thermal stress conditions, according to FCM, there was no significant evolution of subvisible particles.

[0274] Based on ELISA, combined with CEACAM5 Based on the ELISA results, the binding to CEACAM5 is presented in Table 23. The formulations were tested relative to the T0 sample after T3M -20℃, T3M 5℃, and T3M 25℃ (relative potency EC 50T0 / EC 50T3m). Table 23. Binding rates according to ELISA after T3M 5℃, T3M 25℃, and T3M -20℃ [Preparation] [T3M5℃] [T3M25℃] [T3M-20℃] A – Suc 2.5% Man 3.75% PS80 0.01% 96 98 104 B – Suc 10% PS80 0.01% 101 93 102 C – Suc 10% PS80 0.02% 99 98 98 D – Suc 10% PS80 0.04% 100 95 97 E – Sorb 5% PS80 0.01% 102 94 94 F – Sorb 5% PS80 0.04% 99 100 92

[0275] According to ELISA, the relative binding potency to CEACAM5 was stable after T3M -20℃, T3M 5℃, and T3M 25℃. No differences were observed between formulation prototypes under these experimental conditions.

[0276] Shaking pressure Table 24 presents the results of visual observation after shaking pressure. Table 24. Visual observations (particles) after shaking pressure. [Preparation] [T0] [Shaking pressure] A – Suc 2.5% Man 3.75% PS80 0.01% 2+ 2+ B – Suc 10% PS80 0.01% 2+ 2+ C – Suc 10% PS80 0.02% 2+ 2+ D – Suc 10% PS80 0.04% 0 2++ E – Sorb 5% PS80 0.01% 2+ 2+ F – Sorb 5% PS80 0.04% 2+ 2++

[0277] After shaking and pressure, very few particles were observed in all formulations. The particles were most commonly found to be exogenous fibers.

[0278] LO (HIAC) and FCM Tables 25 and 26 show the subvisible particle results based on HIAC and FCM after shaking pressure. Table 25. HIAC results after shaking pressure [Particles] [ / mL] [T0] [After shaking with pressure] [Preparation] [≧] [1.5 µm] [≧]

[10] [µm] [≧]

[25] [µm] [≧] [1.5 µm] [≧] [10 µm] [≧] [25 µm] A – Suc 2.5% Man 3.75% PS80 0.01% 352 twenty two 5 485 11 1 B – Suc 10% PS80 0.01% 114 10 4 222 13 2 C – Suc 10% PS80 0.02% 208 17 1 257 12 2 D – Suc 10% PS80 0.04% 198 twenty four 5 155 13 1 E – Sorb 5% PS80 0.01% 277 19 3 105 7 1 F – Sorb 5% PS80 0.04% 241 34 2 124 6 2 Table 26. FCM Results After Shaking [Grains] [ / mL] [T0] [After shaking pressure] [prototype] [≧] [2 µm] [≧] [10 µm] [≧] [25 µm] [≧] [2 µm] [≧] [10 µm] [≧] [25 µm] A – Suc 2.5% Man 3.75% PS80 0.01% 436 40 4 1337 59 5 B – Suc 10% PS80 0.01% 446 35 1 1165 59 5 C – Suc 10% PS80 0.02% 211 15 2 759 102 12 D – Suc 10% PS80 0.04% 105 6 1 412 25 3 E – Sorb 5% PS80 0.01% 158 7 1 225 12 1 F – Sorb 5% PS80 0.04% 161 8 1 798 33 3

[0279] According to FCM, there is a slight increase in particles ≥10 µM in formulation C, but not according to HIAC. Overall, the stability of all formulations with respect to subvisible particles after shaking pressure is satisfactory.

[0280] In-use simulation

[0281] Visual observation Evaluate three sampling points: · P0: T0 after dilution to 0.8 g / L in 0.9% NaCl · P1: T24h at room temperature after dilution to 0.8 g / L in 0.9% NaCl, without infusion through the infusion line and filter · P2: After T24h at room temperature after dilution to 0.8 g / L in 0.9% NaCl, after infusion through the infusion line and filter.

[0282] After dilution in 0.9% NaCl, all formulations showed visible particles: 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 significantly better than the other formulations. At the P1 sampling point, the following ranking was proposed: F < D < C < E < B < A (the differences between samples were smaller than at P0) At the P2 sampling point, there were far fewer particles in all formulations except prototype E. Prototype E showed a large number of particles; the other prototypes were similar, with only a few particles.

[0283] In summary, dilution in 0.9% NaCl increased the visible particle content in all formulations. After infusion (P2 samples), the particle content returned to a level comparable to the T0 content, except for formulation E with 100 ppm ps80.

[0284] Subvisible particles Tables 27 and 28 depict the subvisible particle results according to HIAC after in-use simulation, expressed as particles / mL and particles / container, respectively. Table 27. HIAC results after in-use simulation [Particles] [ / mL] [T0] [P0] [P1] [P2] [Formulation] [≥] [1.5] [μm] [≥]

[10] [μm] [≥]

[25] [μm] [≥] [1.5] [μm] [≥]

[10] [μm] [≥]

[25] [μm] [≥] [1.5] [μm] [≥]

[10] [[ID=7,8]] [μm] [≥]

[25] [μm] [≥] [1.5] [μm] [≧]

[10] [µm] [≧]

[25] [µm] A – Suc 2.5% Man 3.75% PS80 0.01% 352 22 5 5836 245 5 4782 207 11 160 19 3 B – Suc 10% PS80 0.01% 114 10 4 4072 86 2 1735 52 2 120 8 0 C – Suc 10% PS80 0.02% 208 17 1 2733 37 2 1445 25 1 59 6 1 D – Suc 10% PS80 0.04% 198 24 5 1992 18 1 889 45 3 37 7 1 E – Sorb 5% PS80 0.01% 277 19 3 2676 110 4 935 43 1 260 37 3 F – Sorb 5% PS80 0.04% 241 34 2 881 8 0 1430 32 2 62 4 1 Table 28. HIAC results after simulation, expressed as granules / container (50mL bag). [Grains] [ / ] [container] [T0] [P0] [P1] [P2] [Preparation] [Specifications] [≧]

[10] [µm] [≧]

[25] [µm] [≧]

[10] [µm] [≧]

[25] [µm] [≧]

[10] [µm] [≧]

[25] [µm] [≧]

[10] [µm] [≧]

[25] [µm] A – Suc 2.5% Man 3.75% PS80 0.01% For reference only before infusion. Post-infusion: Number of particles ≥10µm ≤6000 / recipient Particle number ≥ 25µm ≤600 / recipient 1100 250 12250 250 10350 550 950 150 B – Suc 10% PS80 0.01% 500 200 4300 100 2600 100 400 0 C – Suc 10% PS80 0.02% 850 50 1850 100 1250 50 300 50 D – Suc 10% PS80 0.04% 1200 250 900 50 2250 150 350 50 E – Sorb 5% PS80 0.01% 950 150 5500 200 2150 50 1850 150 F – Sorb 5% PS80 0.04% 1700 100 400 0 1600 100 200 50

[0285] As shown in Figure 18 for sampling points P0 and P1, dilution in a saline solution (NaCl 0.9%) resulted in osmotic pressure producing ≥1.5 µm subvisible particles in all formulations. Formulation A had a significantly higher number of ≥1.5 µm particles. Formulations A and E had significantly higher numbers of ≥10 µm particles at sampling point P0. Both formulations A and E had a low PS80 test concentration of 100 ppm.

[0286] After infusion through the infusion line and a 0.2 µm filter (sampling point P2), the subvisible particle count of all formulations was reduced. All formulations met pharmacopoeia standards and were suitable for 50 mL infusion bags. For volumes exceeding 100 mL, formulation E failed to meet pharmacopoeia standards (less than 25 particles / mL ≥ 10 μm and less than 3 particles / mL ≥ 25 μm). At a PS80 concentration of 100 ppm, a higher risk of noncompliance was highlighted.

[0287] Concentration and DAR Figures 19 and 20 depict the results of protein concentration and DAR 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 (including a 0.2 μM pes in-line filter). No differences were highlighted between the prototypes.

[0288] SEC-UPLC Figures 21 and 22 present the results of HMW% and LMW% after in-use simulation. During the in-use simulation, according to SEC, no significant evolution was observed in all prototypes after dilution and storage in bags at room temperature for 24 hours.

[0289] in conclusion

[0290] Based on thermostress, the sorbitol formulations (E & F) showed better performance in terms of free maytansin (T1M40℃), fragmentation (T3M25℃ and T1M40℃), and turbidity (T1M40℃). All other test parameters were identical for all formulations.

[0291] Furthermore, simulations of subsequent shaking pressure during use demonstrate that: All formulations showed satisfactory results under shaking pressure, and no significant differences were highlighted between the prototypes.

[0292] Diluting at 0.8 mg / mL in 0.9% NaCl resulted in the formation of subvisible particles, particularly in formulations containing 100 ppm PS80. According to LO (HIAC), formulation E (5% sorbitol, 100 ppm PS80) did not meet the most stringent pharmacopoeia standards.

[0293] Infusion of diluted huMAb2-3-SPDB-DM4 at 0.8 mg / mL via a PE line and PES filter resulted in a significant decrease in protein concentration and DAR in the first milliliter.

[0294] Based on these results, the initial huMAb2-3-SPDB-DM4 formulation was selected as follows: sodium acetate 10 mM pH 5.5, sorbitol 5% (w / v), and PS80 400ppm (0.04%).

[0295] Example 3. Effect of EDTA addition on huMAb2-3-SPDB-DM4 formulation

[0296] Accelerated degradation of polysorbate esters can be observed in the presence of trace metals. It is well known that iron and other metals (such as copper) are involved in the degradation of sorbitol ester 80 (PS80), even in very low concentrations (ppb). Kranz et al., J Pharm Sci2019 108(6):2022-2032. The addition of the chelating agent disodium EDTA was investigated to assess its ability to limit the oxidation of PS80.

[0297] Research Design

[0298] The aim of this study was to evaluate the protective effect of EDTA at three different concentrations (1, 10, and 50 μM) on huMAb2-3-SPDB-DM4 material containing metal residues (representing achievable levels). A filling platform was used to simulate a laboratory-scale pharmaceutical (DP) filling procedure.

[0299] The stability of the samples was evaluated at 5°C, 25°C, and 40°C for up to 1 month. After formulation locking, additional analyses were performed on selected EDTA concentrations and EDTA-free comparative samples for up to 6 months. Table 29. Test formulations [name] [Buffer] [Target] [pH] [excipient] [EDTA] [content] [Target Concentration] [(g / L)] w / o EDTA Acetate 10 mM 5.5 Sorbitol 5% PS80 0.04% 0 5.0 EDTA 1 µM Acetate 10 mM 5.5 Sorbitol 5% PS80 0.04% 1 µM 5.0 EDTA 10 µM Acetate 10 mM 5.5 Sorbitol 5% PS80 0.04% 10 µM 5.0 EDTA 50 µM Acetate 10 mM 5.5 Sorbitol 5% PS80 0.04% 50 µM 5.0

[0300] Four test formulations were stored at 5°C, 25°C, and 40°C for up to one month prior to formulation lock-in. Supplementary analyses were performed for a limited number of prototypes and analytical methods at 25°C for up to six months (T6M25°C). Table 30 describes the time points, pressure conditions, and analytical methods used in the formulation studies. Table 30. Time Points and Analysis Methods [Time point] [Purpose] [Analysis Methods] [Preparation locked] T0 Initial time point Visual inspection, pH, osmolarity, protein concentration, DAR, SEC, DLS, MFI, EDTA content, CGE, iCIEF, free drug, PS80, PTM T1M5℃ DP storage conditions Visual inspection, pH, osmolarity, protein concentration, DAR, SEC, DLS, MFI, CGE, iCIEF, free drug, PS80 T1M25℃ DP acceleration conditions Visual inspection, pH, osmolarity, protein concentration, DAR, SEC, DLS, MFI, CGE, iCIEF, free drug, PS80 T2W40℃ DP pressure conditions Visual inspection, pH, osmolarity, protein concentration, DAR, SEC, DLS, MFI, CGE, iCIEF, free drug, PS80, PTM T1M40℃ DP pressure conditions Visual inspection, pH, osmolarity, protein concentration, DAR, SEC, DLS, MFI, CGE, iCIEF, free drug, PS80, PTM Supplementary Analysis T3M5℃ Focusing on selected formulations and comparative samples without EDTA Visual inspection, pH, osmolarity, protein concentration, DAR, SEC, DLS, MFI, PS80 T3M25℃ Focusing on selected formulations and comparative samples without EDTA Visual inspection, pH, osmolarity, protein concentration, DAR, SEC, DLS, MFI, PS80 T6M25℃ Focusing on selected formulations and comparative samples without EDTA PS80

[0301] The active pharmaceutical ingredient (DS) used in this study contained 24 ng / mL of iron and 7 ng / mL of Ni. The DS thus provided was prepared in 10 mM acetate, 5% sorbitol, 0.04% PS80, pH 5.5, 5 g / L, and stored at -20°C.

[0302] method

[0303] Preparation of main packaging materials

[0304] Glass vials are manually rinsed with water for injection (WFI) and depyrogenated in an oven before filling. Stoppers are sterilized by autoclaving.

[0305] Preparation of formulation

[0306] Three EDTA stock solutions (50 μM, 500 μM, and 2500 μM) were prepared by dissolving disodium EDTA in a graduated glass flask containing WFI. The resulting solutions were filtered using a 0.22 μm sterile syringe filter.

[0307] The ADC DS were thawed, collected in 10L polycarbonate (PC) bottles, and homogenized by manually inverting the bottles. The ADC DS were then dispensed into six 2L PC bottles using a peristaltic pump at 1.5L (1.527 kg) filling rates.

[0308] The EDTA stock solution was then added to the ADC DS and homogenized by manually inverting the flask. Table 31 lists the calculated volumes of the EDTA stock solution to be added to the ADC DS to obtain the following target concentrations: no EDTA, 1 μM, 10 μM, and 50 μM. Table 31. EDTA Addition Volume [Target] [EDTA] [concentration] [EDTA 50 μM] [EDTA 500 μM] [EDTA 2500 μM] 0 μM 0 0 0 1 μM 30.6 mL 0 0 10 μM 0 30.6 mL 0 50 μM 0 0 30.6 mL

[0309] After preparation, the solution was stored at 5°C until DP filling began. The dilution factor of the initial formulation after EDTA addition was estimated to be approximately 2%.

[0310] Small bottle refill After preparation, the product is filtered in a 10L intermediate bag and then filled into 10R vials on a dedicated filling platform simulating an industrial pharmaceutical (DP) process. The filled vials are then manually plugged and crimped.

[0311] Analytical methods

[0312] Visual inspection The samples were visually examined in the pharmacopeia chamber and under an optical fiber MLC-150C from MOTIC to analyze the presence of visible particles (scoring detailed in Table 32). Table 32. Ranking of Visual Inspections [Particle Detection] [Number of particles] 0 Undetectable by cold light (fiber optics) + Countable (1≦n≦5) 1 Detected using only cold light (fiber optics) ++ Several (n>5) 2 Visible particles on the visual inspection table (European Pharmacopoeia conditions) +++ a lot of

[0313] Microflow Imaging (MFI) For MFI analysis, 1 mL of sample is required, without any dilution. The procedure is as follows: the sample is injected into the cell at a rate of 0.1 mL / min, and recording begins after a 0.2 mL purge. Measurements are performed at room temperature. Rinsing with Hellmanex 3% and MilliQ filtered water is performed before and after each measurement.

[0314] Dynamic light scattering An 80 μL solution was analyzed on a Nanosizer (Zetasizer nano-S, Malvern) using the following parameters: 3 measurements, 11 runs, 10 seconds each, and an incident angle of 173°. No dilution was required. The results are presented as the average of the three measurements.

[0315] pH The pH probe was dropped into a 2 mL sample and measured at room temperature.

[0316] osmotic pressure Osmotic pressure was measured using a freezing point osmoremeter. Measurements were repeated three times, and the results are presented as the average. Each measurement required a volume of 20 μL.

[0317] PS80 content The PS80 content was measured by gas chromatography (GC).

[0318] Results and Discussion

[0319] The results discussed below are limited to pressure conditions of 40°C and 25°C, as no differences or evolution were observed between samples at storage conditions of 5°C. Formulation locking was performed after the T1M results, and it was decided to analyze only the selected 10 μM formulation and the EDTA-free comparison sample at later time points.

[0320] Visual observation

[0321] The results of visual observation are presented in Table 33.

[0322] According to the European Pharmacopoeia method, all formulations except T2W40°C were considered to have essentially no visible particles at all pressure points. The presence of visible particles was not confirmed at T1M at 40°C and was thus attributed to the non-GMP environmental preparation.

[0323] When using the fiber-optic approach (which is more sensitive than the European Pharmacopoeia approach), some particles were observed in all formulations and at all time points, but there was no trend.

[0324] No significant differences and evolutions were observed in the presence or absence of EDTA.

[0325] pH and osmotic pressure

[0326] pH and osmotic pressure values ​​are presented in Table 34 .

[0327] In terms of pH, highlighting no evolution. The values ​​remain unchanged and meet the acceptance criteria (5.2-5.8). Evolution of osmotic pressure values ​​with time was not confirmed for all samples regardless of EDTA concentration.

[0328] Protein Concentration and DAR (UV)

[0329] Protein concentration and DAR results are presented in Table 35 .

[0330] No significant evolution over time was confirmed in protein concentration values ​​for all samples, regardless of EDTA concentration.

[0331] After T2M 40°C and T1M 40°C, the DAR values ​​of all samples decreased with time ( Figure 23 ).

[0332] In the case of samples without EDTA, more decreases were observed with time (-1.2 without EDTA at 40°C for T1M and -0.9 for the three EDTA concentrations at 40°C for T1M). This trend was confirmed at the 3M25°C time point, with more reduction in EDTA-free samples compared to 10 μM EDTA samples. Table 33. Visual observation [T0] [T2W40℃] [T1M40℃] [T1M25℃] [T3M25] [°] [C] EDTA μM 0 1 10 50 0 1 10 50 0 1 10 50 0 1 10 50 0 50 Visual observation 1++ 1++ 1++ 1++ 1+ 2+++ 2++ 2++ 1+ 1+++ 1++ 1++ 1++ 1++ 1+++ 1+++ 1++ 1+ Table 34. pH and Osmotic Pressure [T0] [T2W40℃] [T1M40℃] [T1M25℃] [T3M25] [°] [C] EDTA μM 0 1 10 50 0 1 10 50 0 1 10 50 0 1 10 50 0 50 pH 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 Osm. 267 264 282 272 270 265 282 276 269 264 282 273 264 263 283 270 267 282 Table 35. Protein Concentration and DAR [T0] [T2W40℃] [T1M40℃] [T1M25℃] [T3M25] [°] [C] EDTA μM 0 1 10 50 0 1 10 50 0 1 10 50 0 1 10 50 0 50 concentration mg / mL 4.4 4.3 4.7 4.5 4.5 4.4 4.8 4.6 4.5 4.4 4.8 4.6 4.5 4.4 4.7 4.5 4.6 4.8 DAR 3.8 3.9 3.9 3.9 3.1 3.4 3.5 3.4 2.6 3.0 3.0 3.0 3.8 3.8 3.9 3.8 3.3 3.6 Table 36. Z-mean and PDI% [T0] [T2W40℃] [T1M40℃] [T1M25℃] [T3M25] [°] [C] EDTA μM 0 1 10 50 0 1 10 50 0 1 10 50 0 1 10 50 0 50 Zav (nm) 11.2 11.2 11.2 11.3 11.3 11.2 11.3 11.4 11.3 11.2 11.2 11.2 11.1 11.0 11.2 11.0 11.4 11.2 PDI% 21.7 21.3 20.5 23.9 26.5 36.4 23.2 26.1 24.9 25.5 20.2 23.5 27.5 19.4 18.9 22.0 30.0 21.6 Table 37. MFI Results [T0] [T2W40℃] [T1M40℃] [T1M25℃] [T3M25] [°] [C] EDTA μM 0 1 10 50 0 1 10 50 0 1 10 50 0 1 10 50 0 50 ≧2 μm 329 367 406 292 229 719 1104 310 376 237 906 171 902 714 846 751 1381 1166 ≧10 μm 0 8 0 10 5 30 32 4 0 0 8 9 0 6 53 21 39 15 ≥25 μm 0 2 0 2 0 5 5 0 0 0 2 3 0 2 10 8 6 6 Table 38. Monomers and HMW [T0] [T2W40℃] [T1M40℃] [T1M25℃] [T3M25] [°] [C] EDTA μM 0 1 10 50 0 1 10 50 0 1 10 50 0 1 10 50 0 50 monomer % 96.6 96.6 96.6 96.6 95.4 96.5 96.2 96.2 93.2 95.3 94.8 95.0 97.2 97.5 97.3 97.4 96.1 96.7 HMW% 1.1 1.1 1.2 1.1 2.1 1.8 2.0 1.9 3.1 2.1 2.5 2.3 1.3 1.2 1.3 1.3 1.7 1.5 Table 39. Results of reduced cGE [T0] [T2W40℃] [T1M40] [℃] [T1M25] [℃] EDTA μM 0 1 10 50 0 1 10 50 0 1 10 50 0 1 10 50 The sum of light chains and heavy chains (corrected area%) 98.2 97.8 98.1 98.3 97.8 98.0 97.8 97.9 95.6 97.5 98.2 97.3 98.2 98.1 98.1 97.9 Table 40. Non-reducing cGE results [T0] [T2W40] [℃] [T1M40] [℃] [T1M25] [℃] EDTA μM 0 1 10 50 0 1 10 50 0 1 10 50 0 1 10 50 purity (H2L2, corrected area%) 87.1 87.1 87.3 87.2 85.2 86.9 86.9 86.6 84.8 87.2 86.7 87.0 85.0 86.1 86.2 86.0 Main fragments (RMT 0.96, corrected area%) 9.3 9.3 9.1 9.3 10.0 9.6 9.6 9.7 8.3 7.7 8.0 8.0 10.5 10.4 10.3 10.4 The sum of the other fragments (corrected area%) 3.5 3.5 3.6 3.5 4.8 3.5 3.5 3.5 6.9 5.1 5.2 5.0 4.5 3.5 3.6 3.5 Table 41. PS80 content [T0] [T2W40℃] [T1M40℃] [T1M25℃] [T3M25℃] EDTA μM 0 1 10 50 1 10 50 0 1 10 50 0 1 10 50 0 50 PS80 ppm 310 323 332 317 282 298 329 315 241 311 333 319 300 309 331 331 233 390 (Continued) [T6M25] [℃] EDTA μM 0 1 10 50 PS80 ppm 103 317 333 317 Table 42. Based on the low PI and main isotypes of IiCIEF [T0] [T2W40] [℃] [T1M40℃] [T1M25] [℃] EDTA μM 0 1 10 50 0 1 10 50 0 1 10 50 0 1 10 50 The sum of high pI homotypes (area %) 9 9 9 9 7 8 8 8 4 6 5 7 9 9 9 9 Total of main similar types (area %) 83 83 82 82 77 81 81 81 66 74 73 72 82 82 82 83 The sum of low pI isomorphic (area %) 8 8 9 8 15 11 11 10 30 20 twenty two twenty one 9 9 9 9 Table 43. Total free maytansine [T0] [T2W40] [℃] [T1M40] [℃] [T1M25] [℃] EDTA μM 0 1 10 50 0 1 10 50 0 1 10 50 0 1 10 50 Total free 0.1 0.1 0.1 0.1 1.5 1.4 1.5 1.4 2.9 2.7 2.7 2.6 0.5 0.6 0.6 0.6 Oxidizing substances - - - - - - - - + - - - - - - - Impurity A ND ND ND ND 0.25 0.07 0.10 0.09 0.69 0.41 0.43 0.41 ND ND ND ND The number of other maltannin-like impurities in LOQ 0 0 0 0 1 1 0 0 8 5 4 4 0 0 0 0 Table 44. Oxidation of Met255 and Met431 T0 T2W40 [℃] T1M40 [℃] EDTA μM 0 1 10 50 0 1 10 50 0 1 10 50 Met255 Oxidation (%) 5 4 5 4 8 5 6 5 twenty three 10 12 11 Met431 Oxidation (%) 2 2 1 1 3 2 2 2 10 4 4 4

[0333] Submicron particles (DLS)

[0334] Table 36 presents the values ​​of PdI% and Z average over time.

[0335] For Zav, no trend was observed across all time points and for all EDTA concentrations. Regarding PDI%, a slight increase was observed in most samples at the 25°C and 40°C time points. However, no clear trend was observed in EDTA content.

[0336] Subvisible particles (MFI)

[0337] Table 37 presents the results for subvisible particles.

[0338] No differences were observed between the prototypes at any time point. A very small increase in subvisible particles was observed at T3M 25℃; this increase was similar for both EDTA-free and 10 μM EDTA samples.

[0339] Based on the purity and HMW% of size exclusion chromatography (SEC)

[0340] Table 38 presents the results of SEC (monomer % and HMW %).

[0341] The percentage of monomers in all samples decreased after T1M40℃, with a faster rate in the absence of EDTA (at T1M40℃, the percentage was -3.4% for samples without EDTA and -1.3% / -1.8% for samples with EDTA, see Figure 24).

[0342] This result is confirmed by the increase in HMW% and is even higher for the EDTA-free sample (at T1M40℃, the EDTA-free sample is +2.0%, and the EDTA-containing sample is +1.0 / +1.3%, see Figure 25).

[0343] There was no significant difference in EDTA concentration (monomer % and HMW %) among the three tests.

[0344] Based on the purity and fragmentation of CGE

[0345] Tables 39 and 40 present the CGE results under reducing conditions (total percentage of light and heavy chains) and non-reducing conditions (purity, percentage of major fragments, percentage of other fragments).

[0346] In non-reducing cGE (Table 40, Figures 26 and 27), purity decreased significantly after 1 M 40 °C in the absence of EDTA, which was confirmed by the increase in fragments (the sum of all fragments). No significant evolution was observed at T2W 40 °C, T1M 25 °C, and T1M 40 °C for the three EDTA concentrations.

[0347] In the reduction of cGE (see Table 39 and Figure 28), in the absence of EDTA, the sum of light and heavy chains decreased significantly after 1M 40℃. No significant evolution was observed at T2W 40℃, T1M 25℃, and T1M 40℃ for the three EDTA concentrations.

[0348] Based on the PS80 content obtained by gas chromatography (GC)

[0349] Table 41 presents the results for PS80 content.

[0350] For all EDTA concentrations, no significant decrease in PS80 content was recorded up to T6M25℃. In the absence of EDTA, a significant decrease in PS80 was observed after T1M40℃ and T6M25℃ (see also Figure 29).

[0351] Charge variant analysis based on imaging capillary isoelectric focusing (iCIEF)

[0352] Table 42 summarizes the iCIEF results (sum of major isotype percentage, sum of high pI isotype percentage, and sum of low pI isotype percentage).

[0353] Under pressure conditions of 40°C, the EDTA-containing samples exhibited improved stability in terms of charge isotype. In the absence of EDTA, the sum of low pI isotypes increased, but the decrease in major isotypes was more pronounced (low pI: +22% without EDTA at T1M 40°C, +12-13% with EDTA. Major isotype: -17% without EDTA, -9 / -10% with EDTA; see also Figures 30 and 31). No significant difference was observed in EDTA concentration.

[0354] Free maytansin content

[0355] Table 43 presents the results of the free drug analysis.

[0356] Regarding the total amount of free drug in all samples (with and without EDTA), no significant difference was observed. However, a difference in the percentage of impurity A was observed: after 2 weeks and 1 month at 40°C, the percentage of impurity A was higher in the absence of EDTA compared to the EDTA-containing samples. The three EDTA concentrations could not be distinguished. Furthermore, in the absence of EDTA, oxidized substances appeared after 1M at 40°C, while no oxidized substances were detected in the presence of EDTA.

[0357] Post-translation modification

[0358] This section only discusses the oxidation of Met255 and Met431, as no differences were noted between formulations of other PTMs.

[0359] PTM analysis showed that the oxidation levels at positions M255 and M431 were lower in the presence of EDTA compared to the EDTA-free sample. No significant differences were observed among the three EDTA concentrations (see Table 44).

[0360] in conclusion

[0361] For all test conditions, the three EDTA concentrations provided stabilization for PS80 content and other quality properties that may be affected by the oxidation process, particularly PTM, free maytansine, cGE, iCIEF, and SEC.

[0362]

Claims

1. A pharmaceutical formulation comprising: an antibody-drug conjugate (ADC) comprising an anti-human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5) antibody conjugated to maytansine derivative 4 (DM4) via an N-succinimino-4-(2-pyridyldithio)butyric acid (SPDB) linker; wherein the antibody comprises a heavy chain variable domain (VH) of the amino acid sequence of SEQ ID NO: 6 and a light chain variable domain (VL) of the amino acid sequence of SEQ ID NO: 7, the formulation comprising the following excipients: (i) sodium acetate at a concentration of about 10 mM; (ii) sorbitol at a concentration of about 5% w / v; (iii) polysorbate 80 at a concentration of about 0.04% w / v; and (iv) disodium EDTA at a concentration between about 1 µM and about 50 µM, wherein the formulation has a pH of about 5.

5.

2. The pharmaceutical preparation as claimed in claim 1, wherein the concentration of the antibody-drug conjugate is about 5 mg / mL.

3. The pharmaceutical preparation of claim 1, wherein the concentration of the disodium EDTA is about 1 µM, about 10 µM or about 50 µM.

4. The pharmaceutical formulation of claim 1, wherein the antibody-drug conjugate has a drug-to-antibody ratio ranging from about 1 to about 10.

5. A liquid pharmaceutical formulation comprising: (i) 5 mg / mL of an antibody-drug conjugate (ADC) against human carcinoembryonic antigen-associated cell adhesion molecule 5 (hCEACAM5); (ii) 10 mM sodium acetate; (iii) 5% w / v sorbitol; (iv) 0.04% w / v polysorbate 80; and (v) 10 µM disodium EDTA, wherein the formulation has a pH of 5.5, and wherein the ADC is composed of huMAb2-3-SPDB-DM4.

6. Use of an effective amount of the pharmaceutical preparation as claimed in claim 1 or 5 for the preparation of a medicament for treating cancer.

7. As claimed in claim 6, wherein the cancer manifests as carcinoembryonic antigen-associated cell adhesion molecule 5 (CEACAM5).

8. As claimed in claim 6, wherein the cancer is a cancer characterized by high levels of carcinoembryonic antigen-associated cell adhesion molecule 5 (CEACAM5).

9. As requested in paragraph 6, wherein the cancer is selected from the group consisting of: colorectal cancer, stomach cancer, lung cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, pancreatic cancer, and bladder cancer.

10. As requested in paragraph 6, wherein the cancer is selected from the group consisting of: prostate cancer, pancreatic cancer, lung cancer, and colorectal cancer.

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