Pharmaceutical composition comprising Anti-epcam and CD3 bispecific antibody

Abstract

Provided is a pharmaceutical composition, comprising an anti-EpCAM and CD3 bispecific antibody, a buffer solution, a stabilizer, and a surfactant. A lyophilized formulation capable of stably storing the anti-EpCAM and CD3 bispecific antibody is prepared; the physical and chemical stability of the anti-EpCAM and CD3 bispecific antibody can be improved, and the lyophilized formulation can maintain stability under conditions of high-temperature acceleration, light exposure, and long-term storage, thereby improving clinical use safety.

Description

Anti-EpCAM and CD3 bispecific antibody drug composition Technical Field

[0001] This invention relates to the field of pharmaceutical preparations, specifically to a preparation containing a bispecific antibody, its preparation method, and its application. Background Technology

[0002] Tumors originating from epithelial tissue (including covering epithelium and glandular epithelium) are the most common, with malignant epithelial tumors (carcinomas) posing the greatest threat to humans. Most malignant tumors in the human body originate from epithelial tissue. As malignant tumors grow, infiltrate, and metastasize, they can cause serious adverse effects on the body, such as pain, nutrient deficiency, and organ dysfunction. When they invade the pleural or abdominal cavity, they can lead to the leakage and accumulation of tissue fluid within the pleural / abdominal cavities. Large amounts of fluid accumulation can cause abdominal pain, difficulty breathing, electrolyte imbalance, hypoproteinemia, and secondary infections, severely increasing the patient's disease burden and impacting their quality of life.

[0003] EpCAM (Epithelial Cell Adhesion Molecule) is highly expressed in tumors of epithelial origin, reaching or approaching 100% in common cancers such as esophageal, gastric, colon, prostate, lung, and ovarian cancers, making it a promising tumor targeting target. CD3 (Cluster of Differentiation 3) is the surface antigen of normal human immune cells (T cells) and the primary signal for T cell activation. Recombinant anti-EpCAM and CD3 bispecific antibodies, after entering the body, primarily bridge immune cells and tumor cells through dual-target binding, thereby activating T cells to release substances such as TNF-α, IFN-γ, perforin, and granzyme B to kill tumor cells. Recombinant anti-EpCAM and CD3 bispecific antibodies can be used to treat malignant tumors, malignant pleural effusion, and malignant ascites.

[0004] Recombinant anti-EpCAM and CD3 bispecific antibodies are more structurally complex than natural antibodies, making them more susceptible to various physical and chemical changes during preparation, storage, and use. Protein stability is influenced by intrinsic protein characteristics (e.g., amino acid sequence, glycosylation pattern) and external factors (e.g., temperature, solvent pH, excipients, interface, or shear rate). Antibody degradation is often accompanied by a decrease in monomeric peak purity, an increase in aggregates or small fragments, a decrease in antibody charge variant peaks, and an increase in acidic or basic variants, thereby affecting function, safety, and efficacy. Therefore, therapeutic antibodies require appropriate formulations and preparations to protect protein stability during production, storage, and administration. Summary of the Invention

[0005] The purpose of this invention is to provide a stable therapeutic bispecific antibody pharmaceutical composition and its uses, which can maximally inhibit the occurrence of physical and chemical changes, improve product quality and stability, maintain stability under conditions such as high temperature acceleration, light exposure and long-term storage, improve the safety of clinical use, and the reconstituted pharmaceutical composition is suitable for intravenous, subcutaneous, intramuscular, intrapleural or intraperitoneal administration, and can be used as a drug for clinical use.

[0006] This invention relates to a pharmaceutical composition comprising an anti-EpCAM and CD3 bispecific antibody, a buffer, a stabilizer, and a surfactant, wherein:

[0007] The anti-EpCAM and CD3 bispecific antibody comprises (a) a light chain-heavy chain pair that specifically binds to EpCAM, wherein the light chain is bound to the heavy chain via a disulfide bond; and (b) a fusion peptide that specifically binds to CD3, the fusion peptide comprising a single-chain variable fragment (scFv) and an Fc fragment having a CH2 domain and a CH3 domain, the fusion peptide being bound to the heavy chain via a disulfide bond, the concentration of the anti-EpCAM and CD3 bispecific antibody being about 0.01-5 mg / mL, for example about 0.05-5 mg / mL, 0.1-5 mg / mL, 0.2-5 mg / mL, 0.3-5 mg / mL, 0.4-5 mg / mL, 0.5-4.5 mg / mL, 1-4 mg / mL, 1.5-3.5 mg / mL or 2-3 mg / mL, preferably about 0.4 mg / mL or 0.5 mg / mL;

[0008] The light chain that specifically binds to EpCAM, according to the Kabat, Chothia, AHo, AbM or IMGT numbering system, contains LCDR1-3 in the variable region of the light chain shown in SEQ ID NO:16; the heavy chain that specifically binds to EpCAM contains HCDR1-3 in the variable region of the heavy chain shown in SEQ ID NO:17; and the fusion peptide that specifically binds to CD3 contains HCDR1-3 in the variable region of the heavy chain shown in SEQ ID NO:18 and LCDR1-3 in the variable region of the light chain shown in SEQ ID NO:19.

[0009] The buffer solution is selected from a buffer solution of acetic acid and acetate, a buffer solution of histidine and histidine salt, a buffer solution of citric acid and citrate, or a buffer solution of succinic acid and succinate. The concentration of the buffer solution is about 10-40 mM, preferably about 10-30 mM, 10-20 mM, 15-40 mM, 20-40 mM, 25-35 mM or 10-30 mM, more preferably about 10 mM, 15 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM or 40 mM.

[0010] The stabilizer is selected from sucrose, trehalose, sorbitol, proline, glycine, methionine, or any combination thereof, more preferably trehalose or sucrose, or a combination of trehalose (or sucrose) and proline (or glycine, methionine), preferably with a concentration of about 2%-10%, more preferably about 3%-9%, 4%-8%, 5%-7%, 5%-6%, more preferably about 5%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.06%, 5.7%, 5.8%, 5.9%, 6%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7.5%, or 8%;

[0011] The surfactant is selected from polysorbates, poloxamer, or any combination thereof, preferably polysorbates (such as polysorbate 20 or 80) or poloxamer 188, and the surfactant concentration is about 0.02-0.5% (w / v), preferably about 0.03-0.4%, 0.04-0.3%, 0.05-0.2%, 0.06-0.1%, more preferably about 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, or 0.5%.

[0012] The pH of the pharmaceutical composition is about 4.5-5.5, for example about 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5;

[0013] Preferably, the pH of the acetic acid and acetate buffer is about 4.5-5.0; the pH of the histidine and histidine salt buffer is about 5.0-5.5; the pH of the citric acid and citrate buffer is about 5.0; and the pH of the succinic acid and succinate buffer is about 5.0-5.5.

[0014] In this document, the anti-EpCAM and CD3 bispecific antibody may be included in the pharmaceutical composition in any amount between about 0.01 and 5 mg / mL, such as about 0.05-5 mg / mL, 0.1-5 mg / mL, 0.2-5 mg / mL, 0.3-5 mg / mL, 0.4-5 mg / mL, 0.5-4.5 mg / mL, 1-4 mg / mL, 1.5-3.5 mg / mL or 2-3 mg / mL, such as about 0.2 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 2 mg / mL or 5 mg / mL.

[0015] In this invention, "single-chain variable fragment" or "ScFv" refers to a fusion protein of variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin. In some aspects, these regions are linked by short linker peptides of 10 to about 25 amino acids. The linker may be glycine-rich for flexibility, or contain serine or threonine for solubility, and can link the N-terminus of the VH to the C-terminus of the VL, and vice versa. The protein retains the properties of the original immunoglobulin, except that the constant regions have been removed and linkers have been introduced. ScFv molecules are known in the art, such as those described in U.S. Patent 5,892,019.

[0016] In some embodiments, the Fc fragment of the fusion peptide having CH2 and CH3 domains is derived from IgG antibodies, such as the Fc fragments of IgG1, IgG2, IgG3, IgG4, and IgG5, for example, the fragment is derived from human IgG antibodies.

[0017] In some embodiments, in the EpCAM-specific light-heavy chain pair, the light chain includes a light chain variable region and a light chain constant region, and the heavy chain includes a heavy chain variable region, CH1, CH2, and CH3 domains. In some embodiments, a mortar-and-mortar structure is formed between the CH3 domain of the EpCAM-specific heavy chain and the CH3 domain of the CD3-specific fusion peptide.

[0018] In this invention, the term "histidine and histidine buffer" or "histidine and histidine salt buffer" refers to a solution containing histidine and histidine salt, wherein the "histidine and histidine salt buffer concentration" refers to the total concentration of histidine and histidine salt in the buffer. The histidine and histidine salt buffer may be a mixture of histidine and histidine salt, wherein the pH of the histidine and histidine salt buffer is adjusted to approximately 4.5-5.5, for example, approximately 5.0-5.5, and the final pharmaceutical composition has a pH of approximately 4.5-5.5.

[0019] In this invention, the term "acetic acid and acetate buffer" or "acetic acid and acetate buffer" refers to a solution containing acetic acid and acetate, wherein the "concentration of the acetic acid and acetate buffer" is the total concentration of acetic acid and acetate in the buffer. In one embodiment of the invention, the acetate is selected from the group consisting of sodium acetate, potassium acetate, calcium acetate, or combinations thereof. In one embodiment of the invention, the acetate used is sodium acetate. The pH of the acetic acid and acetate buffer is adjusted to about 4.5-5.5, for example, about 4.5-5.0, and the final pharmaceutical composition has a pH of about 4.5-5.5.

[0020] In this invention, the term "citric acid and citrate buffer" or "citric acid and citrate buffer" refers to a solution containing citric acid and citrate, wherein the "concentration of citric acid and citrate buffer" is the total concentration of citric acid and citrate in the buffer. In one embodiment of the invention, the citrate is selected from the group consisting of sodium citrate, calcium citrate, potassium citrate, diammonium citrate, or combinations thereof. In one embodiment of the invention, the citrate used is sodium citrate. The pH of the citric acid and citrate buffer is adjusted to approximately 5.0, and the final pharmaceutical composition has a pH of approximately 4.5-5.5.

[0021] In this invention, the term "succinic acid and succinate buffer" or "succinic acid and succinate buffer" refers to a solution containing succinic acid and succinate, wherein the "concentration of succinic acid and succinate buffer" is the total concentration of succinic acid and succinate in the buffer. In one embodiment of the invention, the succinate is selected from the group consisting of sodium succinate, potassium succinate, calcium succinate, or combinations thereof. In one embodiment of the invention, the succinate used is sodium succinate. The pH of the succinic acid and succinate buffer is adjusted to a value in the range of about 5.0-5.5, for example, about 5.5, and the final pharmaceutical composition has a pH of about 4.5-5.5.

[0022] Stabilizers are used to stabilize the structure of the active ingredient in a pharmaceutical composition and provide protective properties at room temperature and in lyophilized conditions, such as acting as cryoprotectants and desiccant agents to protect proteins from denaturation that may occur during lyophilization. Sugars and amino acids are commonly used stabilizers for protein drugs. They can also act as cryoprotectants and desiccant agents to protect proteins from denaturation that may occur during lyophilization. In one embodiment, the stabilizer is a sugar, amino acid, alcohol, or a combination thereof. In a preferred embodiment, the stabilizer is selected from sucrose, trehalose, sorbitol, proline, glycine, methionine, and combinations thereof, more preferably trehalose. Too low a stabilizer concentration can reduce the stability of the pharmaceutical composition. In a preferred embodiment, the concentration of the stabilizer in the pharmaceutical composition is about 2-10% (w / v).

[0023] In some embodiments, the pharmaceutical composition comprises any of the following as stabilizers (w / v): about 5%-10% trehalose (e.g., about 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% trehalose), or about 5%-8% sucrose (e.g., about 5%, 5%, 5%, 6.0%, 6.5%, 7%, 7.5%, or 8% sucrose), or about 2% glycine, or about 1.8% sorbitol and about 5.7% trehalose, or about 1.2% proline and about 5.7% trehalose, or about 0.3% methionine and about 5% trehalose, or about 1% glycine and about 5% trehalose, or about 5% sucrose and about 5% trehalose.

[0024] Surfactants are substances that can alter (usually reduce) the surface tension of a liquid or the interfacial tension between two phases. Surfactants are amphiphilic, containing both hydrophilic and hydrophobic groups.

[0025] In some embodiments, the pharmaceutical composition comprises any of the following as a surfactant: about 0.05%-0.1% of polysorbate 80 (Tween 80 or PS80), about 0.02%-0.1% of polysorbate 20 (Tween 20 or PS20), or about 0.05-0.5% of poloxamer 188 (P188). In one embodiment, the pharmaceutical composition comprises about 0.02%, 0.05%, or 0.1% of polysorbate 80. In one embodiment, the pharmaceutical composition comprises about 0.02%, 0.05%, or 0.1% of polysorbate 20. In one embodiment, the pharmaceutical composition comprises about 0.05% or 0.5% of poloxamer 188.

[0026] In some embodiments, the pharmaceutical composition comprises about 0.01-5 mg / mL (e.g., about 0.2-5 mg / mL) of anti-EpCAM and CD3 bispecific antibody, about 10-40 mM of histidine and histidine salt buffer, about 2%-10% of stabilizer and about 0.02%-0.1% of surfactant, and the pH of the pharmaceutical composition is about 4.5-5.5.

[0027] In some embodiments, the pharmaceutical composition comprises about 0.01-5 mg / mL (e.g., 0.2-5 mg / mL) of anti-EpCAM and CD3 bispecific antibody, about 10-40 mM of histidine and histidine salt buffer, about 2%-10% of trehalose and about 0.02%-0.1% of polysorbate 80, and the pH of the pharmaceutical composition is about 4.5-5.5.

[0028] In some embodiments, the pharmaceutical composition is selected from the group consisting of:

[0029] (1) A pharmaceutical composition comprising about 0.2 mg / mL to 5 mg / mL of anti-EpCAM and CD3 bispecific antibody, about 30 mM histidine and histidine salt buffer, about 5.0% sucrose and about 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is about 5.2;

[0030] (2) A pharmaceutical composition comprising about 0.2 mg / mL to 5 mg / mL of anti-EpCAM and CD3 bispecific antibody, about 30 mM histidine and histidine salt buffer, about 10.0% trehalose and about 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is about 5.2;

[0031] (3) A pharmaceutical composition comprising about 0.2 mg / mL to 5 mg / mL of anti-EpCAM and CD3 bispecific antibody, about 30 mM histidine and histidine salt buffer, about 5.0% sucrose, about 5.0% trehalose and about 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is about 5.2;

[0032] (4) A pharmaceutical composition comprising about 0.2 mg / mL to 5 mg / mL of anti-EpCAM and CD3 bispecific antibody, about 30 mM histidine and histidine salt buffer, about 5.0% trehalose, about 0.3% methionine and about 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is about 5.2;

[0033] (5) A pharmaceutical composition comprising about 0.5 mg / mL of anti-EpCAM and CD3 bispecific antibody, about 30 mM acetate and acetate buffer, about 5.0% trehalose and about 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is about 4.5-5.5;

[0034] (6) A pharmaceutical composition comprising about 0.5 mg / mL of anti-EpCAM and CD3 bispecific antibody, about 30 mM histidine and histidine salt buffer, about 5.0% trehalose and about 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is about 4.5-5.5, for example about 5.0-5.5, preferably about 5.2;

[0035] (7) A pharmaceutical composition comprising about 0.5 mg / mL of anti-EpCAM and CD3 bispecific antibody, about 30 mM citrate and citrate buffer, about 5.0% trehalose and about 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is about 5.0;

[0036] (8) A pharmaceutical composition comprising about 0.5 mg / mL of anti-EpCAM and CD3 bispecific antibody, about 30 mM succinic acid and succinate buffer, about 5.0% trehalose and about 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is about 5.0-5.5;

[0037] (9) A pharmaceutical composition comprising about 0.5 mg / mL of anti-EpCAM and CD3 bispecific antibody, about 30 mM histidine and histidine salt buffer, about 8.0% sucrose and about 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is about 4.5-5.5, for example about 5.0-5.5, preferably about 5.2;

[0038] (10) A pharmaceutical composition comprising about 0.5 mg / mL of anti-EpCAM and CD3 bispecific antibody, about 30 mM histidine and histidine salt buffer, about 1.0% glycine and about 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is about 4.5-5.5, for example about 5.0-5.5, preferably about 5.2;

[0039] (11) A pharmaceutical composition comprising about 0.5 mg / mL of anti-EpCAM and CD3 bispecific antibody, about 30 mM histidine and histidine salt buffer, about 1.8% sorbitol, about 5.7% trehalose and about 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is about 4.5-5.5, for example about 5.0-5.5, preferably about 5.2;

[0040] (12) A pharmaceutical composition comprising about 0.5 mg / mL of anti-EpCAM and CD3 bispecific antibody, about 30 mM histidine and histidine salt buffer, about 1.2% proline, about 5.7% trehalose and about 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is about 4.5-5.5, for example about 5.0-5.5, preferably about 5.2;

[0041] (13) A pharmaceutical composition comprising about 0.5 mg / mL of anti-EpCAM and CD3 bispecific antibody, about 30 mM histidine and histidine salt buffer, about 0.3% methionine, about 5% trehalose and about 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is about 4.5-5.5, for example about 5.0-5.5, preferably about 5.2;

[0042] (14) A pharmaceutical composition comprising about 0.5 mg / mL of anti-EpCAM and CD3 bispecific antibody, about 30 mM histidine and histidine salt buffer, about 1% glycine, about 5% trehalose and about 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is about 4.5-5.5, for example about 5.0-5.5, preferably about 5.2;

[0043] (15) A pharmaceutical composition comprising about 0.5 mg / mL of anti-EpCAM and CD3 bispecific antibody, about 30 mM histidine and histidine salt buffer, about 5% trehalose and about 0.02%-0.1% polysorbate 80 or polysorbate 20, wherein the pH of the pharmaceutical composition is about 4.5-5.5, for example about 5.0-5.5, preferably about 5.2;

[0044] (16) A pharmaceutical composition comprising about 0.5 mg / mL of anti-EpCAM and CD3 bispecific antibody, about 30 mM histidine and histidine salt buffer, about 5% trehalose and about 0.05%-0.5% poloxamer 188, wherein the pH of the pharmaceutical composition is about 4.5-5.5, for example about 5.0-5.5, preferably about 5.2;

[0045] (17) A pharmaceutical composition comprising about 0.5 mg / mL of anti-EpCAM and CD3 bispecific antibody, about 30 mM histidine and histidine salt buffer, about 2%-10% trehalose and about 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is about 4.5-5.5, for example about 5.0-5.5, preferably about 5.2;

[0046] (18) A pharmaceutical composition comprising about 0.3 mg / mL of anti-EpCAM and CD3 bispecific antibody, about 30 mM histidine and histidine salt buffer, about 1% glycine, about 5% trehalose and about 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is about 4.5-5.5, for example about 5.0-5.5, preferably about 5.2;

[0047] (19) A pharmaceutical composition comprising about 0.5 mg / mL of anti-EpCAM and CD3 bispecific antibody, about 10-40 mM histidine and histidine salt buffer, about 5% trehalose and about 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is about 4.5-5.5, for example about 5.0-5.5, preferably about 5.2;

[0048] (20) A pharmaceutical composition comprising about 0.2-5 mg / mL of anti-EpCAM and CD3 bispecific antibody, about 30 mM histidine and histidine salt buffer, about 5% trehalose and about 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is about 4.5-5.5, for example about 5.0-5.5, preferably about 5.2;

[0049] (21) A pharmaceutical composition comprising about 0.01-5 mg / mL of anti-EpCAM and CD3 bispecific antibody, about 30 mM histidine and histidine salt buffer, about 5% trehalose and about 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is about 4.5-5.5, for example about 5.0-5.5, preferably about 5.2;

[0050] (22) A pharmaceutical composition comprising about 0.05-5 mg / mL of anti-EpCAM and CD3 bispecific antibody, about 30 mM histidine and histidine salt buffer, about 5% trehalose and about 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is about 4.5-5.5, for example about 5.0-5.5, preferably about 5.2;

[0051] (23) A pharmaceutical composition comprising about 0.1-5 mg / mL of anti-EpCAM and CD3 bispecific antibody, about 30 mM histidine and histidine salt buffer, about 5% trehalose and about 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is about 4.5-5.5, for example about 5.0-5.5, preferably about 5.2;

[0052] (24) A pharmaceutical composition comprising about 0.4 mg / mL of anti-EpCAM and CD3 bispecific antibody, about 30 mM histidine and histidine salt buffer, about 5%-10% trehalose and about 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is about 4.5-5.5, for example about 5.0-5.5, preferably about 5.2.

[0053] In this invention, unless otherwise specified, all “%” represent weight / volume ratio (w / v).

[0054] The pharmaceutical compositions of the present invention may optionally contain antioxidants, which can inhibit the oxidation of the pharmaceutical formulation, improve the stability of the drug, and help extend its shelf life. Examples of antioxidants include, but are not limited to, ascorbic acid, tryptophan, methionine, glutathione, sodium thiosulfate, catalase, or any combination thereof.

[0055] The pharmaceutical compositions of the present invention may optionally contain a preservative that can inhibit the growth and reproduction of microorganisms, including but not limited to m-cresol, phenol, benzyl alcohol, benzalkonium chloride, phenoxyethanol, methylparaben, or any combination thereof.

[0056] The pharmaceutical compositions of the present invention may also contain one or more other excipients known to those skilled in the art, such as isotonic sodium chloride, metal chelating agent EDTA, etc.

[0057] The choice of pH is crucial for the stability of the pharmaceutical composition, and should ensure the stability of the corresponding active substance. Furthermore, the pH should also ensure that other components in the stock solution retain their physical and chemical properties, such as preventing degradation, before lyophilization. As a preferred embodiment, the pH of the pharmaceutical composition is about 4.5-5.5, preferably about 5.0-5.5, for example about 5.2.

[0058] In some embodiments, the anti-EpCAM and CD3 bispecific antibody comprises a variable region sequence as shown below, or an amino acid sequence having at least 85%, 90%, or 95% sequence homology with it and simultaneously binding to EpCAM and CD3:

[0059] VL (SEQ ID NO:16) of the anti-EpCAM arm:

[0060] VH (SEQ ID NO:17) of the anti-EpCAM arm:

[0061] VH against CD3 arm (SEQ ID NO:18):

[0062] VL (SEQ ID NO:19) against CD3 arm:

[0063] Those skilled in the art will understand that, based on a given variable region sequence, the CDR region sequence of an antibody can be determined using known antibody sequence numbering systems in the prior art, such as Kabat, Chothia, AbM, IMGT, or the Honegger numbering scheme (AHo). For example, for sequences contained within the heavy chain variable region, those skilled in the art can determine the sequences of HCDR1-3 regions based on known numbering systems.

[0064] In the specific implementation plan, the CDR sequence of the anti-EpCAM and CD3 bispecific antibody is as follows:

[0065] According to the IMGT numbering system

[0066] According to the Kabat numbering system

[0067] To improve the affinity, immunogenicity, stability, biological activity, or other conventional physicochemical properties of antibodies, sequences with, for example, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology to a given sequence can be screened using conventional methods. In this document, the CDR of the antibody is conserved, and other regions can be altered within the bounds of homology. The bispecific antibody is capable of heterotropically binding to EpCAM and CD3.

[0068] In some embodiments, the anti-EpCAM and CD3 bispecific antibody comprises (a) a light chain-heavy chain pair, wherein the light chain is bound to the heavy chain via a disulfide bond; and (b) a fusion peptide comprising a single-chain variable fragment (scFv) and an Fc fragment having a CH2 domain and a CH3 domain, wherein the fusion peptide is bound to the heavy chain via a disulfide bond.

[0069] Preferably, the light chain-heavy chain pair targets EpCAM, and the fusion peptide targets CD3;

[0070] Preferably, in the bispecific antibody of this application, T366 in one CH3 domain is replaced by a relatively large amino acid residue, such as tyrosine (Y) or tryptophan (W), and Y407 in the other CH3 domain is replaced by a relatively small amino acid residue, such as threonine (T), alanine (A) or valine (V). Preferably, one CH3 domain of the bispecific antibody of this application contains a T366W mutation and the other CH3 domain contains a Y407A mutation, and the mutations form a mortar-and-mortar structure pair.

[0071] Preferably, one of the CH3 domains of the bispecific antibody of this application contains one or more substitutions, which are substitutions of amino acid residues that are positively charged under physiological conditions, while the other CH3 domain contains one or more substitutions, which are substitutions of one or more amino acid residues that are negatively charged under physiological conditions. The positively charged amino acid residues may be arginine (R), histidine (H) or lysine (K), and the negatively charged amino acid residues may be aspartic acid (D) or glutamic acid (E). The amino acid residues that can be substituted include, but are not limited to, D356, L368, K392, D399 and K409. Preferably, the bispecific antibody of this application contains L368R, K392D, D399K and K409D, and these mutations form ionic bonds.

[0072] Furthermore, the anti-EpCAM and CD3 bispecific antibody preferably has a heavy chain of SEQ ID NO:1, a light chain of SEQ ID NO:2, and a single chain of SEQ ID NO:3.

[0073] In some embodiments, the pharmaceutical composition comprises: 30 mM acetate and acetate buffer (e.g., acetate and sodium acetate trihydrate buffer) at pH 4.5, 5.0, or 5.5, 5% trehalose, 0.05% polysorbate 80, and 0.5 mg / mL anti-EpCAM and CD3 bispecific antibody, said antibody comprising the heavy chain of SEQ ID NO:1, the light chain of SEQ ID NO:2, and the single chain of SEQ ID NO:3.

[0074] In some embodiments, the pharmaceutical composition comprises: a 30 mM histidine and histidine salt buffer (e.g., histidine and histidine hydrochloride buffer) at pH 5.0, 5.5, or 6.0, 5% trehalose, 0.05% polysorbate 80, and a 0.5 mg / mL anti-EpCAM and CD3 bispecific antibody, said antibody comprising the heavy chain of SEQ ID NO:1, the light chain of SEQ ID NO:2, and the single chain of SEQ ID NO:3.

[0075] In some embodiments, the pharmaceutical composition comprises: 30 mM citrate and citrate buffer (e.g., citrate and sodium citrate buffer) at pH 5.0, 5% trehalose, 0.05% polysorbate 80, and 0.5 mg / mL anti-EpCAM and CD3 bispecific antibody, said antibody comprising the heavy chain of SEQ ID NO:1, the light chain of SEQ ID NO:2, and the single chain of SEQ ID NO:3.

[0076] In some embodiments, the pharmaceutical composition comprises: 30 mM succinic acid and succinate buffer (e.g., succinic acid and sodium succinate hexahydrate buffer) at pH 5.0 or 5.5, 5% trehalose, 0.05% polysorbate 80, and 0.5 mg / mL anti-EpCAM and CD3 bispecific antibody, said antibody comprising the heavy chain of SEQ ID NO:1, the light chain of SEQ ID NO:2, and the single chain of SEQ ID NO:3.

[0077] In some embodiments, the pharmaceutical composition comprises 0.5 mg / mL anti-EpCAM and CD3 bispecific antibody, 0.05% polysorbate 80, 30 mM histidine and histidine salt buffer (e.g., histidine and histidine hydrochloride buffer), and selected from any of the following: 5% trehalose, or 8% sucrose, or 2.0% glycine, or 1.8% sorbitol and 5.7% trehalose, or 1.2% proline and 5.7% trehalose, or 0.3% methionine and 5% trehalose, or 1% glycine and 5% trehalose, wherein the antibody comprises the heavy chain of SEQ ID NO:1, the light chain of SEQ ID NO:2, and the single chain of SEQ ID NO:3, and the pH of the pharmaceutical composition is 5.2.

[0078] In some embodiments, the pharmaceutical composition comprises 0.5 mg / mL anti-EpCAM and CD3 bispecific antibody, 5% trehalose, 30 mM histidine and histidine salt buffer (e.g., histidine and histidine hydrochloride buffer), and selected from any of the following: 0.02% polysorbate 80, or 0.05% polysorbate 80, or 0.1% polysorbate 80, 0.02% polysorbate 20, or 0.05% polysorbate 20, or 0.1% polysorbate 20, or 0.05% poloxamer 188, or 0.5% poloxamer 188, wherein the antibody comprises the heavy chain of SEQ ID NO:1, the light chain of SEQ ID NO:2, and the single chain of SEQ ID NO:3, and the pH of the pharmaceutical composition is 5.2.

[0079] In some embodiments, the pharmaceutical composition comprises 0.5 mg / mL anti-EpCAM and CD3 bispecific antibody, 0.05% polysorbate 80, 30 mM histidine and histidine salt buffer (e.g., histidine and histidine hydrochloride buffer), and 5%-10% trehalose (e.g., 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% trehalose), wherein the antibody comprises the heavy chain of SEQ ID NO:1, the light chain of SEQ ID NO:2, and the single chain of SEQ ID NO:3, and the pH of the pharmaceutical composition is 5.2.

[0080] In some embodiments, the pharmaceutical composition comprises 0.5 mg / mL anti-EpCAM and CD3 bispecific antibody, 0.05% polysorbate 80, 30 mM histidine and histidine salt buffer (e.g., histidine and histidine hydrochloride buffer), and 5% trehalose, wherein the antibody comprises the heavy chain of SEQ ID NO:1, the light chain of SEQ ID NO:2, and the single chain of SEQ ID NO:3, and the pH of the pharmaceutical composition is 4.5-5.5, for example 4.5, 5.0, 5.2, or 5.5.

[0081] In some embodiments, the pharmaceutical composition comprises 0.5 mg / mL of anti-EpCAM and CD3 bispecific antibody, 0.05% polysorbate 80, 5% trehalose, and 10 mM-50 mM (e.g., 10 mM, 20 mM, 30 mM, 40 mM, or 50 mM) histidine and histidine salt buffer (e.g., histidine and histidine hydrochloride monohydrate buffer), wherein the antibody comprises the heavy chain of SEQ ID NO:1, the light chain of SEQ ID NO:2, and the single chain of SEQ ID NO:3, and the pH of the pharmaceutical composition is 4.5-5.5, for example 4.5, 5.0, 5.2, or 5.5.

[0082] In some embodiments, the pharmaceutical composition comprises 0.2 mg / mL to 5 mg / mL (e.g., 0.2 mg / mL, 0.5 mg / mL, 2 mg / mL, 5 mg / mL) of anti-EpCAM and CD3 bispecific antibody, 0.05% polysorbate 80, 5% trehalose, and 30 mM histidine and histidine salt buffer (e.g., histidine and histidine hydrochloride buffer), wherein the antibody comprises the heavy chain of SEQ ID NO:1, the light chain of SEQ ID NO:2, and the single chain of SEQ ID NO:3, and the pH of the pharmaceutical composition is 4.5-5.5, for example 4.5, 5.0, 5.2, or 5.5.

[0083] In some embodiments, the pharmaceutical composition comprises 0.5 mg / mL anti-EpCAM and CD3 bispecific antibody, 0.05% polysorbate 80, 5.0% trehalose, and 30 mM histidine and histidine salt buffer (e.g., histidine and histidine hydrochloride buffer), wherein the antibody comprises the heavy chain of SEQ ID NO:1, the light chain of SEQ ID NO:2, and the single chain of SEQ ID NO:3, and the pH of the pharmaceutical composition is 5.2.

[0084] In some embodiments, the pharmaceutical composition comprises 0.2 mg / mL to 5 mg / mL (e.g., 0.2 mg / mL, 0.5 mg / mL, 2 mg / mL, 5 mg / mL) of anti-EpCAM and CD3 bispecific antibody, 30 mM histidine and histidine salt buffer (e.g., histidine and histidine hydrochloride buffer), 0.05% polysorbate 80, and selected from any one of: 5.0% sucrose or 10.0% trehalose or 5.0% sucrose and 5.0% trehalose or 5.0% trehalose and 0.3% methionine, wherein the antibody comprises the heavy chain of SEQ ID NO:1, the light chain of SEQ ID NO:2, and the single chain of SEQ ID NO:3, and the pH of the pharmaceutical composition is 5.2.

[0085] In some embodiments, the pharmaceutical composition comprises 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, or 0.2 mg / mL anti-EpCAM and CD3 bispecific antibody, 30 mM histidine and histidine salt buffer (e.g., histidine and histidine hydrochloride buffer), 0.05% polysorbate 80, and 5.0% trehalose, wherein the antibody comprises the heavy chain of SEQ ID NO:1, the light chain of SEQ ID NO:2, and the single chain of SEQ ID NO:3, and the pH of the pharmaceutical composition is 5.2.

[0086] In this document, the final pH of the pharmaceutical composition is 4.5-5.5, for example 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5.

[0087] In some embodiments, the heavy chain terminus of the anti-EpCAM and CD3 bispecific antibody is free of lysine and / or the fusion peptide terminus of the anti-EpCAM and CD3 bispecific antibody is free of lysine.

[0088] In some embodiments, the pharmaceutical composition is a lyophilized formulation or an aqueous formulation. When it is a lyophilized formulation, the concentration of each component in the pharmaceutical composition is the same as the component concentration in the solution before lyophilization, or the component concentration in the solution after reconstitution of the lyophilized formulation.

[0089] In some embodiments, the pharmaceutical composition as described in any of the foregoing is in a form suitable for intravenous, intraperitoneal, intrapleural, urethral, ​​subcutaneous, or intramuscular injection.

[0090] This disclosure also provides a method for inhibiting the formation of aggregates and / or charge heterogeneous components in a bispecific antibody containing anti-EpCAM and CD3, comprising adding a buffer to the antibody, the buffer being selected from acetate and acetate buffer, histidine and histidine salt buffer, citrate and citrate buffer, or succinate and succinate buffer, preferably with a concentration of about 10-40 mM and a pH of 4.5-5.5, preferably, the pH of the acetate and acetate buffer is 4.5-5.0; the pH of the histidine and histidine salt buffer is 5.0-5.5; the pH of the citrate and citrate buffer is 5.0; and the pH of the succinate and succinate buffer is 5.0-5.5, wherein the anti-EpCAM and CD3 bispecific antibody is as defined herein, and the concentration of the anti-EpCAM and CD3 bispecific antibody is about 0.01-5 mg / mL.

[0091] This disclosure also provides a kit comprising at least one container, each container independently containing an anti-EpCAM and CD3 bispecific antibody, a buffer, a stabilizer, or a surfactant as defined in any of the preceding aspects.

[0092] In some embodiments, the present invention also provides a method for preparing a lyophilized powder of an anti-EpCAM and CD3 bispecific antibody pharmaceutical composition, wherein the anti-EpCAM and CD3 bispecific antibody pharmaceutical composition, as defined above, comprises the following steps: mixing the components of the anti-EpCAM and CD3 bispecific antibody pharmaceutical composition; sterile filtration (e.g., through a 0.22 μm PES filter membrane); and freeze-drying in a lyophilizer, the freeze-drying comprising pre-freezing, primary drying, and secondary drying. In some embodiments, the pharmaceutical composition is prepared as a lyophilized powder.

[0093] The features mentioned above in this invention, or the features mentioned in the embodiments, can be combined arbitrarily. All features disclosed in this specification can be used in any compositional form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features. Attached Figure Description

[0094] Figure 1. Schematic diagram of the structure of recombinant anti-EpCAM and CD3 bispecific antibody, wherein the antibody comprises (a) a specific light chain-heavy chain pair against EpCAM and (b) a fusion peptide against CD3, the fusion peptide comprising a light chain variable region (VL), a heavy chain variable region (VH), and CH2 and CH3 domains of the anti-CD3 antibody. Detailed Implementation

[0095] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any methods and materials similar to or equivalent to those described herein may be applied to the methods of the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0096] Example 1. Antibody Preparation

[0097] Recombinant anti-EpCAM and CD3 bispecific antibodies were prepared according to Examples 1 and 2 in WO2023087255A1. The bispecific antibodies comprised an A chain, a B chain, and a C chain, with the specific amino acid sequences as follows:

[0098] Chain A: Antibody heavy chain targeting EpCAM

[0099] B chain: Antibody light chain targeting EpCAM

[0100] C-chain: Single-chain antibody targeting CD3

[0101] Those skilled in the art can prepare lyophilized formulations of bispecific antibodies based on the disclosure herein. It is understood by those skilled in the art that any method for removing moisture from the pharmaceutical compositions of this application is applicable to this application.

[0102] The analytical methods for the formulation are as follows:

[0103] 1. Purity determination: Size exclusion chromatography (SEC-HPLC)

[0104] Polymers in the sample were detected by size exclusion chromatography (SEC-HPLC) using a G3000 SWXL (TOSOH) column. Citrate buffer (50 mmol / L, pH 6.0) containing 100 mmol / L sodium chloride was used as the mobile phase, and the sample was eluted isocratically using a Waters e2695 high-performance liquid chromatograph (detection wavelength: 280 nm, flow rate: 0.5 mL / min, column temperature: 25 °C).

[0105] Among the detected peaks, the peak with the largest area and height is identified as the main peak. Peaks detected earlier than monomers are collectively referred to as polymers or high molecular weight species (HMWS). The peak area percentages of polymer and monomer peaks are recorded.

[0106] This method is more sensitive to the detection of polymers; if the drug aggregates, the proportion of polymer peaks will increase.

[0107] 2. Purity testing: CE-SDS non-reduction purity testing method

[0108] Sample purity was determined by capillary electrophoresis (CE-SDS) using an uncoated capillary column (total length 30.2 cm, effective length 20 cm, inner diameter 50 μm, outer diameter 375 μm). The filling solution was SDS-MW Gel Buffer (pH 8, 0.2% SDS), and the buffer solution was SDS-MW Sample Buffer (0.1 mol / L Tris-HCl, 1% SDS, pH 9.0). Separation and detection were performed using capillary electrophoresis (capillary temperature 25℃, sample chamber temperature 8℃, detection wavelength 214 nm).

[0109] Record the retention time and peak area ratio of each peak in the sample, and report the peak area percentage of the main peak and the peak area percentage of other peaks.

[0110] This method is more sensitive to the detection of small fragments, and the proportion of small fragments will increase if the drug degrades.

[0111] 3. Purity Testing: CE-SDS Reduction Purity Testing Method

[0112] Sample purity was determined by capillary electrophoresis (CE-SDS) using an uncoated capillary column (total length 30.2 cm, effective length 20 cm, inner diameter 50 μm, outer diameter 375 μm). The filling solution was SDS-MW Gel Buffer (pH 8, 0.2% SDS), and the buffer solution was SDS-MW Sample Buffer (0.1 mol / L Tris-HCl, 1% SDS, pH 9.0). Separation and detection were performed using capillary electrophoresis (capillary temperature 25℃, sample chamber temperature 8℃, detection wavelength 214 nm).

[0113] This method uses SDS to reduce recombinant anti-EpCAM and CD3 bispecific antibodies into anti-EpCAM light chain, anti-EpCAM heavy chain, and anti-CD3 single chain. The reduced proteins are separated by capillary electrophoresis, and the retention time and peak area ratio of each sample are recorded. The purity (the sum of the three peak areas obtained after reduction of recombinant anti-EpCAM and CD3 bispecific antibodies) and the peak area percentage of other peaks are reported.

[0114] When proteins undergo abnormal degradation or other chemical reactions, impurity peaks may appear in addition to the three theoretically reduced peaks. This method can be used to determine whether impurity peaks are generated or increased.

[0115] 4. Detection of charge variants: Weak cation exchange chromatography (WCX-HPLC)

[0116] Charge variants in the samples were detected by weak cation exchange chromatography (WCX-HPLC) using a weak cation exchange column (TSKgel CM-STAT column, 7 μm, 4.6 mm × 10 cm, TOSOH), with 20 mmol / L NaH₂PO₄ (pH 6.4) and 20 mmol / L NaH₂PO₄ + 500 mmol / L sodium chloride (pH 6.4) as mobile phases. Separation was achieved using a Waters e2695 HPLC system (detection wavelength: 280 nm, flow rate: 1.0 mL / min, column temperature: 45 °C) with gradient elution.

[0117] This method mainly detects the acidity, alkalinity, and main peak ratio of different charges. If the drug is not stored stably, the ratio of each peak may change.

[0118] 5. Moisture Detection Methods

[0119] The test was conducted according to the 2020 edition of the Chinese Pharmacopoeia, Part III (General Chapter 0832, Method I). A Metrohm 916 Ti-Touch automatic potentiometric titrator was used for the test. After calibrating the Karl Fischer reagent titer, samples were taken for analysis.

[0120] Result calculation:

[0121] Where: S is the volume of Karl Fischer reagent consumed by the test sample, in mL;

[0122] F is the water equivalent factor of the reagent (i.e., the titer of the standardization), in mg / mL;

[0123] W represents the weight of the test sample, expressed in mg.

[0124] 6. Insoluble microparticles

[0125] Insoluble particulate matter was detected using the light obscuration method according to the Chinese Pharmacopoeia (2020 Edition, Part III, General Chapter 0903), and the number of insoluble particulate matter ≥10μm and ≥25μm in each sample was reported. Standard criteria: For injection solutions with a labeled volume of 100mL or more, each mL should contain no more than 25 particles of 10μm or larger and no more than 3 particles of 25μm or larger; for injection solutions with a labeled volume of less than 100mL, each injection solution container should contain no more than 6000 particles of 10μm or larger and no more than 600 particles of 25μm or larger. The labeled volume of the injection solution of this invention is less than 100mL.

[0126] 7. Detection methods for relative antibody binding activity

[0127] The relative binding activity of the antibodies was detected by indirect ELISA using a microplate reader. This method uses CD3 and EpCAM antigens to capture the dual antibodies, which are then bound to rabbit anti-human IgG-HRP. After color development, the OD reading is terminated. 450 nm value. A 4-PL curve was automatically generated by plotting the concentrations (ng / mL) of a series of graded dilutions of the reference / test sample on the x-axis and the absorbance values ​​of the reference / test sample on the y-axis, to obtain the EC50 values ​​of the test sample and the reference sample. 50 The relative binding activity of the test sample is calculated using the EC value of the reference sample. Relative binding activity of the test sample = EC value of the reference sample. 50 Value (ng / mL) / EC of test sample 50 Value (ng / mL) × 100%.

[0128] 8. Methods for detecting antibody biological activity

[0129] Antibody biological activity was detected using a reporter gene assay with an ELISA reader. HCT116 cells were adjusted to the appropriate cell density and seeded onto cell culture plates. After incubation overnight at 37°C with 5% CO2, the supernatant was discarded. Serially diluted test / reference samples and Jurkat-NFAT cells adjusted to the appropriate cell density were added to the cell culture plates and incubated at 37°C with 5% CO2 for 6 hours. Luciferase assay solution was added for color development, and the plates were read using a chemiluminescence immunoassay reader. The final concentrations of the reference / test sample (pg / mL) were plotted on the x-axis, and the chemiluminescence intensity (RLU) was plotted on the y-axis. A four-parameter nonlinear (4-PL) regression was performed to fit a dose-response curve, yielding the EC50 values ​​of the test and reference samples. 50 The value is used to calculate the biological activity of the test sample. The biological activity of the test sample = EC of the reference sample. 50 Value (pg / mL) / EC of test sample 50 Value (pg / mL) × 100%.

[0130] 9. Appearance inspection methods

[0131] Take at least one sample bottle and examine it under natural light against a white and black background. Then, reconstitute the sample with the specified solvent in the specified volume and examine it under natural light against a white and black background.

[0132] 10. Methods for testing clarity

[0133] Referring to the 2020 edition of the Chinese Pharmacopoeia, Part III (General Chapter 0902, Method II), a turbidimeter was used for detection. A small drop of dimethyl silicone oil was placed on the outer wall of the sample vial and allowed to flow to the bottom. The dimethyl silicone oil was then spread evenly with a lint-free paper towel, and excess oil was wiped away, leaving a thin layer of dimethyl silicone oil on the sample vial without leaving any silicone oil residue. The vial was then allowed to stand for 2 minutes. 10 mL of the sample mixture was added to the sample vial up to the white line mark. The sample was then mixed thoroughly using a vortex mixer (20 seconds). After standing for 5 minutes, the turbidimeter was used for detection. The turbidity value 1 was recorded. The sample vial was removed, and its outer wall was wiped with a lint-free paper towel. It was then placed back in the vial for a second reading, and the turbidity value 2 was recorded. The average of the two turbidity values ​​was reported, and it should not exceed 6 NTU.

[0134] Example 2. Effect of buffer solution on anti-EpCAM and CD3 bispecific antibody formulation

[0135] Following the formulations in Table 1, stock solutions containing different buffers (selected from 30 mM acetate and acetate buffer (acetic acid and sodium acetate trihydrate, pH 4.5-5.5), 30 mM histidine and histidine salt buffer (specifically histidine and histidine hydrochloride monohydrate, pH 5.0-6.5), 30 mM citric acid and citrate buffer (citric acid and sodium citrate, pH 5.0-6.0), 30 mM phosphate buffer (disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate monohydrate, pH 6.5-7.5) or 30 mM succinic acid and succinate buffer (succinic acid and sodium succinate hexahydrate, pH 5.0-5.5)) and 0.5 mg / mL bispecific antibody were prepared. Each formulation also contained 5% trehalose (Tre) and 0.05% polysorbate 80 (PS80). The lyophilized formulations were freeze-dried using standard methods, and their high-temperature stability at 40 °C was assessed using SEC-HPLC and WCX-HPLC.

[0136] Table 1: Bispecific antibody formulations containing different buffer solutions

[0137] The SEC-HPLC and WCX-HPLC results of the lyophilized formulations are shown in Table 2. For each formulation, the purity of the main peak detected by SEC on day 0 after lyophilization was significantly reduced in the stock solutions containing phosphate buffer or citrate and citrate buffer at pH 5.5 and pH 6.0, indicating that the bispecific antibodies are highly unstable in phosphate buffer or citrate and citrate buffer at pH 5.5 and pH 6.0. The main peak purity of 30mM acetate and acetate buffer (pH 4.5-5.0), 30mM histidine and histidine salt buffer (pH 5.0-6.0), 30mM citrate and citrate buffer (pH 5.0), and 30mM succinate and succinate buffer (pH 5.0-5.5) showed smaller reductions, exhibiting good stability. Among these, the 30mM histidine and histidine salt buffer (pH 5.0-5.5) showed good stability in both SEC-HPLC and WCX-HPLC detections, indicating good physical and chemical stability.

[0138] Table 2: SEC-HPLC and WCX-HPLC detection results of different bispecific antibody formulations

[0139] N / A: The purity of the main peak of the sample by SEC-HPLC decreased significantly after the solution was changed, and its subsequent stability was not further investigated.

[0140] Example 3. Effect of stabilizers on anti-EpCAM and CD3 bispecific antibody formulations

[0141] According to the formulation in Table 3, solutions containing 30 mM histidine and histidine salt buffer, 0.05% PS80 and different stabilizers of bispecific antibodies (antibody concentration of 0.5 mg / mL) were prepared at pH 5.2, lyophilized, and the high-temperature stability of the lyophilized formulation was investigated at 40 °C. The protective effect of different stabilizers on bispecific antibodies was evaluated by SEC-HPLC and WCX-HPLC.

[0142] Table 3: Bispecific antibody formulations containing different stabilizers

[0143] The results are shown in Table 4. Formulations containing 2.7% arginine hydrochloride or 5% sorbitol showed a collapsed appearance after lyophilization. Formulations containing mannitol showed a significant decrease in the purity of the main peak after lyophilization at 40°C, as detected by SEC-HPLC. Other formulations containing trehalose, sucrose, or combinations of trehalose with sorbitol, proline, methionine, and glycine showed good appearance and stable main peak proportions after lyophilization.

[0144] Table 4: SEC-HPLC and WCX-HPLC detection results of different bispecific antibody formulations

[0145] Example 4. Effects of surfactant type and concentration on anti-EpCAM and CD3 bispecific antibody preparations

[0146] Following the formulations in Table 5, bispecific antibody preparations containing different surfactants were prepared (antibody concentration: 0.5 mg / mL). After lyophilization, the high-temperature stability at 40°C, appearance, SEC-HPLC, and changes in insoluble particles were tested.

[0147] Table 5: Bispecific antibody formulations containing different surfactants

[0148] The results are shown in Table 6. A few protein particles appeared after reconstitution of the lyophilized formulations of bispecific antibodies without surfactants. Formulations containing higher concentrations of surfactants, such as 0.6% PS80 or 0.6% PS20, showed a decrease in the purity of the main peak after lyophilization at 40°C. Lyophilized formulations containing 0.02%-0.1% PS80, 0.02%-0.1% PS20, or 0.05%-0.5% poloxamer 188 showed better overall appearance and stability.

[0149] Table 6: Effects of different surfactants on bispecific antibody formulations

[0150] Example 5. Effect of trehalose concentration on bispecific antibody preparations

[0151] Following the formulations in Table 7, bispecific antibody preparations containing different concentrations of trehalose were prepared (antibody concentration: 0.5 mg / mL). After lyophilization, the high-temperature stability of the lyophilized preparations at 40°C was investigated, and changes in moisture content, SEC-HPLC, and WCX-HPLC were measured.

[0152] Table 7: Bispecific antibody preparations containing different concentrations of trehalose

[0153] The results are shown in Table 8. The lyophilized recombinant bispecific antibody met the moisture requirements within the range of 2%-10% trehalose, and other formulations were within acceptable ranges. SEC purity and charge isomers maintained good stability.

[0154] Table 8: Effects of different concentrations of trehalose on bispecific antibody preparations

[0155] Example 6. Verification of the effect of pH 4.5-6.0 on the stability of anti-EpCAM and CD3 bispecific antibodies.

[0156] Antibody formulations were prepared according to the formulas in Table 9, containing 0.5 mg / mL bispecific antibody, 30 mM histidine and histidine salt buffer, 5.0% trehalose, and 0.05% polysorbate 80, with pH values ​​of 4.5, 5.0, 5.2, 5.5, and 6.0, respectively. After lyophilization, the high-temperature stability of the lyophilized formulation at 40 °C, SEC-HPLC, and charge isomer and relative binding activity were investigated.

[0157] Table 9: Bispecific antibody preparations at different pH values

[0158] The results are shown in Table 10. The bispecific antibody showed good stability in the pH range of 4.5-5.5. The formulation with pH 6.0 showed a decreasing trend of SEC monomer, and the relative binding activity of the EpCAM terminal increased by 108% compared with day 0, which was a significant change. Other formulations were relatively stable.

[0159] Table 10: Effect of pH on bispecific antibody preparations

[0160] Example 7. Effects of histidine and histidine saline concentrations on the stability of anti-EpCAM and CD3 bispecific antibodies.

[0161] Antibody formulations were prepared according to the formula in Table 11, containing 0.5 mg / mL bispecific antibody, 5.0% trehalose, 0.05% PS80, pH 5.2, and histidine and histidine salt buffer concentrations of 10 mM, 20 mM, 30 mM, 40 mM, and 50 mM, respectively. After lyophilization, the high-temperature stability of the lyophilized formulation at 40 °C was investigated, and the non-reducing purity was determined by SEC-HPLC and CE-SDS.

[0162] Table 11: Bispecific antibody preparations with different histidine and histidine saline concentrations

[0163] The results are shown in Table 12. After being placed at 40℃ for 4 weeks, compared with day 0, the monomer purity showed no significant decreasing trend with time in terms of SEC-HPLC.

[0164] Table 12: Effects of different histidine and histidine saline concentrations on bispecific antibody preparations

[0165] Example 8. Effect of antibody content on the stability of anti-EpCAM and CD3 bispecific antibodies

[0166] Antibody formulations were prepared according to the formulas in Table 13, containing 0.2 mg / mL, 0.5 mg / mL, 2 mg / mL, or 5 mg / mL bispecific antibodies, 30 mM histidine and histidine salt buffer, 5.0% trehalose, 0.05% PS80, and pH 5.2. After lyophilization, the high-temperature stability of the lyophilized formulations at 40 °C was investigated, and SEC-HPLC and charge isomers were detected.

[0167] Table 13: Bispecific antibody preparations with different antibody concentrations

[0168] The results are shown in Table 14. The anti-EpCAM and CD3 bispecific antibodies exhibited good stability in the range of 0.2-5 mg / mL.

[0169] Table 14: Effect of different antibody concentrations on bispecific antibody preparations

[0170] Example 9. Stability of anti-EpCAM and CD3 bispecific antibody preparation under accelerated high temperature irradiation at 40°C and long-term storage at 2-8°C.

[0171] A formulation containing 0.5 mg / mL anti-EpCAM and CD3 bispecific antibody, 30 mM histidine and histidine hydrochloride buffer, 5.0% trehalose, 0.05% PS80, and pH 5.2 was prepared. After lyophilization, the stability of the lyophilized formulation was investigated after standing at 40℃ for 4 weeks, under light for 12 days, at 25℃ for 6 months, and at 2-8℃ for 12 months.

[0172] The results are shown in Tables 15-18. The lyophilized formulations of the anti-EpCAM and CD3 bispecific antibodies exhibited excellent stability at 40°C, under light, 25°C, and 2-8°C.

[0173] Table 15: Stability of bispecific antibody preparations under high temperature (40℃±2℃) conditions

[0174] Table 16: Light conditions (25℃±2℃, visible light not less than 1.2×10⁻⁶ after 12 days of irradiation) 6 lux.hr and UV energy not less than 200w.hr / m 2 Stability of bispecific antibody preparations

[0175] N / A: Not detected

[0176] Table 17: Accelerated stability study (25℃±2℃) from 0 to 6 months

[0177] Table 18: Results of long-term (5℃±3℃) stability study from 0 to 12 months

[0178] Example 10. Other formulations

[0179] The stable pharmaceutical formulation provided by this invention comprises an anti-EpCAM and CD3 bispecific antibody (preferably 0.2 mg / mL-5 mg / mL, such as 0.5 mg / mL); and a combination of any of the following:

[0180] (1) 30mM histidine and histidine salt buffer, 5.0% sucrose, 0.05% Tween 80, pH 5.2;

[0181] (2) 30mM histidine and histidine salt buffer, 10.0% trehalose, 0.05% Tween 80, pH 5.2;

[0182] (3) 30mM histidine and histidine salt buffer, 5.0% sucrose, 5.0% trehalose, 0.05% Tween 80, pH 5.2;

[0183] (4) 30mM histidine and histidine salt buffer, 5.0% trehalose, 0.3% methionine, 0.05% Tween 80, pH 5.2.

[0184] The detection steps of Example 9 were repeated, and similar results were obtained in all cases.

[0185] Example 11. Effect of other antibody contents on the stability of anti-EpCAM and CD3 bispecific antibodies

[0186] Antibody formulations were prepared according to the formulas in Table 19, containing 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, and 0.2 mg / mL bispecific antibodies, 30 mM histidine and histidine salt buffer, 5.0% trehalose, 0.05% PS80, and pH 5.2. After lyophilization, the high-temperature stability of the lyophilized formulations at 40 °C was investigated, and SEC-HPLC and charge isomers were detected.

[0187] Table 19: Bispecific antibody preparations with different antibody concentrations

[0188] The results showed that the anti-EpCAM and CD3 bispecific antibodies exhibited good stability in the range of 0.01-0.2 mg / mL.

[0189] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A pharmaceutical composition comprising an anti-EpCAM and CD3 bispecific antibody, a buffer, a stabilizer, and a surfactant, wherein: The anti-EpCAM and CD3 bispecific antibody comprises (a) a light chain-heavy chain pair that specifically binds to EpCAM, wherein the light chain is bound to the heavy chain via a disulfide bond; and (b) a fusion peptide that specifically binds to CD3, the fusion peptide comprising a single-chain variable fragment (scFv) and an Fc fragment having a CH2 domain and a CH3 domain, the fusion peptide being bound to the heavy chain via a disulfide bond, and the concentration of the anti-EpCAM and CD3 bispecific antibody is approximately 0.01-5 mg / mL; The light chain that specifically binds to EpCAM, according to the Kabat, Chothia, AHo, AbM or IMGT numbering system, contains LCDR1-3 in the variable region of the light chain shown in SEQ ID NO:16; the heavy chain that specifically binds to EpCAM contains HCDR1-3 in the variable region of the heavy chain shown in SEQ ID NO:17; and the fusion peptide that specifically binds to CD3 contains HCDR1-3 in the variable region of the heavy chain shown in SEQ ID NO:18 and LCDR1-3 in the variable region of the light chain shown in SEQ ID NO:

19. The buffer solution is selected from a buffer solution of acetic acid and acetate, a buffer solution of histidine and histidine salt, a buffer solution of citric acid and citrate, or a buffer solution of succinic acid and succinate. The concentration of the buffer solution is about 10-40 mM, preferably about 10-30 mM, 10-20 mM, 15-40 mM, 20-40 mM, 25-35 mM or 10-30 mM, more preferably about 10 mM, 15 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM or 40 mM. The stabilizer is selected from sucrose, trehalose, sorbitol, proline, glycine, methionine, or any combination thereof, more preferably trehalose or sucrose, or a combination of trehalose (or sucrose) and sorbitol (or proline, glycine, or methionine). Preferably, the concentration of the sucrose or trehalose is about 2%-10%, more preferably about 3%-9%, 4%-8%, 5%-7%, 5%-6%, more preferably about 5%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.06%, 5.7%, 5.8%, 5.9%, 6%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7.5%, or 8%. The surfactant is selected from polysorbates, poloxamer, or any combination thereof, preferably polysorbates (such as polysorbate 20 or 80) or poloxamer 188, and the surfactant concentration is about 0.02%-0.5%. The pH of the pharmaceutical composition is about 4.5-5.5, for example about 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.

5.

2. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition comprises about 0.01-5 mg / mL, for example about 0.2-5 mg / mL, of an anti-EpCAM and CD3 bispecific antibody, about 10-40 mM of histidine and histidine salt buffer, about 2%-10% of a stabilizer, about 0.02%-0.1% of a surfactant, and the pH of the pharmaceutical composition is about 4.5-5.

5.

3. The pharmaceutical composition according to any one of claims 1-2, wherein the pharmaceutical composition comprises about 0.01-5 mg / mL, for example about 0.2-5 mg / mL, of an anti-EpCAM and CD3 bispecific antibody, about 10-40 mM of histidine and histidine salt buffer, about 2%-10% of trehalose, about 0.02%-0.1% of polysorbate 80, and the pH of the pharmaceutical composition is about 4.5-5.

5.

4. The pharmaceutical composition according to any one of claims 1-3, wherein the pharmaceutical composition is selected from the group consisting of: (1) A pharmaceutical composition comprising 0.2 mg / mL to 5 mg / mL of anti-EpCAM and CD3 bispecific antibody, 30 mM histidine and histidine salt buffer, 5.0% sucrose and 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is 5.2; (2) A pharmaceutical composition comprising 0.2 mg / mL to 5 mg / mL of anti-EpCAM and CD3 bispecific antibody, 30 mM histidine and histidine salt buffer, 10.0% trehalose and 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is 5.2; (3) A pharmaceutical composition comprising 0.2 mg / mL to 5 mg / mL of anti-EpCAM and CD3 bispecific antibody, 30 mM histidine and histidine salt buffer, 5.0% sucrose, 5.0% trehalose and 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is 5.2; (4) A pharmaceutical composition comprising 0.2 mg / mL to 5 mg / mL of anti-EpCAM and CD3 bispecific antibody, 30 mM histidine and histidine salt buffer, 5.0% trehalose, 0.3% methionine and 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is 5.2; (5) A pharmaceutical composition comprising 0.5 mg / mL of anti-EpCAM and CD3 bispecific antibody, 30 mM acetate and acetate buffer, 5.0% trehalose, and 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is 4.5-5.5; (6) A pharmaceutical composition comprising 0.5 mg / mL of anti-EpCAM and CD3 bispecific antibody, 30 mM histidine and histidine salt buffer, 5.0% trehalose, and 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is 4.5-5.5, for example 5.0-5.5, preferably 5.2; (7) A pharmaceutical composition comprising 0.5 mg / mL of anti-EpCAM and CD3 bispecific antibody, 30 mM citrate and citrate buffer, 5.0% trehalose, and 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is 5.0; (8) A pharmaceutical composition comprising 0.5 mg / mL of anti-EpCAM and CD3 bispecific antibody, 30 mM succinic acid and succinate buffer, 5.0% trehalose, and 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is 5.0-5.5; (9) A pharmaceutical composition comprising 0.5 mg / mL of anti-EpCAM and CD3 bispecific antibody, 30 mM histidine and histidine salt buffer, 8.0% sucrose, and 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is 4.5-5.5, for example 5.0-5.5, preferably 5.2; (10) A pharmaceutical composition comprising 0.5 mg / mL of anti-EpCAM and CD3 bispecific antibody, 30 mM histidine and histidine salt buffer, 2% glycine, and 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is 4.5-5.5, for example 5.0-5.5, preferably 5.2; (11) A pharmaceutical composition comprising 0.5 mg / mL of anti-EpCAM and CD3 bispecific antibody, 30 mM histidine and histidine salt buffer, 1.8% sorbitol, 5.7% trehalose and 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is 4.5-5.5, for example 5.0-5.5, preferably 5.2; (12) A pharmaceutical composition comprising 0.5 mg / mL of anti-EpCAM and CD3 bispecific antibody, 30 mM histidine and histidine salt buffer, 1.2% proline, 5.7% trehalose and 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is 4.5-5.5, for example 5.0-5.5, preferably 5.2; (13) A pharmaceutical composition comprising 0.5 mg / mL of anti-EpCAM and CD3 bispecific antibody, 30 mM histidine and histidine salt buffer, 0.3% methionine, 5% trehalose and 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is 4.5-5.5, for example 5.0-5.5, preferably 5.2; (14) A pharmaceutical composition comprising 0.5 mg / mL of anti-EpCAM and CD3 bispecific antibody, 30 mM histidine and histidine salt buffer, 1% glycine, 5% trehalose and 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is 4.5-5.5, for example 5.0-5.5, preferably 5.2; (15) A pharmaceutical composition comprising 0.5 mg / mL of anti-EpCAM and CD3 bispecific antibody, 30 mM histidine and histidine salt buffer, 5% trehalose and 0.02%-0.1% polysorbate 80 or polysorbate 20, wherein the pH of the pharmaceutical composition is 4.5-5.5, for example 5.0-5.5, preferably 5.2; (16) A pharmaceutical composition comprising 0.5 mg / mL of anti-EpCAM and CD3 bispecific antibody, 30 mM histidine and histidine salt buffer, 5% trehalose and 0.05%-0.5% poloxamer 188, wherein the pH of the pharmaceutical composition is 4.5-5.5, for example 5.0-5.5, preferably 5.2; (17) A pharmaceutical composition comprising 0.5 mg / mL of anti-EpCAM and CD3 bispecific antibody, 30 mM histidine and histidine salt buffer, 2%-10% trehalose and 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is 4.5-5.5, for example 5.0-5.5, preferably 5.2; (18) A pharmaceutical composition comprising 0.3 mg / mL of anti-EpCAM and CD3 bispecific antibody, 30 mM histidine and histidine salt buffer, 5% trehalose and 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is 4.5-5.5, for example 5.0-5.5, preferably 5.2; (19) A pharmaceutical composition comprising 0.5 mg / mL of a bispecific antibody against EpCAM and CD3, 10-40 mM histidine and histidine salt buffer, 5% trehalose, and 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is 4.5-5.5, for example 5.0-5.5, preferably 5.2; and (20) A pharmaceutical composition comprising 0.2-5 mg / mL of anti-EpCAM and CD3 bispecific antibody, 30 mM histidine and histidine salt buffer, 5% trehalose and 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is 4.5-5.5, for example 5.0-5.5, preferably 5.2; (21) A pharmaceutical composition comprising 0.01-5 mg / mL of anti-EpCAM and CD3 bispecific antibody, 30 mM histidine and histidine salt buffer, 5% trehalose and 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is 4.5-5.5, for example 5.0-5.5, preferably 5.2; (22) A pharmaceutical composition comprising 0.05-5 mg / mL of anti-EpCAM and CD3 bispecific antibody, 30 mM histidine and histidine salt buffer, 5% trehalose and 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is 4.5-5.5, for example 5.0-5.5, preferably 5.2; (23) A pharmaceutical composition comprising 0.1-5 mg / mL of anti-EpCAM and CD3 bispecific antibody, 30 mM histidine and histidine salt buffer, 5% trehalose and 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is 4.5-5.5, for example 5.0-5.5, preferably 5.2; (24) A pharmaceutical composition comprising 0.4 mg / mL of anti-EpCAM and CD3 bispecific antibody, 30 mM histidine and histidine salt buffer, 5%-10% trehalose and 0.05% polysorbate 80, wherein the pH of the pharmaceutical composition is 4.5-5.5, for example 5.0-5.5, preferably 5.

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5. The pharmaceutical composition according to any one of claims 1-4, wherein the pharmaceutical composition further comprises an antioxidant and / or a preservative.

6. The pharmaceutical composition according to claim 5, wherein the antioxidant is selected from ascorbic acid, tryptophan, methionine, glutathione, sodium thiosulfate, catalase or any combination thereof; or the preservative is selected from m-cresol, phenol, benzyl alcohol, benzalkonium chloride, phenoxyethanol, methylparaben or any combination thereof.

7. The pharmaceutical composition according to any one of claims 1-6, wherein the pharmaceutical composition further comprises an isotonic agent (such as sodium chloride) and / or a metal chelating agent (such as EDTA).

8. The pharmaceutical composition according to any one of claims 1-8, wherein the CDR sequences of the anti-EpCAM and CD3 bispecific antibodies, according to the IMGT numbering system, are as follows: LCDR1-3 of the anti-EpCAM light chain variable region shown in SEQ ID NO:4-6, HCDR1-3 of the anti-EpCAM heavy chain variable region shown in SEQ ID NO:7-9, HCDR1-3 of the anti-CD3 heavy chain variable region shown in SEQ ID NO:10-12, and LCDR1-3 of the anti-CD3 light chain variable region shown in SEQ ID NO:13-15; or According to the Kabat numbering system, the CDR sequences of the anti-EpCAM and CD3 bispecific antibodies are as follows: LCDR1-3 of the anti-EpCAM light chain variable region shown in SEQ ID NO:20-22, HCDR1-3 of the anti-EpCAM heavy chain variable region shown in SEQ ID NO:23-25, HCDR1-3 of the anti-CD3 heavy chain variable region shown in SEQ ID NO:26-28, and LCDR1-3 of the anti-CD3 light chain variable region shown in SEQ ID NO:29-31.

9. The pharmaceutical composition according to any one of claims 1-8, wherein the anti-EpCAM and CD3 bispecific antibody comprises a variable region sequence as shown below, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology with the following sequences and simultaneously binding to EpCAM and CD3: an anti-EpCAM light chain variable region as shown in SEQ ID NO:16, an anti-EpCAM heavy chain variable region as shown in SEQ ID NO:17, an anti-CD3 heavy chain variable region as shown in SEQ ID NO:18, and an anti-CD3 light chain variable region as shown in SEQ ID NO:

19.

10. The pharmaceutical composition according to any one of claims 1-9, wherein the anti-EpCAM and CD3 bispecific antibody comprises (a) a light chain-heavy chain pair, wherein the light chain is bound to the heavy chain via a disulfide bond; and (b) a fusion peptide comprising a single-chain variable fragment (scFv) and an Fc fragment having a CH2 domain and a CH3 domain, wherein the fusion peptide is bound to the heavy chain via a disulfide bond; Preferably, the light chain-heavy chain pair targets EpCAM, and the fusion peptide targets CD3; Preferably, the anti-EpCAM and CD3 bispecific antibody comprises the heavy chain shown in SEQ ID NO:1, the light chain shown in SEQ ID NO:2, and the single chain shown in SEQ ID NO:3; Preferably, the heavy chain terminus of the anti-EpCAM and CD3 bispecific antibody is free of lysine and / or the fusion peptide terminus is free of lysine.

11. The pharmaceutical composition according to any one of claims 1-10, wherein the pharmaceutical composition is a lyophilized formulation or an aqueous formulation, preferably, the formulation is in a form suitable for intravenous injection, intraperitoneal injection, intrapleural injection, urethral injection, subcutaneous injection or intramuscular injection.

12. The composition according to claim 1, wherein the pharmaceutical composition comprises 0.2 mg / mL-5 mg / mL anti-EpCAM and CD3 bispecific antibody, 30 mM histidine and histidine salt buffer (e.g., histidine and histidine hydrochloride buffer), 0.05% polysorbate 80, and 5.0% trehalose, wherein the antibody comprises the heavy chain of SEQ ID NO:1, the light chain of SEQ ID NO:2, and the single chain of SEQ ID NO:3, and the pH of the pharmaceutical composition is 5.

2.

13. A method for inhibiting the formation of aggregates and / or charge heterogeneous components in an anti-EpCAM and CD3 bispecific antibody, comprising adding a buffer to the antibody, the buffer being selected from acetate and acetate buffer, histidine and histidine salt buffer, citrate and citrate buffer, or succinate and succinate buffer, preferably having a concentration of about 10-40 mM and a pH of 4.5-5.5, the anti-EpCAM and CD3 bispecific antibody being as defined in any one of claims 1 or 8-10, and the concentration of the anti-EpCAM and CD3 bispecific antibody being about 0.01-5 mg / mL, preferably, the pH of the acetate and acetate buffer being 4.5-5.0; the pH of the histidine and histidine salt buffer being 5.0-5.5; the pH of the citrate and citrate buffer being 5.0; and the pH of the succinate and succinate buffer being 5.0-5.

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14. A method for preparing a lyophilized powder of the pharmaceutical composition according to any one of claims 1-12, comprising the following steps: mixing the components of the anti-EpCAM and CD3 bispecific antibody pharmaceutical composition; sterile filtration (e.g., filtration through a 0.22 μm PES filter membrane); and freeze-drying in a lyophilizer, wherein the freeze-drying includes pre-freezing, primary drying, and secondary drying.

15. A kit comprising at least one container, said container independently containing an anti-EpCAM and CD3 bispecific antibody as defined in any one of claims 1-12, a buffer, a stabilizer or a surfactant, preferably the kit further comprising instructions.

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