Stable pharmaceutical formulations containing anti-CLDN18.2 antibodies

Abstract

Pharmaceutical formulations are provided. The pharmaceutical formulations comprise a claudin (CLDN) 18.2 antibody, a buffer, a stabilizer, and a surfactant. The pharmaceutical formulations provided herein can maintain the stability of the anti-CLDN18.2 antibody after long-term storage, storage at high temperatures (e.g., 40°C), and / or multiple freeze-thaw cycles. Use of the pharmaceutical formulations in the manufacture of medicaments for preventing and / or treating CLDN18.2-associated diseases, particularly cancers (e.g., CLDN18.2-associated cancers), and methods for preparing the pharmaceutical formulations are further provided.

Description

[Technical Field]

[0001] The present disclosure relates to a pharmaceutical formulation, and in particular to a stable pharmaceutical formulation comprising an anti-CLDN18.2 antibody. The present disclosure also relates to a method for preparing the pharmaceutical formulation and its use. [Background technology]

[0002] Studies have shown that CLDN18.2 belongs to the claudin (CLDN) family and is a subtype of CLDN18. CLDN18.2 is a highly selective gastric lineage marker and is highly expressed in gastric, esophageal, pancreatic, lung, and various other cancer types. The CLDN18.2 protein is a transmembrane protein located on the surface of the cell membrane, containing four transmembrane segments and two extracellular rings. As a surface protein, CLDN18.2 is an ideal target for the development of therapeutic antibodies because its exposed extracellular structure allows it to bind to antibodies. Anti-CLDN18.2 antibodies specifically recognize and bind to CLDN18.2 molecules on the surface of tumor cells, resulting in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), which induces apoptosis and inhibition of cell proliferation, eliminating cancer cells, and controlling disease.

[0003] Antibody molecules have a complex multi-level protein structure and tend to be physically associated, which can trigger unwanted immune responses or pose a risk to patients due to clogging of syringes or pumps during administration. Thus, one of the long-standing problems with liquid formulations of antibodies is stability issues due to aggregation.

[0004] Therefore, there is a need for pharmaceutical formulations of anti-CLDN18.2 antibodies that have stability and consistent quality. Summary of the Invention

[0005] The present disclosure provides stable pharmaceutical formulations comprising anti-CLDN18.2 antibodies that retain homogeneity and stability after long-term storage, treatment (e.g., storage) at elevated temperatures (e.g., 40°C), and / or multiple freeze-thaw cycles.

[0006] In one aspect, the present disclosure provides a pharmaceutical formulation comprising an anti-CLDN18.2 antibody and a buffer, wherein the buffer is an acetate buffer or a histidine buffer and has a pH value of 4.5 to 6.0. In some embodiments, the concentration of the buffer in the pharmaceutical formulation is 5 mM to 50 mM or 10 mM to 30 mM.

[0007] In some embodiments, the pharmaceutical formulation further comprises a stabilizer.

[0008] In some embodiments, the concentration of the stabilizer in the pharmaceutical formulation is between 1% (w / v) and 20% (w / v), or between 1% (w / v) and 10% (w / v).

[0009] In some embodiments, the stabilizer is selected from the group consisting of sucrose, trehalose, and sorbitol.

[0010] In some embodiments, the stabilizer is sucrose or trehalose, and the concentration of sucrose or trehalose in the pharmaceutical formulation is 5% (w / v) to 10% (w / v). In other embodiments, the stabilizer is sorbitol, and the concentration of sorbitol in the pharmaceutical formulation is 2% (w / v) to 8% (w / v).

[0011] In some embodiments, the pharmaceutical formulation further comprises a surfactant.

[0012] In some embodiments, the concentration of surfactant in the pharmaceutical formulation is between 0.005% (w / v) and 0.4% (w / v), or between 0.01% (w / v) and 0.2% (w / v).

[0013] In some embodiments, the surfactant is selected from the group consisting of polysorbate 80 and poloxamer 188.

[0014] In some embodiments, the surfactant is polysorbate 80 and the concentration of polysorbate 80 in the pharmaceutical formulation is 0.01% (w / v) to 0.1% (w / v). In other embodiments, the surfactant is poloxamer 188 and the concentration of poloxamer 188 in the pharmaceutical formulation is 0.05% (w / v) to 0.2% (w / v).

[0015] In some embodiments, the pharmaceutical formulation further comprises a chelating agent.

[0016] In some embodiments, the concentration of the chelating agent in the pharmaceutical formulation is between 30 μM and 350 μM or between 40 μM and 60 μM.

[0017] In some embodiments, the chelating agent is selected from the group consisting of EDTA, DTPA, IDHA, EDDHA, and HBED.

[0018] In some embodiments, the concentration of the anti-CLDN18.2 antibody in the pharmaceutical formulation is between 1 mg / ml and 200 mg / ml.

[0019] In some embodiments, the concentration of the anti-CLDN18.2 antibody in the pharmaceutical formulation is 20 mg / ml to 40 mg / ml.

[0020] In some embodiments, the anti-CLDN18.2 antibody comprises a heavy chain CDR1 (HCDR1) set forth in SEQ ID NO: 1, an HCDR2 set forth in SEQ ID NO: 2, and an HCDR3 set forth in SEQ ID NO: 3, and a light chain CDR1 (LCDR1) set forth in SEQ ID NO: 4, an LCDR2 set forth in SEQ ID NO: 5, and an LCDR3 set forth in SEQ ID NO: 6.

[0021] In some embodiments, the anti-CLDN18.2 antibody comprises CDR1, CDR2, and CDR3 of a heavy chain variable region set forth in SEQ ID NO:7, and CDR1, CDR2, and CDR3 of a light chain variable region set forth in SEQ ID NO:8.

[0022] In some embodiments, the anti-CLDN18.2 antibody comprises a heavy chain variable region set forth in SEQ ID NO:7 and a light chain variable region set forth in SEQ ID NO:8.

[0023] In some embodiments, the anti-CLDN18.2 antibody comprises a heavy chain set forth in SEQ ID NO:9 and a light chain set forth in SEQ ID NO:10.

[0024] In some embodiments, the pharmaceutical formulation comprises an anti-CLDN18.2 antibody, a buffer, a stabilizer, and a surfactant, wherein the buffer is an acetate buffer, the stabilizer is sucrose or trehalose, the surfactant is polysorbate 80, and the pH is about 4.5 to 6.0.

[0025] In some embodiments, the concentration of the anti-CLDN18.2 antibody in the pharmaceutical formulation is 20 mg / ml to 40 mg / ml, the concentration of the acetate buffer in the pharmaceutical formulation is 10 mM to 30 mM, the concentration of sucrose or trehalose in the pharmaceutical formulation is 5% (w / v) to 10% (w / v), and / or the concentration of polysorbate 80 in the pharmaceutical formulation is 0.01% (w / v) to 0.2% (w / v).

[0026] In some embodiments, the concentration of the anti-CLDN18.2 antibody in the pharmaceutical formulation is about 30 mg / ml, the concentration of the acetate buffer in the pharmaceutical formulation is about 20 mM, the concentration of sucrose or trehalose in the pharmaceutical formulation is about 9% (w / v), the concentration of polysorbate 80 in the pharmaceutical formulation is 0.01% (w / v) to 0.1% (w / v), and the pH value is about 5.0 to 5.5.

[0027] In some embodiments, the stabilizer is sucrose, the concentration of polysorbate 80 in the pharmaceutical formulation is about 0.05% (w / v), and the pH value is about 5.3.

[0028] In some embodiments, the pharmaceutical formulation further comprises EDTA, wherein the concentration of EDTA in the pharmaceutical formulation is between 40 μM and 60 μM.

[0029] In some embodiments, the concentration of EDTA in the pharmaceutical formulation is about 50 μm.

[0030] In some embodiments, the pharmaceutical formulation comprises an anti-CLDN18.2 antibody, a buffer, a stabilizer, and a surfactant, wherein the buffer is a histidine buffer, the stabilizer is sucrose or sorbitol, and the surfactant is polysorbate 80 or poloxamer 188.

[0031] In some embodiments, the stabilizer is sucrose and the concentration of sucrose in the pharmaceutical formulation is 6% (w / v) to 12% (w / v), hi other embodiments, the stabilizer is sorbitol and the concentration of sorbitol in the pharmaceutical formulation is 2% (w / v) to 8% (w / v).

[0032] In some embodiments, the surfactant is polysorbate 80 and the concentration of polysorbate 80 in the pharmaceutical formulation is 0.01% (w / v) to 0.1% (w / v). In other embodiments, the surfactant is poloxamer 188 and the concentration of poloxamer 188 in the pharmaceutical formulation is 0.05% (w / v) to 0.2% (w / v).

[0033] In another aspect, the present disclosure provides a method for preparing a pharmaceutical formulation, the method comprising: (1) providing a formulation solvent and an anti-CLDN18.2 antibody stock solution, wherein the formulation solvent comprises a buffer, a stabilizer, and optionally a surfactant; and (2) subjecting the anti-CLDN18.2 antibody stock solution to solvent exchange with the formulation solvent to obtain a pharmaceutical formulation described herein.

[0034] In some embodiments, the anti-CLDN18.2 antibody stock solution used in the methods described herein contains a chelating agent (e.g., EDTA), the formulation solvent does not contain a chelating agent, and the pharmaceutical formulation is substantially free of a chelating agent (e.g., after filtration). In other embodiments, the formulation solvent contains a buffer and a stabilizer, but does not contain a surfactant.

[0035] In other embodiments, the formulation medium used in the methods described herein comprises a buffer, a stabilizer, and a chelating agent (eg, EDTA).

[0036] In other embodiments, the surfactant is added after the anti-CLDN18.2 antibody stock solution is subjected to solvent exchange with the formulation solvent to obtain the pharmaceutical formulation described herein.

[0037] In some embodiments, the acetate buffer used in the methods described herein is an acetic acid-sodium acetate buffer; and the histidine buffer is a histidine-histidine hydrochloride buffer.

[0038] In another aspect, the present disclosure provides the use of a pharmaceutical formulation described herein in the manufacture of a medicament for preventing and / or treating a CLDN18.2-associated disease.

[0039] In some embodiments, the CLDN18.2-associated disease is selected from the group consisting of gastric cancer, adenocarcinoma of the gastroesophageal junction, lung cancer, bronchogenic carcinoma, bone cancer, perihilar cholangiocarcinoma, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, bladder cancer, head and neck cancer, spinal cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, anal cancer, esophageal cancer, gastrointestinal cancer, skin cancer, prostate cancer, pituitary cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, or adenocarcinoma. [Brief explanation of the drawings]

[0040] [Figure 1] FIG. 1 shows protein stability data measured by SEC in surfactant screening experiments using poloxamer 188 as surfactant at three different concentrations each (formulations F14, F15, and F16). [Figure 2]FIG. 2 shows anti-CLDN18.2 antibody protein stability data measured by CEX in surfactant screening experiments using poloxamer 188 as surfactant at three different concentrations each (formulations F14, F15, and F16). [Figure 3] FIG. 3 shows protein stability data measured by CE-SDS in surfactant screening experiments using poloxamer 188 as surfactant at three different concentrations each (formulations F14, F15, and F16). [Figure 4] FIG. 4 shows protein stability data measured by SEC in excipient screening experiments using sorbitol and sucrose as stabilizers (formulations F21 and F22, respectively). [Figure 5] Figure 5 shows protein stability data measured by CEX in excipient screening experiments using sorbitol and sucrose as stabilizers (formulations F21 and F22, respectively). [Figure 6] FIG. 6 shows protein stability data measured by CE-SDS in excipient screening experiments using sorbitol and sucrose as stabilizers (formulations F21 and F22, respectively). [Figure 7] FIG. 7 shows the protective effect of edetate disodium (EDTA disodium) in preventing the degradation of polysorbate 80 (PS80). [Figure 8] FIG. 8 shows the protective effect of edetate disodium (EDTA disodium) on protein stability as measured by NR CE-SDS. DETAILED DESCRIPTION OF THE INVENTION

[0041] The following description of the present disclosure is intended solely to describe various embodiments of the present disclosure. The specific examples described should not be construed as limiting the scope of the present disclosure. It should be understood that various equivalent substitutions, modifications, or variations may be made by those skilled in the art without departing from the spirit and essence of the present disclosure, and all such equivalent embodiments are also included herein. All documents cited herein, including publications, patents, and patent applications, are incorporated herein by reference. When a method referred to in this disclosure includes two or more eligible steps, the specified steps may be performed in any order or simultaneously (unless the context precludes this possibility). Furthermore, the method may include one or more of the other steps, which may be performed before any specified step, between two specified steps, or after all specified steps (unless the context precludes this possibility).

[0042] References herein to "about" (a value or range of values) include examples of that value or range of values. For example, "about X" includes "X". In general, the term "about" refers to the value of a variable, any value within the experimental error range of that variable (e.g., within a 95% confidence interval of the mean), or any value within 10% of that variable, based on a larger value. For example, "about X" includes "110% x X", "109% x X", "108% x X", "107% x X", "106% x X", "105% x X", "104% x X", "103% x X", "102% x X", "101% x X", "99% x X", "98% x X", "97% x X", "96% x X", "95% x X", "94% x X", "93% x X", "92% x X", "91% x X", or "90% x X".

[0043] The term "at least" followed by a number is used herein to indicate the beginning of a range beginning with that number (which may be a range with or without an upper limit, depending on the variable specified). For example, "at least 1" refers to a value of 1 or greater than 1. The term "at most" followed by a number is used herein to indicate the end of a range ending with that number (which may be a range with a lower limit of 1 or 0, or a range without a lower limit, depending on the variable specified). For example, "at most 4" refers to a value of 4 or less than 4, and "at most 40%" refers to a value of 40% or less than 40%. In the present disclosure, when a range is stated as "from (a first number) to (a second number)" or "from (a first number) to (a second number)," this indicates that the lower limit of the range is the first number and the upper limit is the second number. For example, 5 to 50 mg / mL refers to a range having a lower limit of 5 mg / mL and an upper limit of 50 mg / mL. As used herein, the terms "less than" or "greater than" a value are inclusive of the value in question.

[0044] antibody As used herein, the term "antibody" includes any immunoglobulin, monoclonal, polyclonal, multivalent, bivalent, monovalent, multispecific, or bispecific antibody that binds to a specific antigen. A complete antibody comprises two heavy chains and two light chains. Each heavy chain comprises a heavy chain variable region (V H ), heavy chain first constant region (C H1 ), heavy chain second constant region (C H2 ) and the third constant region of the heavy chain (C H3 Each light chain consists of a light chain variable region (V L ) and the light chain constant region (C L ) V of the heavy chain H Region and V of the light chain LEach region has three complementary determinant regions (CDRs) interposed between flanking stretches known as framework regions (FRs). The framework regions are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. The six CDRs of one heavy chain and one light chain together constitute the antigen-binding site of an antibody and determine the specificity of the antibody. The antibodies described herein also include fragments or derivatives that retain the antigen-binding function of the intact antibody. The fragment or derivative has the same antigen-binding specificity as the intact antibody, but the binding affinity of the fragment or derivative for a specific antigen may be the same as or different from the binding affinity of the intact antibody.

[0045] In some embodiments, the antibodies described herein comprise antigen-binding fragments. An antigen-binding fragment refers to one or more types of antibody fragments that retain binding specificity for an antigen. Examples of antigen-binding fragments include: (i) V L , V H , C L and C H1 (ii) a Fab' fragment, which is a monovalent fragment consisting of a Fab domain; (iii) a F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments connected by disulfide bonds in the hinge region; and (iv) a V H and C H1 (v) a single arm V of an antibody; L and V H (vi) dAb fragments containing a single variable domain (Ward et al., Nature 341:544-546 (1989); PCT Publication WO 90 / 05144); (vii) isolated CDRs; and (viii) V fragments containing a V domain, either directly or via a peptide chain. L and V H These include, but are not limited to, single-chain Fv fragments, which are monovalent fragments formed by the connection between domains (Huston JS et al., Proc Natl Acad Sci USA, 85:5879 (1988)).

[0046] In some embodiments, the antibodies described herein include chimeric antibodies having portions of the heavy and / or light chains that are identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, and the remaining portions of the chains that are identical or homologous to corresponding sequences in antibodies from another class or belonging to another antibody class or subclass, and fragments thereof, provided that they possess the desired functional activity.

[0047] In some embodiments, the antibodies described herein include humanized antibodies. Humanized forms of non-human (e.g., murine) antibodies can be chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding sequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. In some examples, humanized antibodies replace the amino acid sequences of human CDRs with those of non-human V. H and V L A humanized antibody may be a CDR-grafted antibody, in which amino acid residues of a CDR of a human immunoglobulin have been introduced into the human immunoglobulin (i.e., acceptor antibody) to replace the amino acid sequence of the corresponding non-human CDR. In other examples, the majority of the amino acid sequence of a humanized antibody can be derived from a human immunoglobulin (i.e., acceptor antibody), with the amino acid residues of the acceptor antibody's CDR replaced by amino acid residues of the CDR of a non-human (e.g., mouse, rat, or rabbit) antibody having the desired specificity, affinity, and capacity. Typically, a humanized antibody contains at least one, and generally two, variable domains, with all or substantially all of the CDR sequences derived from a non-human immunoglobulin and all or substantially all of the framework region (FR) sequences derived from a human immunoglobulin. In some examples, residues in the framework regions of the human immunoglobulin variable regions are replaced by corresponding non-human residues. Furthermore, a humanized antibody can contain residues that are found neither in the original antibody nor in the imported CDR or framework region sequences.

[0048] An anti-CLDN18.2 antibody as described herein refers to an antibody that can specifically bind to the CLDN18.2 protein.

[0049] The CLDN18.2 protein described herein refers to claudin-18 splice variant 2 derived from mammals such as primates (e.g., humans and monkeys) and rodents (e.g., mice). In some embodiments, the CLDN18.2 is human CLDN18.2. An exemplary sequence of human CLDN18.2 includes human CLDN18.2 protein (NCBI Ref Seq No. NP_001002026.1). CLDN18.2 can be expressed in cancer cells. In one embodiment, CLDN18.2 is expressed on the surface of cancer cells.

[0050] In some embodiments, the anti-CLDN18.2 antibodies described herein comprise a heavy chain variable region (V H ) containing the heavy chain variable region (V H ) comprises the CDR1 set forth in SEQ ID NO: 1, the CDR2 set forth in SEQ ID NO: 2 and / or the CDR3 set forth in SEQ ID NO: 3.

[0051] In some embodiments, the anti-CLDN18.2 antibodies described herein comprise a light chain variable region (V L ) containing the light chain variable region (V L ) comprises a CDR1 set forth in SEQ ID NO:4, a CDR2 set forth in SEQ ID NO:5 and / or a CDR3 set forth in SEQ ID NO:6.

[0052] In some embodiments, the anti-CLDN18.2 antibodies described herein comprise a heavy chain variable region (V H ) and the light chain variable region (V L ) containing the heavy chain variable region (V H ) comprises a CDR1 having the amino acid sequence shown in SEQ ID NO: 1, a CDR2 having the amino acid sequence shown in SEQ ID NO: 2, and / or a CDR3 having the amino acid sequence shown in SEQ ID NO: 3, and a light chain variable region (V L) comprises a CDR1 having the amino acid sequence set forth in SEQ ID NO: 4, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 5, and / or a CDR3 having the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the anti-CLDN18.2 antibody comprises a heavy chain CDR1 (HCDR1) set forth in SEQ ID NO: 1, an HCDR2 set forth in SEQ ID NO: 2, and an HCDR3 set forth in SEQ ID NO: 3, and a light chain CDR1 (LCDR1) set forth in SEQ ID NO: 4, an LCDR2 set forth in SEQ ID NO: 5, and an LCDR3 set forth in SEQ ID NO: 6. In some embodiments, the anti-CLDN18.2 antibody comprises CDR1, CDR2, and CDR3 of a heavy chain variable region set forth in SEQ ID NO: 7, and CDR1, CDR2, and CDR3 of a light chain variable region set forth in SEQ ID NO: 8.

[0053] In some embodiments, the anti-CLDN18.2 antibodies described herein comprise a heavy chain variable region (V) having the amino acid sequence set forth in SEQ ID NO:7. H In some embodiments, the anti-CLDN18.2 antibodies described herein comprise a light chain variable region (V) having the amino acid sequence set forth in SEQ ID NO: 8. L In some embodiments, the anti-CLDN18.2 antibodies described herein comprise a heavy chain variable region (V) having the amino acid sequence set forth in SEQ ID NO:7. H ), and a light chain variable region (V L In some embodiments, the anti-CLDN18.2 antibody comprises a heavy chain variable region set forth in SEQ ID NO:7 and a light chain variable region set forth in SEQ ID NO:8.

[0054] In some embodiments, the anti-CLDN18.2 antibodies described herein further comprise an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region comprises a heavy chain constant region and / or a light chain constant region. The heavy chain constant region comprises a C H1 , C H1 ~C H2 or C H1 ~C H3 The light chain constant region comprises the C L Includes the area.

[0055] In some embodiments, the anti-CLDN18.2 antibodies described herein comprise a heavy chain having the amino acid sequence set forth in SEQ ID NO: 9 and a light chain having the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the anti-CLDN18.2 antibodies comprise a heavy chain set forth in SEQ ID NO: 9 and a light chain set forth in SEQ ID NO: 10.

[0056] Exemplary amino acid sequences for use in some embodiments are listed in Table A below.

[0057] [Table A]

[0058] The present disclosure relates to pharmaceutical formulations comprising an anti-CLDN18.2 antibody (e.g., an anti-CLDN18.2 antibody described herein). In some embodiments, the concentration of the anti-CLDN18.2 antibody in the pharmaceutical formulation of the present disclosure is 1 mg / ml to 200 mg / ml, 1 mg / ml to 190 mg / ml, 10 mg / ml to 190 mg / ml, 20 mg / ml to 180 mg / ml, 20 mg / ml to 170 mg / ml, 20 mg / ml to 160 mg / ml, 20 mg / ml to 150 mg / ml, 20 mg / ml to 14 ... The concentration of the anti-CLDN18.2 antibody may be 130 mg / ml, 20 mg / ml to 120 mg / ml, 20 mg / ml to 110 mg / ml, 20 mg / ml to 100 mg / ml, 20 mg / ml to 90 mg / ml, 20 mg / ml to 80 mg / ml, 20 mg / ml to 70 mg / ml, 20 mg / ml to 60 mg / ml, 20 mg / ml to 50 mg / ml, 20 mg / ml to 40 mg / ml, or 20 mg / ml to 30 mg / ml. In some embodiments, the concentration of the anti-CLDN18.2 antibody is any concentration value within the above-mentioned ranges. For example, depending on the requirement, the concentration of the anti-CLDN18.2 antibody in the pharmaceutical formulation can be at least 5 mg / ml, at least 10 mg / ml, at least 20 mg / ml, at least 30 mg / ml, at least 40 mg / ml, at least 50 mg / ml, at least 60 mg / ml, at least 70 mg / ml, at least 80 mg / ml, at least 90 mg / ml, at least 100 mg / ml, at least 110 mg / ml, at least 120 mg / ml, at least 130 mg / ml, at least 140 mg / ml, at least 150 mg / ml, at least 160 mg / ml, at least 170 mg / ml, at least 180 mg / ml, at least 190 mg / ml and / or at most 200 mg / ml.

[0059] buffer The term "buffer" generally refers to a buffered solution that resists changes in pH through the action of its acid-base conjugate components. As used herein, "buffer" refers to a complex solution known to be safe when used in pharmaceutical formulations to maintain or control the pH of the formulation within a desired range. Acceptable buffers capable of controlling the pH within a range from slightly acidic to slightly alkaline pH values (e.g., pH values between 4.5 and 8.0) include, but are not limited to, succinate buffer, citrate buffer, phosphate buffer, acetate buffer, arginine buffer, 2-amino-2-hydroxymethyl-1,3-propanediol (TRIS) buffer, histidine buffer, and others, either alone or in any combination.

[0060] The buffer in the formulation solvent can be prepared using any suitable method known in the art. In some embodiments, the buffer of the present disclosure can be prepared using a specific acid-base ion pair. In one example, the excipient of the acid-base ion pair can be accurately weighed and added to pure water that is approximately 60% of the volume of the target buffer, mixed uniformly, and then the pH value of the resulting solution can be determined. If the pH value deviates from the target value, the pH value can be adjusted using an appropriate ion pair. The solution is then diluted with pure water to reach the target weight or volume. Finally, the conductivity, osmolality, and pH value of the solution are measured for verification.

[0061] The stable pharmaceutical formulations of the present disclosure can include a buffer such that the pharmaceutical formulation has a pH value of 4.5 to 8.0, such as a pH value of 4.5 to 6.0, 6.0 to 7.0, or 7.0 to 8.0. In some embodiments, a suitable buffer is used such that the pharmaceutical formulation has a pH value of 4.5 to 6.0. In particular, the pH value of the pharmaceutical formulations of the present disclosure can be any pH value within the pH ranges listed above, such as 4.5, 4.6, 4.7, 4.8, 4.9, 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, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0.

[0062] Examples of buffers that can control the pH value of pharmaceutical formulations within a desired range include acetate buffers, histidine buffers, citrate buffers, and other organic acid buffers or inorganic acid buffers. Any one of these buffers can be used alone, or two or more of these buffers can be combined for use. Preferably, the pharmaceutical formulation of the present disclosure contains acetate buffers and / or histidine buffers. More preferably, the pharmaceutical formulation of the present disclosure contains acetate buffers.

[0063] "Acetate buffer" refers to a buffer containing acetate radical ions. Acetate buffers can include one or more of acetic acid (e.g., glacial acetic acid), potassium acetate, sodium acetate (e.g., sodium acetate trihydrate), and others. In some embodiments, the acetate buffer is an acetic acid-sodium acetate buffer, such as glacial acetic acid-sodium acetate trihydrate buffer. In some embodiments, the pH value of the acetate buffer can be any pH value within the range of 4.5 to 6.0, such as 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0.

[0064] "Histidine buffer" refers to a buffer containing histidine radical ions. Histidine buffers can include one or more of histidine, histidine hydrochloride (e.g., histidine hydrochloride monohydrate), histidine acetate, histidine phosphate, histidine sulfate, and the like. In some embodiments, the histidine buffer can be a histidine-histidine hydrochloride buffer. In some embodiments, the pH value of the histidine buffer can be any pH value within the range of 4.5 to 6.0, such as 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0.

[0065] "Citrate buffer" refers to a buffer containing citrate radical ions. Citrate buffers can include one or more of citric acid, monosodium citrate, disodium citrate, trisodium citrate, monopotassium citrate, dipotassium citrate, tripotassium citrate, sodium chloride, potassium chloride, and the like. In some embodiments, the citrate buffer is a citric acid-trisodium citrate buffer. In some embodiments, the pH value of the citrate buffer can be any pH value within the range of 4.5 to 6.0, such as 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0.

[0066] In some embodiments, the buffer used in the pharmaceutical formulation of the present disclosure is an acetate buffer or a histidine buffer, and the pH value of the pharmaceutical formulation of the present disclosure is 4.5 to 6.0. Without wishing to be bound by any theory, acetate buffers and histidine buffers are thought to be superior to citrate buffers. For example, when the buffer of the pharmaceutical formulation of the present disclosure is an acetate buffer or a histidine buffer, the anti-CLDN18.2 antibody in the pharmaceutical formulation is more stable (e.g., more stable at high temperatures), the force between antibody molecules is repulsive rather than attractive, and the risk of molecular aggregation is lower.

[0067] The buffer concentration described herein refers to the concentration of buffer ions in the buffer. In some embodiments, the concentration of a buffer suitable for use in a pharmaceutical formulation of the present disclosure may be 5 mmol / L to 100 mmol / L, 5 mmol / L to 90 mmol / L, 5 mmol / L to 80 mmol / L, 5 mmol / L to 70 mmol / L, 5 mmol / L to 60 mmol / L, 5 mmol / L to 50 mmol / L, 10 mmol / L to 50 mmol / L, 10 mmol / L to 40 mmol / L, 10 mmol / L to 30 mmol / L, or 10 mmol / L to 20 mmol / L. In some embodiments, the buffer concentration is any concentration value within the above ranges. For example, depending on the requirements, the concentration of the buffer can be 5 mmol / L, at least 10 mmol / L, at least 15 mmol / L, at least 20 mmol / L, at least 25 mmol / L, at least 30 mmol / L, at least 35 mmol / L, at least 40 mmol / L, at least 45 mmol / L, at least 50 mmol / L, at least 55 mmol / L, at least 60 mmol / L, at least 65 mmol / L, at least 70 mmol / L, at least 75 mmol / L, at least 80 mmol / L, at least 85 mmol / L, at least 90 mmol / L, at least 95 mmol / L and / or at most 100 mmol / L, depending on the particular buffer and the stability required for the pharmaceutical formulation.

[0068] In some embodiments, the buffer used in the pharmaceutical formulations of the present disclosure is an acetate buffer, such as an acetic acid-sodium acetate buffer, and the concentration may be 5 mmol / L to 100 mmol / L. In some embodiments, the concentration of the acetate buffer may be 5 mmol / L to 100 mmol / L, 5 mmol / L to 90 mmol / L, 5 mmol / L to 80 mmol / L, 5 mmol / L to 70 mmol / L, 5 mmol / L to 60 mmol / L, 5 mmol / L to 50 mmol / L, 5 mmol / L to 45 mmol / L, 5 mmol / L to 40 mmol / L, 5 mmol / L to 35 mmol / L, 5 mmol / L to 30 mmol / L, 5 mmol / L to 25 mmol / L, 5 mmol / L to 20 mmol / L, 5 mmol / L to 15 mmol / L, or 5 mmol / L to 10 mmol / L.

[0069] In some embodiments, the buffer used in the pharmaceutical formulations of the present disclosure is a histidine buffer, such as a histidine-histidine hydrochloride buffer, and can have a concentration of 5 mmol / L to 100 mmol / L. In some embodiments, the concentration of the histidine buffer can be 5 mmol / L to 100 mmol / L, 5 mmol / L to 90 mmol / L, 5 mmol / L to 80 mmol / L, 5 mmol / L to 70 mmol / L, 5 mmol / L to 60 mmol / L, 5 mmol / L to 50 mmol / L, 5 mmol / L to 45 mmol / L, 5 mmol / L to 40 mmol / L, 5 mmol / L to 35 mmol / L, 5 mmol / L to 30 mmol / L, 5 mmol / L to 25 mmol / L, 5 mmol / L to 20 mmol / L, 5 mmol / L to 15 mmol / L, or 5 mmol / L to 10 mmol / L.

[0070] In some embodiments, the buffer used in the pharmaceutical formulation of the present disclosure is an acetate buffer or a histidine buffer, and has a concentration of 5 mM to 50 mM. In some embodiments, the buffer used in the pharmaceutical formulation of the present disclosure is an acetate buffer or a histidine buffer, and has a concentration of 10 mM to 30 mM. In some embodiments, the buffer used in the pharmaceutical formulation of the present disclosure is an acetate buffer or a histidine buffer, and has a concentration of about 20 mM. In some embodiments, the buffer used in the pharmaceutical formulation of the present disclosure is an acetate buffer, and has a concentration of about 20 mM.

[0071] stabilizers As used herein, the term "stabilizer" refers to an agent capable of preventing or reducing the chemical and / or physical instability of a protein of interest when bound to the protein. Examples of stabilizers include sugars, alcohols, acids, salts, polymers, and the like. Examples of sugars include glucose, sucrose, trehalose, lactose, glucans, and the like. Examples of alcohols include sorbitol, and the like. Examples of acids include citric acid, phosphoric acid, tartaric acid, amino acids, ethylenediaminetetraacetic acid, and the like. Examples of salts include sodium sulfate, sodium glutamate, sodium chloride, potassium chloride, ammonium acetate, and the like. Examples of polymers include polyethylene glycol, povidone, and the like.

[0072] In some embodiments, the stabilizer used in the pharmaceutical formulation of the present disclosure is selected from sugars.In some embodiments, the stabilizer used in the pharmaceutical formulation of the present disclosure is selected from alcohols.In some embodiments, the stabilizer used in the pharmaceutical formulation of the present disclosure is selected from sucrose, trehalose, sorbitol, or a combination thereof.

[0073] In some embodiments, the concentration of stabilizer used in the pharmaceutical formulation is between 1% (w / v) and 20% (w / v), between 1% (w / v) and 19% (w / v), between 1% (w / v) and 18% (w / v), between 1% (w / v) and 17% (w / v), between 1% (w / v) and 16% (w / v), between 1% (w / v) and 15% (w / v), between 1% (w / v) and 14% (w / v), depending on the particular stabilizer and the stability desired for the pharmaceutical formulation. (w / v), 1% (w / v) to 13% (w / v), 1% (w / v) to 12% (w / v), 1% (w / v) to 11% (w / v), 1% (w / v) to 10% (w / v), 2% (w / v) to 10% (w / v), 5% (w / v) to 10% (w / v), 2% (w / v) to 8% (w / v), 2% (w / v) to 7% (w / v), 2% (w / v) to 6% (w / v), or 2 to 5% (w / v). In some embodiments, the concentration of the stabilizer is any concentration value within the above range.

[0074] In some embodiments, the stabilizer used in the pharmaceutical formulation of the present disclosure is sucrose, and the concentration of sucrose in the pharmaceutical formulation may be 1% (w / v) to 20% (w / v). In some embodiments, the concentration of sucrose in the pharmaceutical formulation may be 1% (w / v) to 10% (w / v), 10% (w / v) to 20% (w / v), or 5% (w / v) to 15% (w / v). In some embodiments, the concentration of sucrose in the pharmaceutical formulation is 5% (w / v) to 10% (w / v). In some embodiments, the concentration of sucrose in the pharmaceutical formulation is about 9% (w / v).

[0075] In some embodiments, the stabilizer used in the pharmaceutical formulation of the present disclosure is trehalose, and the concentration of trehalose in the pharmaceutical formulation may be 1% (w / v) to 20% (w / v). In some embodiments, the concentration of trehalose in the pharmaceutical formulation may be 1% (w / v) to 10% (w / v), 10% (w / v) to 20% (w / v), or 5% (w / v) to 15% (w / v). In some embodiments, the concentration of trehalose in the pharmaceutical formulation is 5% (w / v) to 10% (w / v). In some embodiments, the concentration of trehalose in the pharmaceutical formulation is about 9% (w / v).

[0076] In some embodiments, the stabilizer used in the pharmaceutical formulation of the present disclosure is sorbitol, and the concentration of sorbitol in the pharmaceutical formulation may be 1% (w / v) to 20% (w / v). In some embodiments, the concentration of sorbitol in the pharmaceutical formulation may be 1% (w / v) to 10% (w / v), 10% (w / v) to 20% (w / v), or 5% (w / v) to 15% (w / v). In some embodiments, the concentration of sorbitol in the pharmaceutical formulation is 2% (w / v) to 8% (w / v). In some embodiments, the concentration of sorbitol in the pharmaceutical formulation is about 5% (w / v).

[0077] surfactant As used herein, the term "surfactant" refers to an organic substance having an amphiphilic structure, possessing both hydrophilic and hydrophobic properties; in other words, the organic substance contains groups with opposite solubility tendencies, such as oil-soluble hydrocarbon chains and water-soluble ionic groups. Depending on the charge of the surfactant moiety, surfactants can include anionic surfactants, cationic surfactants, and nonionic surfactants.

[0078] Exemplary surfactants include polysorbates (e.g., polysorbate 80), poloxamers (e.g., poloxamer 188), Triton, polyethylene glycol, polypropylene glycol, and copolymers of ethylene glycol and propylene glycol (e.g., Pluronic, PF68, etc.). In some embodiments, the surfactant used in the pharmaceutical formulations of the present disclosure is selected from polysorbate 80 (also known as PS80 or Tween 80), poloxamer 188, or a combination thereof.

[0079] In some embodiments, the concentration of surfactant used in the pharmaceutical formulations of the present disclosure can be 0.005% (w / v) to 0.4% (w / v), 0.01% (w / v) to 0.3% (w / v), 0.01% (w / v) to 0.2% (w / v), 0.05% (w / v) to 0.2% (w / v), 0.05% (w / v) to 0.1% (w / v), or 0.01% (w / v) to 0.1% (w / v), depending on the particular surfactant and the stability desired for the pharmaceutical formulation. In some embodiments, the surfactant concentration is any concentration value within the above ranges.

[0080] In some embodiments, the surfactant used in the pharmaceutical formulation of the present disclosure is polysorbate 80, and the concentration of polysorbate 80 in the pharmaceutical formulation may be 0.005% (w / v) to 0.4% (w / v). In some embodiments, the concentration of polysorbate 80 in the pharmaceutical formulation may be 0.005% (w / v) to 0.1% (w / v) or 0.1% (w / v) to 0.4% (w / v). In some embodiments, the concentration of polysorbate 80 in the pharmaceutical formulation may be 0.01% (w / v) to 0.1% (w / v). In some embodiments, the concentration of polysorbate 80 in the pharmaceutical formulation may be about 0.05% (w / v).

[0081] In some embodiments, the surfactant used in the pharmaceutical formulations of the present disclosure is poloxamer 188, and the concentration of poloxamer 188 in the pharmaceutical formulation may be 0.005% (w / v) to 0.4% (w / v). In some embodiments, the concentration of poloxamer 188 in the pharmaceutical formulation may be 0.005% (w / v) to 0.1% (w / v) or 0.1% (w / v) to 0.4% (w / v). In some embodiments, the concentration of poloxamer 188 in the pharmaceutical formulation may be 0.05% (w / v) to 0.2% (w / v).

[0082] chelating agents As used herein, the term "chelating agent" refers to a compound capable of reacting with certain metal ions (e.g., manganese(II), copper(II), iron(III), and cobalt(II)) to form stable, water-soluble metal complexes. Chelation refers to the formation or presence of two or more separate bonds between a ligand and a single central atom. Exemplary chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), N-(1,2-dicarboxylethyl)-D,L-aspartic acid (IDHA), ethylenediamine-N,N'-bis(EDDHA), and N,N-bis(2-hydroxyphenyl)ethylenediamine-N,N'-diacetic acid (HBED), or salt forms thereof, such as EDTA-2Na and EDTA-4Na. In some embodiments, the chelating agent used in the pharmaceutical formulations of the present disclosure may be a chelating salt of EDTA (e.g., the disodium salt of EDTA, i.e., EDTA-2Na; or the tetrasodium salt of EDTA, i.e., EDTA-4Na).

[0083] In some embodiments, the concentration of chelating agent used in the pharmaceutical formulation of the present disclosure may range from 30 μmol / L to 350 μmol / L, 30 μmol / L to 330 μmol / L, 30 μmol / L to 300 μmol / L, 30 μmol / L to 270 μmol / L, 30 μmol / L to 250 μmol / L, 30 μmol / L to 23 ... In some embodiments, the concentration of the chelating agent may be any concentration value within the above ranges.

[0084] In some embodiments, the chelating agent used in the pharmaceutical formulations of the present disclosure is edetate disodium (EDTA-Na2) at a concentration of 30 μmol / L to 350 μmol / L or 40 μmol / L to 60 μmol / L. In some embodiments, the chelating agent used in the pharmaceutical formulations of the present disclosure is edetate disodium (EDTA-Na2) at a concentration of about 50 μmol / L.

[0085] In another aspect, the present disclosure provides the use of a chelating agent in preventing the degradation of a surfactant (e.g., polysorbate 80) in a pharmaceutical formulation (e.g., a pharmaceutical formulation provided by the present disclosure).

[0086] In another aspect, the present disclosure provides the use of a chelating agent in preventing reduced stability of a protein (e.g., an antibody) in a pharmaceutical formulation (e.g., a pharmaceutical formulation provided by the present disclosure).

[0087] Other materials In certain embodiments, the pharmaceutical formulations of the present disclosure may further comprise other excipients, such as, but not limited to, isotonicity agents, diluents, and the like.

[0088] The term "isotonicity agent" refers to a compound or composition that provides a drug with an appropriate osmotic tension to prevent the net flow of water through the cell membrane in contact with the drug. In some embodiments, the pharmaceutical formulation of the present disclosure has the same osmotic pressure as human blood. Suitable isotonicity agents include, but are not limited to, glycerol, amino acids or proteins (e.g., glycine or albumin), salts (e.g., sodium chloride), and sugars (e.g., glucose, mannitol, sucrose, and lactose).

[0089] The term "diluent" refers to a pharmaceutically acceptable reagent that can be used to dilute the pharmaceutical formulation of the present disclosure. Typical diluents include water, saline, bacteriostatic agents for injection, pH buffers, sterile salt solutions, Ringer's solution, or glucose solutions.

[0090] formulation In one aspect, the present disclosure provides a stable pharmaceutical formulation comprising an anti-CLDN18.2 antibody (e.g., a specific anti-CLDN18.2 antibody provided herein), a buffer, a stabilizer, and a surfactant. The pharmaceutical formulation has a pH value of 4.5 to 8.0. In some embodiments, the pH value is 4.5 to 6.0 to achieve sufficient stability.

[0091] In some embodiments, the pharmaceutical formulation of the present disclosure comprises: (i) an anti-CLDN18.2 antibody (e.g., a specific anti-CLDN18.2 antibody provided herein), wherein the concentration of the anti-CLDN18.2 antibody is 1 mg / ml to 200 mg / ml, preferably 20 mg / ml to 40 mg / ml, and more preferably about 30 mg / ml; (ii) a buffer, which is preferably an acetate buffer or a histidine buffer, more preferably an acetate buffer, and which has a concentration of 5 mmol / L to 100 mmol / L, preferably 5 mmol / L to 50 mmol / L or 10 mmol / L to 30 mmol / L, more preferably about 20 mmol / L; (iii) a stabilizer, wherein the stabilizer is preferably sucrose, trehalose, or sorbitol; (a) the stabilizer is preferably sucrose or trehalose, and the concentration of the stabilizer in the pharmaceutical formulation is 1% (w / v) to 20% (w / v), preferably 1% (w / v) to 10% (w / v) or 5% (w / v) to 10% (w / v), and more preferably about 9% (w / v); or (b) a stabilizer, wherein the stabilizer is preferably sorbitol, and the concentration of the stabilizer in the pharmaceutical formulation is 1% (w / v) to 20% (w / v), preferably 1% (w / v) to 10% (w / v), or 2% (w / v) to 8% (w / v), and more preferably about 5% (w / v); (iv) a surfactant, preferably polysorbate 80 or poloxamer 188; (a) the surfactant is preferably polysorbate 80, and the concentration of the surfactant in the pharmaceutical formulation is 0.005% (w / v) to 0.4% (w / v), preferably 0.01% (w / v) to 0.2% (w / v), more preferably 0.01% (w / v) to 0.1% (w / v), more preferably about 0.05% (w / v); or (b) a surfactant, preferably poloxamer 188, having a concentration of 0.005% (w / v) to 0.4% (w / v), preferably 0.01% (w / v) to 0.2% (w / v), more preferably 0.05% (w / v) to 0.2% (w / v); and v) optionally, a chelating agent, which is preferably EDTA or a salt thereof (e.g., EDTA-2Na), and the concentration of the chelating agent in the pharmaceutical formulation is 30 μM to 350 μM or 40 μM to 60 μM; The pharmaceutical preparation has a pH value of about 4.5 to 6.0, preferably about 5.0 to 5.5.

[0092] In some embodiments, the pharmaceutical formulation of the present disclosure comprises an anti-CLDN18.2 antibody (e.g., a specific anti-CLDN18.2 antibody provided by the present disclosure) having a concentration of about 1 mg / ml to 200 mg / ml and an acetate buffer or histidine buffer having a concentration of about 5 mmol / L to 100 mmol / L, and the pH of the pharmaceutical formulation is 4.5 to 6.0. In some embodiments, the pharmaceutical formulation comprises an anti-CLDN18.2 antibody having a concentration of about 20 mg / ml to 40 mg / ml. In some embodiments, the pharmaceutical formulation comprises an acetate buffer or histidine buffer having a concentration of about 10 to 30 mmol / L.

[0093] In some embodiments, the pharmaceutical formulation of the present disclosure comprises an anti-CLDN18.2 antibody (e.g., a specific anti-CLDN18.2 antibody provided by the present disclosure) having a concentration of about 1 mg / ml to 200 mg / ml, an acetate buffer or histidine buffer having a concentration of about 5 mmol / L to 100 mmol / L, and sucrose, trehalose, or sorbitol having a concentration of about 1% (w / v) to 20% (w / v), and the pH of the pharmaceutical formulation is 4.5 to 6.0. In some embodiments, the pharmaceutical formulation comprises an anti-CLDN18.2 antibody having a concentration of about 20 mg / ml to 40 mg / ml. In some embodiments, the pharmaceutical formulation comprises an acetate buffer or histidine buffer having a concentration of about 10 mmol / L to 30 mmol / L. In some embodiments, the pharmaceutical formulation comprises sucrose or trehalose having a concentration of about 5% (w / v) to 10% (w / v). In some embodiments, the pharmaceutical formulation comprises sorbitol having a concentration of about 2% (w / v) to 8% (w / v).

[0094] In some embodiments, the pharmaceutical formulation of the present disclosure comprises an anti-CLDN18.2 antibody (e.g., a specific anti-CLDN18.2 antibody provided by the present disclosure) having a concentration of about 1 mg / ml to 200 mg / ml, an acetate buffer or histidine buffer having a concentration of about 5 mmol / L to 100 mmol / L, sucrose, trehalose, or sorbitol having a concentration of about 1% (w / v) to 20% (w / v), and polysorbate 80 or poloxamer 188 having a concentration of about 0.005% (w / v) to 0.4% (w / v), and the pH of the pharmaceutical formulation is 4.5 to 6.0. In some embodiments, the pharmaceutical formulation comprises an anti-CLDN18.2 antibody having a concentration of about 20 mg / ml to 40 mg / ml. In some embodiments, the pharmaceutical formulation comprises an acetate buffer or histidine buffer having a concentration of about 10 mmol / L to 30 mmol / L. In some embodiments, the pharmaceutical formulation comprises sucrose or trehalose at a concentration of about 5% (w / v) to 10% (w / v), or sorbitol at a concentration of about 2% (w / v) to 8% (w / v), or polysorbate 80 at a concentration of about 0.01% (w / v) to 0.1% (w / v), or poloxamer 188 at a concentration of about 0.05% (w / v) to 0.2% (w / v).

[0095] In some embodiments, the pharmaceutical formulation of the present disclosure comprises an anti-CLDN18.2 antibody (e.g., a specific anti-CLDN18.2 antibody provided by the present disclosure) having a concentration of about 1 mg / ml to 200 mg / ml, an acetate buffer or histidine buffer having a concentration of about 5 mmol / L to 100 mmol / L, sucrose, trehalose, or sorbitol having a concentration of about 1% (w / v) to 20% (w / v), polysorbate 80 or poloxamer 188 having a concentration of about 0.005% (w / v) to 0.4% (w / v), and a chelating agent having a concentration of about 30 μmol / L to 350 μmol / L, and the pH of the pharmaceutical formulation is 4.5 to 6.0. In some embodiments, the pharmaceutical formulation comprises an anti-CLDN18.2 antibody having a concentration of about 20 mg / ml to 40 mg / ml. In some embodiments, the pharmaceutical formulation comprises an acetate buffer or histidine buffer at a concentration of about 10 mmol / L to 30 mmol / L. In some embodiments, the pharmaceutical formulation comprises sucrose or trehalose at a concentration of about 5% (w / v) to 10% (w / v), or sorbitol at a concentration of about 2% (w / v) to 8% (w / v). In some embodiments, the pharmaceutical formulation comprises polysorbate 80 at a concentration of about 0.01% (w / v) to 0.1% (w / v), or poloxamer 188 at a concentration of about 0.05% (w / v) to 0.2% (w / v). In some embodiments, the pharmaceutical formulation comprises a chelating agent (e.g., EDTA and EDTA-2Na) at a concentration of about 40 μmol / L to 60 μmol / L. In some other embodiments, the pharmaceutical formulation of the present disclosure is substantially free of a chelating agent.

[0096] In some embodiments, the pharmaceutical formulation of the present disclosure comprises an anti-CLDN18.2 antibody having a concentration of about 20 mg / ml to 40 mg / ml, an acetate buffer having a concentration of about 10 mmol / L to 30 mmol / L, sucrose or trehalose having a concentration of about 5% (w / v) to 10% (w / v), and polysorbate 80 having a concentration of about 0.01% (w / v) to 0.2% (w / v), and the pH value of the pharmaceutical formulation is about 4.5 to 6.0 or about 5.0 to 5.5.

[0097] In some embodiments, the pharmaceutical formulation of the present disclosure comprises an anti-CLDN18.2 antibody having a concentration of about 30 mg / ml, an acetate buffer having a concentration of about 20 mM, sucrose having a concentration of about 9% (w / v), and polysorbate 80 having a concentration of about 0.05% (w / v), and has a pH value of about 5.3.

[0098] In some embodiments, the pharmaceutical formulation of the present disclosure comprises an anti-CLDN18.2 antibody having a concentration of about 20 mg / ml to 40 mg / ml, an acetate buffer having a concentration of about 10 mmol / L to 30 mmol / L, sucrose or trehalose having a concentration of about 5% (w / v) to 10% (w / v), polysorbate 80 having a concentration of about 0.01% (w / v) to 0.2% (w / v), and EDTA or EDTA-2Na having a concentration of about 40 μmol / L to 60 μmol / L, and the pH of the pharmaceutical formulation is about 4.5 to 6.0 or about 5.0 to 5.5.

[0099] In some embodiments, the pharmaceutical formulation of the present disclosure comprises an anti-CLDN18.2 antibody having a concentration of about 30 mg / ml, an acetate buffer having a concentration of about 20 mM, sucrose having a concentration of about 9% (w / v), polysorbate 80 having a concentration of about 0.05% (w / v), and EDTA or EDTA-2Na having a concentration of about 50 μmol / L, and a pH value of about 5.3.

[0100] In some embodiments, the pharmaceutical formulation of the present disclosure comprises an anti-CLDN18.2 antibody having a concentration of about 20 mg / ml to 40 mg / ml, an acetate buffer having a concentration of about 10 mmol / L to 30 mmol / L, sorbitol having a concentration of about 2% (w / v) to 8% (w / v), and polysorbate 80 having a concentration of about 0.01% (w / v) to 0.1% (w / v), and the pH value of the pharmaceutical formulation is about 4.5 to 6.0 or about 5.0 to 5.5.

[0101] In some embodiments, the pharmaceutical formulation of the present disclosure comprises an anti-CLDN18.2 antibody having a concentration of about 20 mg / ml to 40 mg / ml, an acetate buffer having a concentration of about 10 mmol / L to 30 mmol / L, sorbitol having a concentration of about 2% (w / v) to 8% (w / v), polysorbate 80 having a concentration of about 0.01% (w / v) to 0.1% (w / v), and EDTA having a concentration of about 40 μmol / L to 60 μmol / L, and the pH value of the pharmaceutical formulation is about 4.5 to 6.0 or about 5.0 to 5.5.

[0102] In some embodiments, the pharmaceutical formulation of the present disclosure comprises an anti-CLDN18.2 antibody having a concentration of about 20 mg / ml to 40 mg / ml, a histidine buffer having a concentration of about 10 mmol / L to 30 mmol / L, sucrose having a concentration of about 6% (w / v) to 12% (w / v), and poloxamer 188 having a concentration of about 0.05% (w / v) to 0.2% (w / v), and the pH of the pharmaceutical formulation is about 4.5 to 6.0 or about 5.0 to 5.5.

[0103] In some embodiments, the pharmaceutical formulation of the present disclosure comprises an anti-CLDN18.2 antibody having a concentration of about 20 mg / ml to 40 mg / ml, a histidine buffer having a concentration of about 10 mmol / L to 30 mmol / L, sorbitol having a concentration of about 2% (w / v) to 8% (w / v), and poloxamer 188 having a concentration of about 0.05% (w / v) to 0.2% (w / v), and the pH value of the pharmaceutical formulation is about 4.5 to 6.0 or about 5.0 to 5.5.

[0104] In some embodiments, the pharmaceutical formulation of the present disclosure comprises an anti-CLDN18.2 antibody having a concentration of about 20 mg / ml to 40 mg / ml, a histidine buffer having a concentration of about 10 mmol / L to 30 mmol / L, sucrose having a concentration of about 6% (w / v) to 12% (w / v), and polysorbate 80 having a concentration of about 0.01% (w / v) to 0.1% (w / v), and the pH value of the pharmaceutical formulation is about 4.5 to 6.0 or about 5.0 to 5.5.

[0105] In some embodiments, the pharmaceutical formulation of the present disclosure comprises an anti-CLDN18.2 antibody having a concentration of about 20 mg / ml to 40 mg / ml, a histidine buffer having a concentration of about 10 mmol / L to 30 mmol / L, sorbitol having a concentration of about 2% (w / v) to 8% (w / v), and polysorbate 80 having a concentration of about 0.01% (w / v) to 0.1% (w / v), and the pH value of the pharmaceutical formulation is about 4.5 to 6.0 or about 5.0 to 5.5.

[0106] In some embodiments, the pharmaceutical formulation of the present disclosure comprises an anti-CLDN18.2 antibody having a concentration of about 20 mg / ml to 40 mg / ml, a histidine buffer having a concentration of about 10 mmol / L to 30 mmol / L, sucrose or trehalose having a concentration of about 6% (w / v) to 12% (w / v), poloxamer 188 having a concentration of about 0.05% (w / v) to 0.2% (w / v), and EDTA having a concentration of about 40 μmol / L to 60 μmol / L, and the pH value of the pharmaceutical formulation is about 4.5 to 6.0 or about 5.0 to 5.5.

[0107] Polymer formation due to chemical degradation or aggregation of antibody molecules, or deglycosylation, glycosylation modification, oxidation, or other structural modifications of antibody molecules, which can reduce at least one functional activity of the monomeric protein, can result in instability of antibody formulations. For pharmaceutical formulations containing anti-CLDN18.2 antibodies, the anti-CLDN18.2 antibodies can be chemically degraded during storage of the pharmaceutical formulation, resulting in a decrease in antibody concentration. Anti-CLDN18.2 antibodies can aggregate to form polymers that may be insoluble in the form of polymer molecules containing multiple antibody molecules, resulting in a decrease in the content of monomers containing single antibody molecules. Therefore, an increase in the content of polymeric antibodies will result in a decrease in the purity of the monomeric antibody. Furthermore, the formation of insoluble polymers can increase the turbidity of pharmaceutical formulations.

[0108] In some embodiments, pharmaceutical formulations comprising the anti-CLDN18.2 antibodies of the present disclosure can remain stable after long-term storage, treatment (e.g., storage) at high temperatures, and / or multiple freeze-thaw cycles, in which case the physical and / or chemical stability and / or functional activity of the anti-CLDN18.2 antibody remain relatively constant over time. In some embodiments, antibody protein concentration, protein purity, protein activity, pH value of the formulation, osmolality of the formulation, appearance of the formulation, insoluble particles in the formulation, and the like can be used as indicators of the stability of the pharmaceutical formulation. Various analytical techniques for determining protein stability are available in the art and are described in Peptide and Protein Drug Delivery, 247-301, edited by Vincent Lee, Marcel Dekker Inc., New York, New York Press (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993).

[0109] In some embodiments, the stability of a pharmaceutical formulation can be determined over a selected period of time under selected conditions by methods known in the art. Exemplary methods include, but are not limited to, dynamic light scattering (DLS), size exclusion chromatography (SEC), cation exchange chromatography (CEX), non-reducing capillary electrophoresis (NR CE-SDS), pH determination, protein concentration determination, and visual inspection.

[0110] As shown in the examples of the present disclosure, pharmaceutical formulations comprising the anti-CLDN18.2 antibodies provided herein have high stability by visual inspection, for example, high stability under long-term storage, high stability at high temperatures (e.g., 40°C), and high stability after freeze-thawing.

[0111] In some embodiments, a stable pharmaceutical formulation refers to one in which, in a DLS test for the pharmaceutical formulation, the protein particle size does not change significantly during storage; the KD value is positive, in other words, the force of interaction between single antibody molecules is repulsive, and aggregation of single antibody molecules does not occur. As shown in the examples of the present disclosure, pharmaceutical formulations comprising anti-CLDN18.2 antibodies provided herein have high stability as shown in DLS tests, for example, high stability under long-term storage, high stability at high temperatures (e.g., 40°C), and high stability after freeze-thawing.

[0112] In some embodiments, a stable pharmaceutical formulation refers to a pharmaceutical formulation in which, in an SEC test for the pharmaceutical formulation, only a small amount of protein is degraded during storage of the pharmaceutical formulation, and the content of high polymers or oligomers increases slowly. As shown in the examples of the present disclosure, the pharmaceutical formulations comprising the anti-CLDN18.2 antibodies provided herein have high stability as shown in SEC tests, for example, high stability under long-term storage, high stability at high temperatures (e.g., 40°C), and high stability after freeze-thawing.

[0113] In some embodiments, a stable pharmaceutical formulation refers to a pharmaceutical formulation in which the charge heterogeneity of the pharmaceutical formulation does not change significantly during storage, and the resolution of the acidic and alkaline peaks does not change significantly, as determined by a CEX test for the pharmaceutical formulation. As shown in the Examples of the present disclosure, pharmaceutical formulations comprising anti-CLDN18.2 antibodies provided herein have high stability as determined by a CEX test, for example, high stability under long-term storage, high stability at high temperatures (e.g., 40°C), and high stability after freeze-thawing.

[0114] In some embodiments, a stable pharmaceutical formulation refers to a pharmaceutical formulation in which only a small amount of protein is degraded during storage, and the content of high polymers or oligomers increases slowly, in an NR CE-SDS test for the pharmaceutical formulation. As shown in the examples of the present disclosure, the pharmaceutical formulations comprising the anti-CLDN18.2 antibodies provided herein have high stability in an NR CE-SDS test, for example, high stability under long-term storage, high stability at high temperatures (e.g., 40°C), and high stability after freeze-thawing.

[0115] In some embodiments, a stable pharmaceutical formulation refers to one in which, upon visual inspection of the pharmaceutical formulation, no obvious change in appearance of the pharmaceutical formulation is observed during storage, and the pharmaceutical formulation remains a clear, colorless liquid.

[0116] In some embodiments, a stable pharmaceutical formulation refers to a protein concentration test for the pharmaceutical formulation, wherein the protein concentration in the pharmaceutical formulation varies by no more than + / −20%, no more than + / −19%, no more than + / −18%, no more than + / −17%, no more than + / −16%, no more than + / −15%, no more than + / −14%, no more than + / −13%, no more than + / −12%, no more than + / −11%, no more than + / −10%, no more than + / −9%, no more than + / −8%, no more than + / −7%, no more than + / −6%, no more than + / −5%, no more than + / −4%, no more than + / −3%, no more than + / −2%, no more than + / −1%, or no more than + / −0.5%, and wherein the protein concentration can be determined by ultraviolet-visible spectrophotometry in accordance with General Rule 0401 of Chinese Pharmacopoeia (2010 edition), Volume III.

[0117] Preparation of formulations In another aspect, the present disclosure provides a method for preparing a pharmaceutical formulation, comprising: 1) providing a formulation solvent and an anti-CLDN18.2 antibody stock solution, the formulation solvent including a buffer, a stabilizer, and optionally a surfactant; 2) subjecting the anti-CLDN18.2 antibody stock solution to solvent exchange with a formulation solvent to obtain the pharmaceutical formulation described herein; In some embodiments, solvent exchange refers to buffer exchange, such as buffer exchange by dialysis. In some embodiments, solvent exchange refers to solvent exchange by filtration. In some embodiments, filtration refers to ultrafiltration, infiltration, gel filtration, and / or other filtration methods well known to those of skill in the art.

[0118] The present disclosure further provides a method for preparing a pharmaceutical formulation, comprising: 1) providing a formulation solvent and an anti-CLDN18.2 antibody stock solution, the formulation solvent including a buffer and a stabilizer, and the anti-CLDN18.2 antibody stock solution including a chelating agent (e.g., EDTA and EDTA-2Na); 2) subjecting the anti-CLDN18.2 antibody stock solution to solvent exchange with a formulation solvent to obtain a product, wherein the product obtained after solvent exchange is substantially free of chelating agents; 3) adding a surfactant to the product obtained after solvent exchange (i.e., the formulation solvent containing the anti-CLDN18.2 antibody) to obtain the pharmaceutical formulation described herein; The present invention provides a method comprising:

[0119] In some embodiments, the surfactant comprises polysorbate 80.

[0120] In some embodiments, the buffer in the formulation solvent is acetate buffer having a concentration of about 20 mM, the stabilizer is sucrose having a concentration of about 9% (w / v), and the surfactant is polysorbate 80 having a concentration of about 0.05% (w / v).

[0121] In some embodiments, solvent exchange refers to buffer replacement. In some embodiments, an anti-CLDN18.2 antibody stock solution is subjected to solvent exchange by dialysis with a formulation solvent. For example, a certain volume of sample can be placed in a dialysis bag (e.g., a Snake Skin® dialysis bag), the dialysis bag can be sealed, and placed in a target buffer with a volume 100 times or more, and continuously stirred to promote the replacement. Dialysis can be performed for an appropriate number of times (e.g., 3 times), for an appropriate period (e.g., 4 hours, 4 hours, and overnight), and under stirring at an appropriate speed (e.g., 150 rpm).

[0122] In some embodiments, solvent exchange refers to liquid exchange by filtration. In some embodiments, filtration refers to ultrafiltration, permeation, gel filtration, and / or other filtration methods familiar to those skilled in the art.

[0123] As used herein, the term "ultrafiltration" refers to the process of separating different substances (e.g., solvents and solutes) in a mixture (e.g., an anti-CLDN18.2 antibody stock solution) by moving them through a membrane (e.g., an ultrafiltration membrane) at different rates in response to an applied pressure driving force.

[0124] As used herein, the term "permeation" refers to the process of separating components in a mixture (e.g., an anti-CLDN18.2 antibody stock solution) based on the molecular size of the components in the mixture using a filter, such as a permeable membrane. Typically, the protein stock solution permeates through a membrane that retains the protein and allows the buffer to be exchanged. Thus, the protein-containing stock solution is replaced with fresh buffer over time.

[0125] As used herein, the term "gel filtration" refers to a process in which a gel is used to separate larger molecules (e.g., proteins) from smaller molecules by repelling molecules larger than the resin pores so that they can pass through the solid phase more quickly than smaller molecules that are retained by and move more slowly through the solid phase by diffusing into the resin pores.

[0126] In another aspect, the disclosure provides a method for preparing a pharmaceutical formulation, comprising mixing a high concentration anti-CLDN18.2 antibody stock solution with high concentrations of formulation excipients (e.g., buffers, stabilizers, and optionally surfactants), followed by dilution to a target concentration.

[0127] Applicable In another aspect, the present disclosure further provides a method for treating a disease in a subject in need thereof, the method comprising administering a therapeutically effective amount of a pharmaceutical formulation provided herein to the subject, wherein the subject has or is suspected of having a disease requiring treatment with an antibody against CLDN18.2.

[0128] As used herein, the term "treatment" or "treating" refers to reducing or alleviating the severity and / or duration of a disease state or one or more of its symptoms, inhibiting or preventing the progression of a disease state, reducing or terminating the symptoms associated with the condition, and inhibiting or preventing the recurrence, onset, onset, or progression of one or more symptoms associated with a disease state. A subject in need thereof includes a subject who has had the disease.

[0129] The term "therapeutically effective amount" refers to the minimum measurable concentration required to treat (eg, ameliorate or prevent) a particular disease state.

[0130] The pharmaceutical formulations of the present disclosure can be used to treat CLDN18.2-associated diseases, such as chronic and acute diseases. CLDN18.2-associated diseases include cancer and others. In some embodiments, CLDN18.2-associated diseases refer to cancers that express CLDN18.2. Examples of cancers that express CLDN18.2 include lung cancer (e.g., small cell lung cancer, non-small cell lung cancer (NSCLC), lung adenocarcinoma, or lung squamous cell carcinoma), gastric cancer (e.g., gastrointestinal cancer), pancreatic cancer, esophageal cancer, liver cancer (e.g., hepatocellular carcinoma / hepatocellular carcinoma), squamous cell carcinoma, peritoneal cancer, brain tumors (e.g., glioblastoma / glioblastoma multiforme (GBM), non-glioblastoma brain tumors or meningeal tumors), gliomas (e.g., ependymoma, astrocytoma, anaplastic astrocytoma, oligodendroglioma, or mixed gliomas, e.g., oligoastrocytoma [o ligodendroastrocytoma), cervical cancer, ovarian cancer, liver cancer (e.g., hepatoblastoma, hepatocellular carcinoma / hepatocellular tumor and liver cancer), bladder cancer (e.g., urothelial carcinoma), breast cancer, colon cancer, colorectal cancer, rectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer (e.g., renal rhabdomyoma), prostate cancer, vulvar cancer, penile cancer, anal cancer (e.g., squamous cell carcinoma of the anus), thyroid cancer, head and neck cancer (e.g., nasopharyngeal carcinoma), skin cancer (e.g., melanoma or squamous cell carcinoma), Osteosarcoma, Ewing's sarcoma, chondrosarcoma, soft tissue sarcomas (e.g., rhabdomyosarcoma, fibrosarcoma, and Kaposi's sarcoma), carcinoid, ocular cancer (e.g., retinoblastoma), mesothelioma, lymphocytic / lymphoblastic leukemia (e.g., acute lymphocytic / lymphoblastic leukemia (ALL) and chronic lymphocytic / lymphoblastic leukemia (CLL) of the T-cell and B-cell precursor lineages), acute myeloid / myeloblastic leukemia (AML) including mast cell leukemia, chronic myeloid / myelogenous / myeloblastic leukemia (CML), hairy Hair cell leukemia (HCL), Hodgkin's disease, non-Hodgkin's lymphoma, chronic myelocytic monocytic leukemia (CMML), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLCL), mantle cell lymphoma (MCL), Burkitt lymphoma (BL), mycosis fungoides, Cezari syndrome, cutaneous T-cell lymphoma, mast cell tumor, medulloblastoma, nephroblastoma, isolated plasma cell neoplasm, myelodysplastic syndrome, chronic and non-chronic myelodysplastic disease, central nervous system tumors,These include, but are not limited to, pituitary adenoma, vestibular schwannoma, primitive neuroectodermal tumor, ependymoma, chorioplexus papilloma, polycythemia vera, thrombocythemia, gallbladder cancer, idiopathic myelofibrosis, and childhood cancers, such as childhood sarcomas (e.g., neuroblastoma, rhabdomyosarcoma, and osteosarcoma).

[0131] The pharmaceutical formulations of the present disclosure can be administered to a subject by any suitable route. For example, the pharmaceutical formulations can be administered to a subject intravenously.

[0132] In another aspect, the present disclosure provides the use of the pharmaceutical formulation in the manufacture of a medicament for preventing and / or treating a CLDN18.2-associated disease. [Example]

[0133] Experimental method summary: In the following examples, formulation screening experiments were carried out on formulations of anti-CLDN18.2 antibodies. The anti-CLDN18.2 antibodies used in the following examples comprise a heavy chain variable region set forth in SEQ ID NO: 7 and a light chain variable region set forth in SEQ ID NO: 8. The anti-CLDN18.2 antibodies used in the following examples comprise a heavy chain set forth in SEQ ID NO: 9 and a light chain set forth in SEQ ID NO: 10.

[0134] The formulations of anti-CLDN18.2 antibodies tested in the following examples of this disclosure were prepared by the following method:

[0135] 1. Preparation of target formulation buffer: A buffer was prepared using a specific acid-base ion pair. The acid-base ion pair excipient was accurately weighed and added to Milli-Q water, approximately 60% of the volume of the target buffer, and mixed uniformly. The pH value of the resulting solution was then determined. If the pH value deviated from the target value, the pH value could be adjusted with the appropriate ion pair. The solution was then diluted with Milli-Q water to reach the target weight or volume. Finally, the conductivity, osmolality, and pH value of the solution were measured for verification.

[0136] 2. Sample preparation (by dialysis or direct dilution method): a) Dialysis method: A buffer containing anti-CLDN18.2 antibody (i.e., stock solution, also abbreviated as DS in the following examples) was exchanged into a target formulation buffer by dialysis. Specifically, a certain volume of DS sample was placed in a dialysis bag (e.g., a Snake Skin® dialysis bag), which was sealed and placed in a target buffer with a volume 100 times or more, and continuous stirring was performed to promote the exchange. Dialysis was performed three times, i.e., for 4 hours, 4 hours, and overnight, respectively, and the stirring speed was 150 rpm.

[0137] b) Direct dilution method: A high concentration excipient stock solution and a high concentration surfactant stock solution were added to a high concentration DS, which was then diluted with the target buffer system to reach the target concentration.

[0138] The anti-CLDN18.2 antibody formulations tested in the following examples of this disclosure were analyzed by the following analytical methods: 1. Dynamic Light Scattering (DLS): Protein particle size and distribution were determined by dynamic light scattering (DLS), the parameters of the method were as follows: collection for 5 seconds per measurement, 20 times in total, and measurements were performed at a temperature of 25°C. 2. Size Exclusion Chromatography (SEC): Protein aggregation was determined by SEC using an Agilent 1260 system and a TSKgel G3000SWXL column (300 × 7.8 mm, 5 μm). The mobile phase contained 50 mM sodium phosphate buffer and 300 mM NaCl, with a pH value of 6.8 + 0.1. The flow rate was 1.0 mL / min. The sample was diluted to 10 mg / mL and detected in a volume of 10 μL at a wavelength of 280 nm. 3. Cation exchange chromatography (CEX): Protein charge heterogeneity was determined by CEX using a Thermo Propac Elite WCX-10 4 mm × 150 mm 5 μm column on an Agilent 1260 Infinity system. The sample was diluted to 2.00 mg / mL with a mixture of mobile phases A and B. 4. Non-reducing capillary electrophoresis (NR CE-SDS): Protein fragments were determined by the CE-SDS (NR) method. Standard or test samples were diluted to 4 mg / mL with phosphate-citrate buffer, and then 25 μL of the sample was vortex-mixed with 75 μL of SDS sample buffer and 5 μL of NEM (100 mM N-ethylmaleimide), followed by denaturation. The denatured sample was centrifuged, followed by incubation at 70 + 2 °C for 10 + 2 minutes, cooling to room temperature, and centrifugation again. Separation was performed on a PA800 plus using an SDS separation gel kit and uncoated fused silica capillaries. 5. pH: The pH of the samples was measured by using a Seven Compact pH meter equipped with an Inlab® ExpertPro electrode. 6. Protein Concentration: Protein concentrations were obtained by determining absorbance at 280 nm using a Nano Drop 2000 spectrophotometer. The extinction coefficient (E1%) used throughout this study was 1.511 L / g-cm. Each sample was measured twice with a loading volume of 2.0 μL. The average concentration was reported. 7. Visual inspection: The appearance of the sample was inspected with a YB-2 clarity detector under a black background. Clarity and color were reported.

[0139] The sources of the reagents used in the experiments are as follows, and other reagents are conventional in the art.

[0140] [Table B]

[0141] [Example 1] pH screening experiment 1. Experimental Design A pH screening experiment was carried out on the formulation of anti-CLDN18.2 antibody. The anti-CLDN18.2 antibody protein was exchanged by dialysis into the formulation buffer as shown in Table 1, and then diluted with the corresponding formulation buffer to 30.0 mg / mL. Finally, the samples were filtered through a 0.22 μm PVDF membrane and loaded into a 2 mL vial packaging container system by pipette, respectively. The samples were incubated under different conditions listed in Table 1 and then tested.

[0142] [Table 1]

[0143] 2. Analysis of Results (1) At TO, formulations F7, F8, and F9 appeared as slightly opalescent colorless liquids, while the other formulations were clear and colorless liquids. After 14 days of incubation at 40°C, no significant changes were observed in the appearance of any of the other formulations except for formulations F7, F8, and F9.

[0144] [Table 2]

[0145] (2) At TO, the KD values of formulations F7, F8, and F9 were negative, indicating that the force of interaction between single antibody molecules was attractive. The KD values of the other formulas were positive, indicating that the force of interaction between single antibody molecules was repulsive. At TO, the radii of single antibody molecules in formulations F7, F8, and F9 were obviously larger than those in the other formulas. Therefore, formulations F1, F2, F3, F4, F5, and F6 all met the requirements for KD value and particle size at TO.

[0146] [Table 3]

[0147] (3) SEC testing was performed on formulations F1, F2, F3, F4, F5, and F6. As shown in Table 4, after 14 days of incubation at 40°C, formulations F1, F2, F3, F4, F5, and F6 had a small amount of fragments (LMW) formed by degradation of the main peak (MP), and a gradual increase in high molecular weight (HMW). Therefore, the acceptance criteria under such testing conditions were met, and all formulations met the stability requirements.

[0148] [Table 4]

[0149] (4) CEX test was performed. After 14 days of incubation at 40°C, all formulations had degradation of the main peak and formation of an acidic peak. Therefore, the acceptance criteria under such test conditions were met. There was no obvious difference between the formulations.

[0150] [Table 5]

[0151] (5) NR CE-SDS test was performed. After 14 days of incubation at 40°C, all formulations had a small amount of major peak degradation and fragment formation, thus meeting the acceptance criteria under such test conditions.

[0152] [Table 6]

[0153] In conclusion, after incubation at 40°C, the citrate system was excluded based on the DLS and appearance results because the force of interaction between single antibody molecules was attractive and the single antibody molecules had a large radius, indicating that the single antibody molecules had a risk of aggregation during long-term storage. Meanwhile, the formulations in the acetate and histidine systems (i.e., formulations F1, F2, F3, F4, F5, and F6) were relatively stable, so as to meet the stability requirements of protein formulations.

[0154] [Example 2] Surfactant screening experiment-1 1. Experimental Design A surfactant screening experiment was performed on the anti-CLDN18.2 antibody protein. The anti-CLDN18.2 antibody protein was filtered through a 0.22 μm PVDF membrane, and a sucrose solution and a polysorbate 80 solution were added. The anti-CLDN18.2 antibody protein was then diluted with 20 mM acetate buffer and milli-Q water at a pH of 5.5 to prepare the following formulations (F10, F11, F12, and F13). Acetic acid and sucrose were used as buffer and stabilizer, respectively. The final concentration of the anti-CLDN18.2 antibody protein was 30.0 mg / mL. Finally, the samples were filtered through a 0.22 μm PVDF membrane and loaded into a 2 mL vial packaging container system by pipette, followed by the stability study listed in Table 7. The samples were examined after incubation.

[0155] [Table 7]

[0156] 2. Analysis of Results The 40°C data (as shown in Table 8) and vibration data (as shown in Table 9) indicated that polysorbate 80 with a concentration range of 0.01% (w / v) to 0.1% (w / v) had a clear protective effect on the stability of single antibody molecules. All formulations met the testing requirements, and polysorbate 80 with a concentration of 0.05% (w / v) was used for further evaluation in subsequent experiments.

[0157] [Table 8]

[0158] [Table 9]

[0159] [Example 3] Surfactant screening experiment-2 1. Experimental Design A surfactant screening experiment was performed on the anti-CLDN18.2 antibody protein. The anti-CLDN18.2 antibody protein was filtered through a 0.22 μm PES membrane, and a sucrose solution and a poloxamer 188 solution were added. The anti-CLDN18.2 antibody protein was then diluted with 20 mM histidine buffer with a pH value of 5.5 to prepare the following formulation: Histidine and sucrose were used as a buffer and a stabilizer, respectively. The final concentration of the anti-CLDN18.2 antibody protein was 30.0 mg / mL. Finally, the sample was filtered through a 0.22 μm PES membrane, followed by the stability study listed in Table 10. The sample was examined after incubation.

[0160] [Table 10]

[0161] 2. Analysis of Results According to the SEC, CE-SDS, and CEX data, no obvious differences were observed among formulations F14, F15, and F16 (Figures 1-3). Therefore, poloxamer 188 surfactant with concentrations of 0.05% (w / v) to 0.20% (W / V) also meets the formulation requirements.

[0162] [Example 4] Excipient screening experiments 1. Experimental Design An excipient screening experiment was carried out on the anti-CLDN18.2 antibody protein. The anti-CLDN18.2 antibody protein was filtered through a 0.22 μm PVDF membrane, and an excipient solution and a polysorbate 80 solution were added. The anti-CLDN18.2 antibody protein was then prepared into the following formulation: the formulation was diluted with 20 mM acetic acid-sodium acetate buffer with a pH value of 5.5 and water. The final concentration of the anti-CLDN18.2 antibody protein was 30.0 mg / mL. Finally, the samples were filtered through a 0.22 μm PVDF membrane and loaded into 2 mL vial packaging container systems by pipette, followed by the stability studies listed in Table 11.

[0163] [Table 11]

[0164] 2. Analysis of Results (1) Freeze-thaw test The results are shown in Table 12. At TO, formulations F17 and F20 appeared as colorless liquids with a slight opalescence, while formulations F18 and F19 were clear and colorless liquids. After five freeze-thaw cycles, no obvious changes were observed in the protein concentration or appearance of formulations F18 and F19. At TO, the KD values of formulations F17 and F20 were negative, indicating that the force of interaction between single antibody molecules was attractive. The KD values of formulations F18 and F19 were positive, indicating that the force of interaction between single antibody molecules was repulsive. At TO, the radii of single antibody molecules in formulations F17 and F20 were significantly larger than those in the other formulations. After five freeze-thaw cycles, the radii of single antibody molecules in formulations F18 and F19 remained essentially unchanged, achieving a unimodal distribution. No significant differences or changes were observed in the SEC, NR CE-SDS, and CEX main peaks. No significant differences or changes in particles not visible to the naked eye are observed. Thus, formulations F18 and F19 meet the experimental requirements.

[0165] [Table 12]

[0166] (2) High temperature inspection The results are shown in Table 13. After 7 and 14 days of incubation at 40°C, no significant changes were observed in the protein concentration, appearance, or protein particle size of formulations F18 and F19. A small amount of the SEC, NR CE-SDS, and CEX major peaks were degraded. However, acceptable standards were met, and there was no significant difference between the formulations.

[0167] [Table 13]

[0168] (3) Conclusion Based on the DLS and appearance results, excipients such as sodium chloride and arginine hydrochloride were excluded because the force of interaction between single antibody molecules changed from repulsive to attractive after the addition of such excipients, resulting in the risk of molecular aggregation during long-term storage, which was not conducive to long-term storage of the formulation.The results of the 40°C study and the freeze-thaw study showed that sucrose and trehalose had essentially the same protective effect on the stability of single antibody molecules, and both could meet the needs of developing stable formulations.

[0169] [Example 5] Excipient screening experiment-2 1. Experimental Design An excipient screening experiment was carried out on the anti-CLDN18.2 antibody protein. The anti-CLDN18.2 antibody protein was filtered through a 0.22 μm PES membrane, and an excipient solution and a polysorbate 80 solution were added. The anti-CLDN18.2 antibody protein was then prepared into the following formulation, which was diluted with a 20 mM histidine buffer with a pH value of 5.5. The final concentration of the anti-CLDN18.2 antibody protein was 30.0 mg / mL. Finally, the sample was filtered through a 0.22 μm PES membrane, followed by the stability study listed in Table 14.

[0170] [Table 14]

[0171] 2. Analysis of Results According to the stirring experiments and the SEC, CEX and CE-SDS results, both formulations F21 and F22 meet the requirements (as shown in Figures 4-6 and Table 15).

[0172] [Table 15]

[0173] Therefore, both sucrose and sorbitol meet the requirements.

[0174] [Example 6] Formulation validation experiment 1. Experimental Design Based on the above experiments, two formulations that met the requirements were selected to verify the optimal formulation.

[0175] The surfactant solution and excipient solution were added to the buffer system containing the anti-CLDN18.2 antibody protein to adjust the anti-CLDN18.2 antibody protein concentration to 30.0 mg / mL to prepare the following formulation: The samples were filtered through a 0.22 μm PVDF membrane and loaded separately into a 2 mL vial packaging container system and a 5 mL polycarbonate flask by pipette, followed by a stability study.

[0176] [Table 16]

[0177] 2. Analysis of Results In accelerated testing (as shown in Table 17), protein purity (measured by both SEC and NR CE-SDS) decreased slightly due to degradation in the acetate buffer system (Formulation F23) and the histidine buffer system (Formulation F22), with protein degraded to a lesser extent in the acetate buffer system (Formulation F23). CEX purity did not change significantly in the acetate buffer system, but did change significantly in the histidine buffer system. In long-term testing (as shown in Table 18) and low-temperature testing (as shown in Table 19), the acetate buffer system (Formulation F23) and the histidine buffer system (Formulation F22) did not change significantly in appearance, pH, concentration, DLS, and purity (including SEC, NR CE-SDS, and CEX). Considering that the tendency of protein degradation under long-term storage conditions can be better simulated by the conditions of the accelerated test, the acetate buffer system (formulation F23), which was more stable in the accelerated test, was selected as the final formulation. The final formulation contained 30.0 mg / mL of anti-CLDN18.2 antibody protein, 20 mM acetic acid / sodium acetate, 9% (w / v) sucrose, and 0.05% (w / v) polysorbate 80, and had a pH of 5.3.

[0178] [Table 17]

[0179] [Table 18]

[0180] [Table 19]

[0181] [Example 7] Investigating the protective effect of edetate disodium (EDTA-Na2) 1. Experimental Design The following samples shown in the table below were prepared to investigate the protective effect of edetate disodium in preventing the degradation of polysorbate 80 (PS80) and maintaining protein quality in formulations F23 and F24 (for F24, EDTA was added to the stock solution, shaken to mix uniformly with the stock solution, EDTA was removed by dialysis, and the final formulation was formulated) and formulation F25. Formulation F24 was prepared by the following method: EDTA was added to the antibody stock solution, and the antibody stock solution was subjected to solvent exchange with a formulation solvent containing a buffer and stabilizer, simultaneously achieving the effects of EDTA removal and solvent exchange by filtration, and finally, a surfactant was added to obtain the final formulation.

[0182] [Table 20]

[0183] 2. Analysis of Results Under forced experimental conditions at 40°C, it can be seen from Figure 7 that the degradation of PS80 can be effectively prevented by either adding EDTA during downstream purification (formulation F24) or to the formulation (formulation F25). It can be seen from Figure 8 that the degradation of the main peak of the protein was reduced by formulations F24 and F25.

Claims

1. A pharmaceutical formulation comprising an anti-CLDN18.2 antibody and a buffer, wherein the buffer is an acetate buffer or a histidine buffer, and the pH value of the pharmaceutical formulation is 4.5 to 6.

0.

2. 2. The pharmaceutical formulation of claim 1, wherein the concentration of the buffer in the pharmaceutical formulation is 5 mM to 50 mM or 10 mM to 30 mM.

3. 3. The pharmaceutical formulation of claim 1 or 2, further comprising a stabilizer.

4. 4. The pharmaceutical formulation of claim 3, wherein the concentration of the stabilizer in the pharmaceutical formulation is 1% (w / v) to 20% (w / v), or 1% (w / v) to 10% (w / v).

5. 5. The pharmaceutical formulation of claim 3, wherein the stabilizer is selected from the group consisting of sucrose, trehalose and sorbitol.

6. (a) the stabilizer is sucrose or trehalose, and the concentration of the sucrose or trehalose in the pharmaceutical formulation is 5% (w / v) to 10% (w / v); or (b) the stabilizer is sorbitol, and the concentration of the sorbitol in the pharmaceutical formulation is 2% (w / v) to 8% (w / v); The pharmaceutical formulation of claim 5.

7. 10. The pharmaceutical formulation according to any one of the preceding claims, further comprising a surfactant.

8. 8. The pharmaceutical formulation of claim 7, wherein the concentration of the surfactant in the pharmaceutical formulation is 0.005% (w / v) to 0.4% (w / v), or 0.01% (w / v) to 0.2% (w / v).

9. 9. The pharmaceutical formulation of claim 7 or 8, wherein the surfactant is selected from the group consisting of polysorbate 80 and poloxamer 188.

10. (a) the surfactant is polysorbate 80, and the concentration of polysorbate 80 in the pharmaceutical formulation is 0.01% (w / v) to 0.1% (w / v); or (b) the surfactant is poloxamer 188, and the concentration of the poloxamer 188 in the pharmaceutical formulation is 0.05% (w / v) to 0.2% (w / v); 10. The pharmaceutical formulation of claim 9.

11. 10. The pharmaceutical formulation of any one of the preceding claims, further comprising a chelating agent.

12. 12. The pharmaceutical formulation of claim 11, wherein the concentration of the chelating agent in the pharmaceutical formulation is 30 μM to 350 μM or 40 μM to 60 μM.

13. 13. The pharmaceutical formulation of claim 11 or 12, wherein the chelating agent is selected from the group consisting of EDTA, DTPA, IDHA, EDDHA and HBED.

14. 10. The pharmaceutical formulation of any one of the preceding claims, wherein the concentration of the anti-CLDN18.2 antibody in the pharmaceutical formulation is from 1 mg / ml to 200 mg / ml.

15. The pharmaceutical formulation of claim 14, wherein the concentration of the anti-CLDN18.2 antibody in the pharmaceutical formulation is 20 mg / ml to 40 mg / ml.

16. The pharmaceutical formulation of any one of the preceding claims, wherein the anti-CLDN18.2 antibody comprises a heavy chain CDR1 (HCDR1) as set forth in SEQ ID NO: 1, a HCDR2 as set forth in SEQ ID NO: 2, and a HCDR3 as set forth in SEQ ID NO: 3, and a light chain CDR1 (LCDR1) as set forth in SEQ ID NO: 4, a LCDR2 as set forth in SEQ ID NO: 5, and a LCDR3 as set forth in SEQ ID NO:

6.

17. The pharmaceutical formulation of any one of the preceding claims, wherein the anti-CLDN18.2 antibody comprises CDR1, CDR2 and CDR3 of the heavy chain variable region set forth in SEQ ID NO: 7, and CDR1, CDR2 and CDR3 of the light chain variable region set forth in SEQ ID NO:

8.

18. The pharmaceutical formulation of any one of the preceding claims, wherein the anti-CLDN18.2 antibody comprises a heavy chain variable region set forth in SEQ ID NO: 7 and a light chain variable region set forth in SEQ ID NO:

8.

19. 10. The pharmaceutical formulation of any one of the preceding claims, wherein the anti-CLDN18.2 antibody comprises a heavy chain as set forth in SEQ ID NO: 9 and a light chain as set forth in SEQ ID NO:

10.

20. 10. The pharmaceutical formulation of any one of the preceding claims, comprising an anti-CLDN18.2 antibody, a buffer, a stabilizer and a surfactant, wherein the buffer is an acetate buffer, the stabilizer is sucrose or trehalose, the surfactant is polysorbate 80, and the pH value of the pharmaceutical formulation is about 4.5 to 6.

0.

21. The concentration of the anti-CLDN18.2 antibody in the pharmaceutical formulation is 20 mg / ml to 40 mg / ml, the concentration of the acetate buffer in the pharmaceutical formulation is 10 mM to 30 mM, the concentration of the sucrose or trehalose in the pharmaceutical formulation is 5% (w / v) to 10% (w / v), and / or the concentration of the polysorbate 80 in the pharmaceutical formulation is 0.01% (w / v) to 0.2% (w / v). The pharmaceutical formulation of claim 20.

22. The concentration of the anti-CLDN18.2 antibody in the pharmaceutical formulation is about 30 mg / ml, the concentration of the acetate buffer in the pharmaceutical formulation is about 20 mM, the concentration of the sucrose or trehalose in the pharmaceutical formulation is about 9% (w / v), the concentration of the polysorbate 80 in the pharmaceutical formulation is 0.01% (w / v) to 0.1% (w / v), and the pH value is about 5.0 to 5.

5. The pharmaceutical formulation of claim 21.

23. 23. The pharmaceutical formulation of claim 22, wherein the stabilizer is sucrose, the concentration of the polysorbate 80 in the pharmaceutical formulation is about 0.05% (w / v), and the pH value is about 5.

3.

24. 24. The pharmaceutical formulation according to any one of claims 20 to 23, further comprising EDTA, wherein the concentration of said EDTA in said pharmaceutical formulation is from 40 μM to 60 μM.

25. 25. The pharmaceutical formulation of claim 24, wherein the concentration of the EDTA in the pharmaceutical formulation is about 50 μM.

26. The pharmaceutical formulation of any one of the preceding claims, comprising an anti-CLDN18.2 antibody, a buffer, a stabilizer and a surfactant, wherein the buffer is a histidine buffer, the stabilizer is sucrose or sorbitol, and the surfactant is polysorbate 80 or poloxamer 188.

27. 27. The pharmaceutical formulation of claim 26, wherein the stabilizer is sucrose and the concentration of the sucrose in the pharmaceutical formulation is 6% (w / v) to 12% (w / v).

28. 27. The pharmaceutical formulation of claim 26, wherein the stabilizer is sorbitol, and the concentration of the sorbitol in the pharmaceutical formulation is 2% (w / v) to 8% (w / v).

29. 29. The pharmaceutical formulation according to any one of claims 26 to 28, wherein the surfactant is polysorbate 80 and the concentration of polysorbate 80 in the pharmaceutical formulation is 0.01% (w / v) to 0.1% (w / v).

30. 29. The pharmaceutical formulation according to any one of claims 26 to 28, wherein the surfactant is poloxamer 188 and the concentration of the poloxamer 188 in the pharmaceutical formulation is 0.05% (w / v) to 0.2% (w / v).

31. 1. A method for preparing a pharmaceutical formulation, comprising: 1) Providing a formulation solvent and an anti-CLDN18.2 antibody stock solution, wherein the formulation solvent comprises a buffer, a stabilizer, and optionally a surfactant; 2) subjecting the anti-CLDN18.2 antibody stock solution to solvent exchange with the formulation solvent to obtain the pharmaceutical formulation according to any one of claims 1 to 30; A method comprising:

32. 32. The method of claim 31, wherein the anti-CLDN18.2 antibody stock solution contains a chelating agent (e.g., EDTA), the formulation solvent does not contain a chelating agent, and the pharmaceutical formulation is substantially free of chelating agents after filtration.

33. 33. The method of claim 32, wherein the formulation solvent comprises a buffer and a stabilizer and is surfactant-free.

34. 32. The method of claim 31, wherein the formulation medium comprises a buffer, a stabilizer, and a chelating agent (e.g., EDTA).

35. The method of any one of claims 33 to 34, wherein the surfactant is added after the anti-CLDN18.2 antibody stock solution is subjected to solvent exchange with the formulation solvent to obtain the pharmaceutical formulation of any one of claims 1 to 30.

36. 10. The pharmaceutical formulation of any one of the preceding claims, wherein the acetate buffer is an acetic acid-sodium acetate buffer and the histidine buffer is a histidine-histidine hydrochloride buffer.

37. Use of a pharmaceutical preparation according to any one of claims 1 to 36 in the manufacture of a medicament for preventing and / or treating a CLDN18.2-related disease.

38. 38. The pharmaceutical preparation of claim 37, wherein the CLDN18.2-related disease is selected from the group consisting of gastric cancer, adenocarcinoma of the gastroesophageal junction, lung cancer, bronchogenic carcinoma, bone cancer, perihilar cholangiocarcinoma, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, bladder cancer, head and neck cancer, spinal cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, anal cancer, esophageal cancer, gastrointestinal cancer, skin cancer, prostate cancer, pituitary cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, or adenocarcinoma.

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