Stable pharmaceutical formulations comprising anti-CLDN18.2 antibodies

Abstract

The invention discloses a pharmaceutical preparation. The pharmaceutical preparation comprises an anti-Claudin (CLDN) 18.2 antibody, a buffer solution, a stabilizing agent and a surface active agent. The pharmaceutical preparation can still maintain the stability of the anti-CLDN18.2 antibody after long-term storage, high temperature (for example, 40 DEG C) and / or multiple freeze thawing. The invention also discloses an application of the medicinal preparation in preparation of medicines for preventing and / or treating CLDN18.2 related diseases, especially cancers (for example, CLDN18.2 related cancers), and a method for preparing the medicinal preparation.

Description

Technical Field

[0001] The present application relates to a pharmaceutical preparation, in particular to a stable pharmaceutical preparation containing an anti-CLDN18.2 antibody. The present application also relates to a preparation method of the pharmaceutical preparation and use thereof. Background Art

[0002] Studies have shown that CLDN18.2 belongs to the Claudin (CLDN) family and is one of the subtypes of CLDN18. CLDN18.2 is a highly selective gastric lineage marker and is highly expressed in a variety of cancer types, including gastric cancer, esophageal cancer, pancreatic cancer, and lung cancer. The CLDN18.2 protein structure includes four transmembrane regions and two extracellular loops. It is a transmembrane protein located on the surface of the cell membrane. As a cell membrane surface protein, CLDN18.2 has an exposed extracellular structure that allows antibody binding and is an ideal target for the development of therapeutic antibodies. Anti-CLDN18.2 antibodies can specifically recognize and bind to CLDN18.2 molecules on the surface of tumor cells, thereby inducing antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), apoptosis, and inhibition of cell proliferation, eliminating cancer cells and controlling the disease.

[0003] Since antibody molecules have a complex multi-level protein structure and are prone to physically stick together, which may cause undesirable immune responses or may clog syringes or pumps during administration and be unsafe for patients, one of the problems that liquid formulations of antibodies have long faced is the stability problem caused by aggregation.

[0004] Therefore, there is a need for pharmaceutical formulations of anti-CLDN18.2 antibodies with stability and consistent quality. SUMMARY OF THE INVENTION

[0006] The present application provides a stable pharmaceutical formulation comprising an anti-CLDN18.2 antibody that remains homogeneous and stable after long-term storage, high temperature (eg, 40° C.) and / or multiple freeze-thaw cycles.

[0007] In one aspect, the present application provides a pharmaceutical preparation comprising an anti-CLDN18.2 antibody and a buffer, wherein the buffer is an acetate buffer or a histidine buffer, and the pH value is 4.5-6.0. In some embodiments, the concentration of the buffer in the pharmaceutical preparation is 5-50 mM, or 10-30 mM.

[0008] In some embodiments, the pharmaceutical formulation further comprises a stabilizer.

[0009] In some embodiments, the concentration of the stabilizer in the pharmaceutical formulation is 1-20% (w / v), or 1-10% (w / v).

[0010] In some embodiments, the stabilizer is selected from the group consisting of: sucrose, trehalose, and sorbitol.

[0011] In some embodiments, the stabilizer is sucrose or trehalose, and the concentration of sucrose or trehalose in the pharmaceutical preparation is 5-10% (w / v). In other embodiments, the stabilizer is sorbitol, and the concentration of sorbitol in the pharmaceutical preparation is 2-8% (w / v).

[0012] In some embodiments, the pharmaceutical formulation further comprises a surfactant.

[0013] In some embodiments, the concentration of the surfactant in the pharmaceutical preparation is 0.005% (w / v) to 0.4% (w / v), or 0.01% (w / v) to 0.2% (w / v).

[0014] In some embodiments, the surfactant is selected from the group consisting of: polysorbate 80 and poloxamer 188.

[0015] In some embodiments, the surfactant is polysorbate 80, and the concentration of polysorbate 80 in the pharmaceutical preparation 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 preparation is 0.05% (w / v) to 0.2% (w / v).

[0016] In some embodiments, the pharmaceutical formulation further comprises a chelating agent.

[0017] In some embodiments, the concentration of the chelating agent in the pharmaceutical formulation is 30-350 μM, or 40-60 μM.

[0018] In some embodiments, the chelating agent is selected from the group consisting of: EDTA, DTPA, IDHA, EDDHA, HBED.

[0019] In some embodiments, the concentration of the anti-CLDN18.2 antibody in the pharmaceutical preparation is 1 to 200 mg / ml.

[0020] In some embodiments, the concentration of the anti-CLDN18.2 antibody in the pharmaceutical preparation is 20-40 mg / ml.

[0021] In some embodiments, the anti-CLDN18.2 antibody comprises a heavy chain CDR1 (HCDR1) as shown in SEQ ID NO:1, a HCDR2 as shown in SEQ ID NO:2, and a HCDR3 as shown in SEQ ID NO:3, and a light chain CDR1 (LCDR1) as shown in SEQ ID NO:4, a LCDR2 as shown in SEQ ID NO:5, and a LCDR3 as shown in SEQ ID NO:6.

[0022] In some embodiments, the anti-CLDN18.2 antibody comprises CDR1, CDR2, and CDR3 of the heavy chain variable region as shown in SEQ ID NO:7, and CDR1, CDR2, and CDR3 of the light chain variable region as shown in SEQ ID NO:8.

[0023] In some embodiments, the anti-CLDN18.2 antibody comprises a heavy chain variable region as shown in SEQ ID NO:7 and a light chain variable region as shown in SEQ ID NO:8.

[0024] In some embodiments, the anti-CLDN18.2 antibody comprises a heavy chain as shown in SEQ ID NO:9 and a light chain as shown in SEQ ID NO:10.

[0025] In some embodiments, the pharmaceutical preparation 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 value is about 4.5-6.0.

[0026] In some embodiments, the concentration of the anti-CLDN18.2 antibody in the pharmaceutical preparation is 20-40 mg / ml, the concentration of the acetate buffer in the pharmaceutical preparation is 10-30 mM, the concentration of sucrose or trehalose in the pharmaceutical preparation is 5-10% (w / v), and / or the concentration of polysorbate 80 in the pharmaceutical preparation is 0.01% (w / v) to 0.2% (w / v).

[0027] 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.

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

[0029] In some embodiments, the pharmaceutical preparation 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.

[0030] In some embodiments, the stabilizer is sucrose, and the concentration of the sucrose in the pharmaceutical preparation is 6-12% (w / v). In other embodiments, the stabilizer is sorbitol, and the concentration of the sorbitol in the pharmaceutical preparation is 2-8% (w / v).

[0031] In some embodiments, the surfactant is polysorbate 80, and the concentration of polysorbate 80 in the pharmaceutical preparation 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 preparation is 0.05% (w / v) to 0.2% (w / v).

[0032] On the other hand, the present application provides a method for preparing a pharmaceutical preparation, the method comprising: 1) providing a preparation solvent and an anti-CLDN18.2 antibody stock solution, wherein the preparation solvent comprises a buffer and a stabilizer and optionally a surfactant; 2) using the preparation solvent to replace the anti-CLDN18.2 antibody stock solution, thereby obtaining the pharmaceutical preparation as described herein.

[0033] In some embodiments, the anti-CLDN18.2 antibody stock solution in the methods described herein contains a chelating agent (e.g., EDTA), the formulation medium does not contain a chelating agent, and the pharmaceutical formulation contains almost no chelating agent. In other embodiments, the formulation medium contains a buffer and a stabilizer and does not contain a surfactant.

[0034] In other embodiments, the formulation vehicle in the methods described herein comprises a buffer, a stabilizer, and a chelating agent (eg, EDTA).

[0035] In other embodiments, a surfactant is added after the anti-CLDN18.2 antibody stock solution is replaced with the formulation vehicle, thereby obtaining a pharmaceutical formulation as described herein.

[0036] In some embodiments, in the formulations or methods described herein, the acetate buffer is an acetic acid-sodium acetate buffer; and the histidine buffer is a histidine-histidine hydrochloride buffer.

[0037] In another aspect, the present application provides use of the pharmaceutical formulation described herein in the preparation of a medicament for preventing and / or treating a CLDN18.2-related disease.

[0038] In some embodiments, the CLDN18.2-related disease is selected from gastric cancer, gastroesophageal junction adenocarcinoma, lung cancer, bronchial cancer, bone cancer, hepatobiliary cancer, 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 DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The stability data of the protein measured by SEC in a surfactant screening experiment in which three different concentrations of Poloxamer 188 (formulations F14, F15 and F16) were selected as surfactants are shown.

[0040] Figure 2 The stability data of the anti-CLDN18.2 antibody protein measured by CEX in a surfactant screening experiment in which three different concentrations of Poloxamer 188 (formulations F14, F15 and F16) were selected as surfactants are shown.

[0041] Figure 3 The graph shows the protein stability data measured by CE-SDS in a surfactant screening experiment in which three different concentrations of Poloxamer 188 (formulations F14, F15 and F16) were selected as surfactants.

[0042] Figure 4 The protein stability data measured by SEC in the excipient screening experiment where sorbitol and sucrose were selected as stabilizers (formulations F21 and F22, respectively) are shown.

[0043] Figure 5 The protein stability data measured by CEX are shown in the excipient screening experiment where sorbitol and sucrose were selected as stabilizers (formulations F21 and F22, respectively).

[0044] Figure 6 The protein stability data measured by CE-SDS are shown in the excipient screening experiment in which sorbitol and sucrose were selected as stabilizers (formulations F21 and F22, respectively).

[0045] Figure 7The protective effect of disodium edetate (disodium EDTA) on polysorbate 80 (PS80) against degradation is shown.

[0046] Figure 8 Shown is the protective effect of disodium edetate (disodium EDTA) on protein stability as measured by NR CE-SDS. DETAILED DESCRIPTION

[0047] The following description of the application is only intended to illustrate each embodiment of the application. The specific embodiments described should not be construed as limiting the scope of the application. Without departing from the spirit and essence of the application, those skilled in the art may perform various equivalent substitutions, changes or variations, and should be understood that these equivalent embodiments are also included herein. All documents cited herein, including publications, patents and patent applications, are incorporated herein by reference in their entirety. When the method mentioned in the present disclosure includes the situation of two or more limiting steps, the defined steps may be performed in any order or simultaneously (unless the context excludes this possibility), and the method may include one or more other steps, and the step may be performed before any defined step, between two defined steps or after all defined steps (unless the context excludes this possibility).

[0048] References to "about" (a certain value or a certain numerical range) herein include embodiments for the value or the numerical range itself, for example, "about X" includes "X". In general, the term "about" refers to the numerical value of a certain variable and all numerical values ​​within the experimental error range of the variable (for example, within the 95% confidence interval of the mean value) or all numerical values ​​within 10% of the variable, whichever is greater. For example, "about X" includes "110% × X", "109% × X", "108% × X", "107% × X", "106% × X", "105% × X", "104% × X", "103% × X", "102% × X", "101% × X", "99% × X", "98% × X", "97% × X", "96% × X", "95% × X", "94% × X", "93% × X", "92% × X", "91% × X" or "90% × X".

[0049] The term "at least" followed by a number is used herein to indicate the beginning of a range that begins with that number (which can be a range with an upper limit or without an upper limit, depending on the variables defined). For example, "at least 1" means 1 or greater than 1. The term "at most" followed by a number is used herein to indicate the end of a range that ends with that number (which can be a range with 1 or 0 as a lower limit, or a range with no lower limit, depending on the variables defined). For example, "at most 4" means 4 or less than 4, and "at most 40%" means 40% or less than 40%. In the present disclosure, when a range is set to "(first number) to (second number)" or "(first number) to (second number)", this means that the lower limit of the range is the first number and the upper limit is the second number. For example, It refers to a range with a lower limit of 5 mg / mL and an upper limit of 50 mg / mL. The term "less than" or "greater than" a certain value includes the value in the present invention.

[0050] Antibody

[0051] As used herein, the term "antibody" includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody or bispecific antibody that binds to a specific antigen. A complete antibody comprises two heavy chains and two light chains. Each heavy chain consists of a heavy chain variable region (V H ) and the first, second and third heavy chain constant regions (C H1 , C H2 , C H3 ), and each light chain consists of a light chain variable region (V L ) and the light chain constant region (C L ) is composed of heavy and light chains. H and V L There are also three complementary determining regions (CDRs) in each region, and the three CDRs are separated by a continuous portion called a framework region (FR), which is more highly conserved than the CDR and forms a scaffold to support the hypervariable loop. The six CDRs of a heavy chain and a light chain together constitute the antigen binding site of the antibody, which determines the specificity of the antibody. The antibodies described herein also include fragments or derivatives with antigen binding function of the complete antibody, and the fragments or derivatives have the same antigen binding specificity as the complete antibody, but the affinity of the fragments or derivatives to their specific antigens may be the same or different from that of the complete antibody.

[0052] In some embodiments, the antibodies described herein include antigen-binding fragments. Antigen-binding fragments refer to one or more antibody fragments that retain the ability to specifically bind to an antigen. Examples of antigen-binding fragments include, but are not limited to (i) Fab fragments, which refer to fragments consisting of V L 、VH , C L and C H1 (ii) a Fab' fragment, which refers to a Fab fragment containing a portion of the hinge region; (iii) a F(ab')2 fragment, which refers to a bivalent fragment containing two Fab fragments connected by a disulfide bond in the hinge region; (iv) a fragment consisting of V H and C H1 The Fd fragment consists of a single-arm V L and V H (vi) dAb fragments (Ward et al., Nature 341: 544-546 (1989); PCT Publication WO 90 / 05144), which contain a single variable domain; (vii) isolated CDRs; (viii) single-chain Fv fragments, which refer to V L and V H The domains are directly linked or connected through a peptide chain to form a monovalent fragment (Huston JS et al., Proc Natl Acad Sci USA, 85: 5879 (1988)).

[0053] In some embodiments, the antibodies described herein include chimeric antibodies, a portion of which the heavy and / or light chains are identical or homologous to the corresponding sequences of antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remaining chains are identical or homologous to the corresponding sequences of antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired functional activity.

[0054] In some embodiments, the antibodies described herein include humanized antibodies. The humanized form of a non-human (e.g., mouse) antibody can be a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding sequence of an antibody) containing minimal sequences obtained from a non-human immunoglobulin. In some examples, a humanized antibody can be a CDR-grafted antibody, in which the amino acid sequence of a human CDR is introduced into a non-human V H and V LThe amino acid sequence of a humanized antibody is replaced by an amino acid sequence of a corresponding non-human CDR. In other examples, most of the amino acid sequence of a humanized antibody can be derived from a human immunoglobulin (receptor antibody), wherein the amino acid residues of the CDR of the receptor are replaced by amino acid residues of the CDR of a non-human (e.g., mouse, rat, rabbit) antibody having the desired specificity, affinity, and ability. Usually, a humanized antibody substantially comprises at least one, generally comprising two variable domains, wherein all or substantially all of the CDR regions correspond to the sequence of a non-human immunoglobulin, and all or substantially all of the framework (FR) regions are sequences of human immunoglobulins. In some examples, the framework region residues of the variable region of a human immunoglobulin are replaced by corresponding non-human residues. Moreover, a humanized antibody may include residues that are not present in both the original antibody and the introduced CDR or framework region sequences.

[0055] The anti-CLDN18.2 antibody described herein refers to an antibody that can specifically bind to the CLDN18.2 protein.

[0056] The CLDN18.2 protein described herein refers to Claudin-18 splice variant 2 derived from mammals, such as primates (e.g., humans, monkeys) and rodents (e.g., mice). In certain embodiments, CLDN18.2 is human CLDN18.2. Exemplary sequences of human CLDN18.2 include human CLDN18.2 protein (NCBI Ref Seq No. NP_001002026.1). CLDN18.2 can be expressed in cancer cells. In one embodiment, the CLDN18.2 is expressed on the surface of cancer cells.

[0057] In some embodiments, the anti-CLDN18.2 antibodies described herein include a heavy chain variable region V H , the heavy chain variable region V H It has a CDR1 as shown in SEQ ID NO:1, a CDR2 as shown in SEQ ID NO:2, and / or a CDR3 as shown in SEQ ID NO:3.

[0058] In some embodiments, the anti-CLDN18.2 antibodies described herein include a light chain variable region V L , the light chain variable region V L It has a CDR1 as shown in SEQ ID NO:4, a CDR2 as shown in SEQ ID NO:5, and / or a CDR3 as shown in SEQ ID NO:6.

[0059] In some embodiments, the anti-CLDN18.2 antibodies described herein include a heavy chain variable region V H and light chain variable region V L, the heavy chain variable region V H comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 1, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 2, and / or a CDR3 having an amino acid sequence as shown in SEQ ID NO: 3, wherein the light chain variable region V L It comprises a CDR1 having an amino acid sequence as shown in SEQ ID NO: 4, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 5, and / or a CDR3 having an amino acid sequence as shown in SEQ ID NO: 6. In some embodiments, the anti-CLDN18.2 antibody comprises a heavy chain CDR1 (HCDR1) as shown in SEQ ID NO: 1, a HCDR2 as shown in SEQ ID NO: 2, and a HCDR3 as shown in SEQ ID NO: 3, and a light chain CDR1 (LCDR1) as shown in SEQ ID NO: 4, a LCDR2 as shown in SEQ ID NO: 5, and a LCDR3 as shown in SEQ ID NO: 6. In some embodiments, the anti-CLDN18.2 antibody comprises CDR1, CDR2, and CDR3 of the heavy chain variable region as shown in SEQ ID NO: 7, and CDR1, CDR2, and CDR3 of the light chain variable region as shown in SEQ ID NO: 8.

[0060] In some embodiments, the anti-CLDN18.2 antibodies described herein include a heavy chain variable region V having an amino acid sequence as shown in SEQ ID NO: 7. H In some embodiments, the anti-CLDN18.2 antibodies described herein include a light chain variable region V having an amino acid sequence as shown in SEQ ID NO: 8. L In some embodiments, the anti-CLDN18.2 antibodies described herein include a heavy chain variable region V having an amino acid sequence as shown in SEQ ID NO: 7. H and a light chain variable region V having an amino acid sequence as shown in SEQ ID NO: 8 L In some embodiments, the anti-CLDN18.2 antibody comprises a heavy chain variable region as shown in SEQ ID NO:7 and a light chain variable region as shown in SEQ ID NO:8.

[0061] In some embodiments, the anti-CLDN18.2 antibodies described herein further include an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region includes a heavy chain and / or a light chain constant region. The heavy chain constant region includes C H1 , C H1 -C H2 or C H1 -CH3 region, the light chain constant region comprising C L district.

[0062] In some embodiments, the anti-CLDN18.2 antibody described herein comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO: 9 and a light chain having an amino acid sequence as shown in SEQ ID NO: 10. In some embodiments, the anti-CLDN18.2 antibody comprises a heavy chain as shown in SEQ ID NO: 9 and a light chain as shown in SEQ ID NO: 10.

[0063] Exemplary amino acid sequences in some embodiments are listed in Table A below:

[0064] Table A Exemplary amino acid sequences

[0065]

[0066]

[0067] The present application relates to a pharmaceutical preparation 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 preparation of the present application may be 1-200 mg / ml, 1-190 mg / ml, 10-190 mg / ml, 20-180 mg / ml, 20-170 mg / ml, 20-160 mg / ml, 20-150 mg / ml, 20-140 mg / ml, 20-130 mg / ml, 20-120 mg / ml, 20-110 mg / ml, 20-100 mg / ml, 20-90 mg / ml, 20-80 mg / ml, 20-70 mg / ml, 20-60 mg / ml, 20-50 mg / ml, 20-40 mg / ml, 20-30 mg / ml. In some embodiments, the concentration of the anti-CLDN18.2 antibody is any concentration value within the above range. For example, as required, the concentration of the anti-CLDN18.2 antibody in the pharmaceutical preparation may 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.

[0068] Buffer

[0069] The term "buffer" generally refers to a buffer solution that tolerates changes in pH through the action of its acid-base conjugate components. "Buffer" as used herein refers to a compound solution that is known to be safely used in pharmaceutical preparations and has the ability to maintain or control the pH of the preparation within a desired range. Acceptable buffers that can control the pH in the range of mildly acidic pH to mildly alkaline pH (e.g., pH 4.5 to 8.0) include, but are not limited to, one or a combination of any of succinate buffer, citrate buffer, phosphate buffer, acetate buffer, arginine buffer, 2-amino-2-hydroxymethyl-1,3-propanediol (TRIS) buffer, histidine buffer, etc.

[0070] The buffer in the formulation solvent can be prepared using any appropriate method known in the art. In some embodiments, specific acidic and alkaline ion pairs can be used to prepare the buffer described in the present application. In one example, the excipients of the acidic and alkaline ion pairs can be accurately weighed, and pure water of about 60% of the target buffer volume can be added. After mixing evenly, the pH of the solution is measured. If the pH value deviates from the target value, the corresponding ion pair can be used to adjust the pH. The solution is then diluted with pure water to the target weight or target volume. Finally, the conductivity, osmotic pressure and pH of the solution are measured for verification.

[0071] The stable pharmaceutical formulation of the present application may contain a buffer solution that allows the pharmaceutical formulation to have 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 solution allows the pharmaceutical formulation of the present application to have a pH value of 4.5 to 6.0. In particular, the pH value of the pharmaceutical formulation of the present application can be any pH value within the pH ranges listed above, for example 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.

[0072] Examples of buffers that can control the pH value of the pharmaceutical preparation within the desired range include acetate buffer, histidine buffer, citrate buffer and other organic acid or inorganic acid buffers. These buffers can be used alone or in combination of two or more. Preferably, the pharmaceutical preparation of the present application comprises acetate buffer and histidine buffer. More preferably, the pharmaceutical preparation of the present application comprises acetate buffer.

[0073] "Acetate buffer" is a buffer containing acetate ions. The acetate buffer may contain one or more of acetic acid (e.g., glacial acetic acid), potassium acetate, sodium acetate (e.g., sodium acetate trihydrate), etc. In some embodiments, the acetate buffer is an acetic acid-sodium acetate buffer, specifically a glacial acetic acid-sodium acetate trihydrate buffer. In some embodiments, the pH value of the acetate buffer may be any pH value in 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.

[0074] "Histidine buffer" is a buffer containing histidine ions. The histidine buffer may include one or more of histidine, histidine hydrochloride (e.g., histidine hydrochloride monohydrate), histidine acetate, histidine phosphate, histidine sulfate, etc. In some embodiments, the histidine buffer may be a histidine-histidine hydrochloride buffer. In some embodiments, the pH value of the histidine buffer may be any pH value in 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.

[0075] "Citrate buffer" is a buffer containing citrate ions. The citric acid buffer may contain one or more of citric acid, monosodium citrate, disodium citrate, trisodium citrate, monopotassium citrate, dipotassium citrate, tripotassium citrate, sodium chloride, potassium chloride, etc. In some embodiments, the citric acid buffer is a citric acid-trisodium citrate buffer. In some embodiments, the pH value of the citric acid buffer may be any pH value in 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.

[0076] In some embodiments, the buffer used in the pharmaceutical preparation of the present application is an acetate buffer or a histidine buffer, and the pH value of the pharmaceutical preparation of the present application is 4.5 to 6.0. Without wishing to be bound by theory, it is believed that acetate buffer and histidine buffer are superior to citric acid buffer. For example, when the buffer of the pharmaceutical preparation of the present application is an acetate buffer or a histidine buffer, the anti-CLDN18.2 antibody in the pharmaceutical preparation is more stable (for example, more stable at high temperatures), the force between the antibody molecules is repulsive rather than attractive, and there is a smaller risk of intermolecular aggregation.

[0077] The concentration of the buffer described herein refers to the concentration of the buffer ions in the buffer. In some embodiments, the concentration of the suitable buffer for the pharmaceutical preparation of the present application may be 5-100mmol / L, 5-90mmol / L, 5-80mmol / L, 5-70mmol / L, 5-60mmol / L, 5-50mmol / L, 10-50mmol / L, 10-40mmol / L, 10-30mmol / L, 10-20mmol / L. In some embodiments, the concentration of the buffer is any concentration value within the above range. For example, as desired, 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 or and / or up to 100 mmol / L, depending on the specific buffer and the desired stability of the pharmaceutical formulation.

[0078] In some embodiments, the buffer used in the pharmaceutical preparation of the present application is an acetate buffer, including an acetic acid-sodium acetate buffer, and its concentration may be 5 to 100 mmol / L. In some embodiments, the concentration of the acetate buffer may be 5 to 100 mmol / L, 5 to 90 mmol / L, 5 to 80 mmol / L, 5 to 70 mmol / L, 5 to 60 mmol / L, 5 to 50 mmol / L, 5 to 45 mmol / L, 5 to 40 mmol / L, 5 to 35 mmol / L, 5 to 30 mmol / L, 5 to 25 mmol / L, 5 to 20 mmol / L, 5 to 15 mmol / L, 5 to 10 mmol / L.

[0079] In some embodiments, the buffer used in the pharmaceutical preparation of the present application is a histidine buffer, including a histidine-histidine hydrochloride buffer, and its concentration may be 5 to 100 mmol / L. In some embodiments, the concentration of the histidine buffer may be 5 to 100 mmol / L, 5 to 90 mmol / L, 5 to 80 mmol / L, 5 to 70 mmol / L, 5 to 60 mmol / L, 5 to 50 mmol / L, 5 to 45 mmol / L, 5 to 40 mmol / L, 5 to 35 mmol / L, 5 to 30 mmol / L, 5 to 25 mmol / L, 5 to 20 mmol / L, 5 to 15 mmol / L, 5 to 10 mmol / L.

[0080] In some embodiments, the buffer used in the pharmaceutical preparation of the present application is an acetate buffer or a histidine buffer, and the concentration is 5 to 50 mM. In some embodiments, the buffer used in the pharmaceutical preparation of the present application is an acetate buffer or a histidine buffer, and the concentration is 10 to 30 mM. In some embodiments, the buffer used in the pharmaceutical preparation of the present application is an acetate buffer or a histidine buffer, and the concentration is about 20 mM. In some embodiments, the buffer used in the pharmaceutical preparation of the present application is an acetate buffer, and the concentration is about 20 mM.

[0081] Stabilizer

[0082] As used herein, the term "stabilizer" refers to an agent that, when combined with a protein of interest, prevents or reduces the chemical and / or physical instability of the protein. Examples of stabilizers include sugars, alcohols, acids, salts, polymers, and the like. Examples of sugars include glucose, sucrose, trehalose, lactose, dextran, 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.

[0083] In some embodiments, the stabilizer for the pharmaceutical preparation of the present application is selected from sugars. In some embodiments, the stabilizer for the pharmaceutical preparation of the present application is selected from alcohols. In some embodiments, the stabilizer for the pharmaceutical preparation of the present application is selected from sucrose, trehalose, sorbitol, or a combination thereof.

[0084] In some embodiments, the concentration of the stabilizer used in the pharmaceutical preparation of the present application in the pharmaceutical preparation may be 1% (w / v) to 20% (w / v), 1% (w / v) to 19% (w / v), 1% (w / v) to 18% (w / v), 1% (w / v) to 17% (w / v), 1% (w / v) to 16% (w / v), 1% (w / v) to 15% (w / v), 1% (w / v) to 14% (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% (w / v) to 5% (w / v), depending on the specific stabilizer and the stability required for the pharmaceutical formulation. In some embodiments, the concentration of the stabilizer is any concentration value within the above range.

[0085] In some embodiments, the stabilizer used in the pharmaceutical preparation of the present application is sucrose, and its concentration in the pharmaceutical preparation may be 1% (w / v) to 20% (w / v). In some embodiments, the concentration of sucrose in the pharmaceutical preparation may be 1% (w / v) to 10% (w / v), 10% (w / v) to 20% (w / v), or 5 to 15% (w / v). In some embodiments, the concentration of sucrose in the pharmaceutical preparation is 5% (w / v) to 10% (w / v). In some embodiments, the concentration of sucrose in the pharmaceutical preparation is about 9% (w / v).

[0086] In some embodiments, the stabilizer used in the pharmaceutical preparation of the present application is trehalose, and its concentration in the pharmaceutical preparation may be 1% (w / v) to 20% (w / v). In some embodiments, the concentration of trehalose in the pharmaceutical preparation 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 preparation is 5% (w / v) to 10% (w / v). In some embodiments, the concentration of trehalose in the pharmaceutical preparation is about 9% (w / v).

[0087] In some embodiments, the stabilizer used in the pharmaceutical preparation of the present application is sorbitol, and its concentration in the pharmaceutical preparation may be 1% (w / v) to 20% (w / v). In some embodiments, the concentration of sorbitol in the pharmaceutical preparation 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 preparation is % (w / v) 2 to 8% (w / v). In some embodiments, the concentration of sorbitol in the pharmaceutical preparation is about 5% (w / v).

[0088] Surfactants

[0089] As used herein, the term "surfactant" refers to organic substances having both hydrophilic and hydrophobic structures, i.e., they contain groups with opposite solubility tendencies, e.g., oil-soluble hydrocarbon chains and water-soluble ionic groups. Surfactants can be divided into anionic, cationic and nonionic surfactants depending on the charge of the surface active part.

[0090] Examples of surfactants include polysorbates (e.g., polysorbate 80), poloxamers (e.g., poloxamer 188), Triton, and polyethylene glycol, polypropylene glycol, and copolymers of ethylene glycol and propylene glycol (e.g., Pluronics, PF68, etc.). In some embodiments, the surfactant of the pharmaceutical formulation of the present application is selected from polysorbate 80 (also known as polysorbate 80, PS 80, or Tween 80), poloxamer 188, or a combination thereof.

[0091] In some embodiments, the concentration of the surfactant in the pharmaceutical preparation of the present application may 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 specific surfactant and the stability required for the pharmaceutical preparation. In some embodiments, the concentration of the surfactant is any concentration value within the above range.

[0092] In some embodiments, the surfactant of the pharmaceutical preparation of the present application is polysorbate 80, and its concentration in the pharmaceutical preparation may be 0.005% (w / v) to 0.4% (w / v). In some embodiments, the concentration of polysorbate 80 in the pharmaceutical preparation 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 preparation may be 0.01% (w / v) to 0.1% (w / v). In some embodiments, the concentration of polysorbate 80 in the pharmaceutical preparation may be about 0.05% (w / v).

[0093] In some embodiments, the surfactant of the pharmaceutical preparation of the present application is poloxamer 188, and its concentration in the pharmaceutical preparation may be 0.005% (w / v) to 0.4% (w / v). In some embodiments, the concentration of poloxamer 188 in the pharmaceutical preparation may be 0.005% (w / v) to 0.1% (w / v) or 0.1% (w / v) to 0.4%. In some embodiments, the concentration of poloxamer 188 in the pharmaceutical preparation may be 0.05% (w / v) to 0.2% (w / v).

[0094] Chelating agents

[0095] As used herein, the term "chelator" refers to a compound that can react with certain metal ions (e.g., manganese (II), copper (II), iron (III) and cobalt (II)) to form a stable, water-soluble metal complex. Chelation is the formation or presence of two or more separate bonds between a ligand and a single central atom. Examples of chelators include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), triaminepentaacetic acid (DTPA), N-(1,2-dicarboxyethyl)-D,L-aspartic acid (IDHA), ethylenediamine-N,N'-bis (EDDHA), N,N-di(2-hydroxyphenyl)ethylenediamine-N,N'-diacetic acid (HBED) or a salt form thereof, such as EDTA-2Na, EDTA-4Na. In some embodiments, the chelator of the pharmaceutical formulation of the present application can be a chelated salt of EDTA (e.g., disodium salt of EDTA, i.e., EDTA-2Na; or tetrasodium salt of EDTA, i.e., EDTA-4Na).

[0096] In some embodiments, the concentration of the chelating agent in the pharmaceutical preparation of the present application may be 30-350 μmol / L, 30-330 μmol / L, 30-300 μmol / L, 30-270 μmol / L, 30-250 μmol / L, 30-230 μmol / L, 30-200 μmol / L, 30-170 μmol / L, 30-150 μmol / L, 30-130 μmol / L, 40-100 μmol / L, 40-90 μmol / L, 40-80 μmol / L, 40-70 μmol / L, 40-60 μmol / L or 40-50 μmol / L, depending on the specific chelating agent and the stability required for the pharmaceutical preparation. In some embodiments, the concentration of the chelating agent is any concentration value within the above range.

[0097] In some embodiments, the chelating agent of the pharmaceutical preparation of the present application is edetate disodium (EDTA-Na2), and the concentration is 30-350 μmol / L or 40-60 μmol / L. In some embodiments, the chelating agent of the pharmaceutical preparation of the present application is edetate disodium (EDTA-Na2), and the concentration is about 50 μmol / L.

[0098] In another aspect, the present application provides use of a chelating agent for preventing degradation of a surfactant (eg, polysorbate 80) in a pharmaceutical formulation (eg, a pharmaceutical formulation provided herein).

[0099] In another aspect, the present application provides use of a chelating agent for preventing a protein (eg, an antibody) from decreasing in stability in a pharmaceutical preparation (eg, a pharmaceutical preparation provided herein).

[0100] Other Materials

[0101] The pharmaceutical preparation of the present application may also optionally contain other materials, such as but not limited to isotonicity agents, diluents, and the like.

[0102] The term "isotonic agent" refers to a compound or composition that can impart a suitable osmotic tension to a drug to avoid a net flow of water across a cell membrane in contact with the drug. In some embodiments, the formulation of the present application has an osmotic pressure substantially the same as human blood. Suitable isotonic agents include, but are not limited to, glycerol, amino acids or proteins (such as glycine or albumin), salts (such as sodium chloride), and sugars (such as glucose, mannitol, sucrose, and lactose).

[0103] The term "diluent" is a pharmaceutically acceptable agent that can be used to dilute the pharmaceutical preparation of the present application. Typical diluents include water, physiological saline, antibacterial agents for injection, pH buffer, sterile saline solution, Ringer's solution or glucose solution.

[0104] preparation

[0105] In one aspect, the present application 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 of 4.5 to 8.0, and in some embodiments, a pH of 4.5 to 6.0, to achieve sufficient stability.

[0106] In some embodiments, the pharmaceutical preparation of the present application comprises:

[0107] i) an anti-CLDN18.2 antibody (e.g., a specific anti-CLDN18.2 antibody provided herein), at a concentration of 1 to 200 mg / ml, preferably 20 to 40 mg / ml, more preferably about 30 mg / ml;

[0108] ii) a buffer, which is preferably an acetate buffer or a histidine buffer, more preferably an acetate buffer, and the concentration of the buffer in the pharmaceutical preparation is 5 to 100 mmol / L, preferably 5 to 50 mmol / L or 10 to 30 mmol / L, more preferably about 20 mmol / L;

[0109] iii) a stabilizer, which is preferably sucrose, trehalose or sorbitol,

[0110] a) is preferably sucrose or trehalose, and the concentration of the stabilizer in the pharmaceutical preparation is 1 to 20% (w / v), preferably 1 to 10% (w / v) or 5 to 10% (w / v), more preferably about 9% (w / v), or

[0111] b) preferably sorbitol, and the concentration of the stabilizer in the pharmaceutical preparation is 1 to 20% (w / v), preferably 1 to 10% (w / v) or 2 to 8% (w / v), more preferably about 5% (w / v);

[0112] iv) a surfactant, which is preferably polysorbate 80 or poloxamer 188,

[0113] a) more preferably polysorbate 80, and the concentration of the surfactant in the pharmaceutical preparation 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 0.05% (w / v), or

[0114] b) more preferably poloxamer 188, and the concentration of the surfactant in the pharmaceutical preparation is 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);

[0115] v) optionally, a chelating agent, preferably EDTA or a salt thereof (e.g., EDTA-2Na), and the concentration of the chelating agent in the pharmaceutical preparation is 30-350 μM or 40-60 μM, and

[0116] The pharmaceutical preparation has a pH value of about 4.5 to 6.0, preferably about 5.0 to 5.5.

[0117] In some embodiments, the pharmaceutical preparation of the present application comprises an anti-CLDN18.2 antibody (e.g., a specific anti-CLDN18.2 antibody provided herein) at a concentration of about 1 to 200 mg / ml, and an acetate buffer or a histidine buffer at a concentration of about 5 to 100 mmol / L, wherein the pH of the pharmaceutical preparation is 4.5 to 6.0. In some embodiments, the above-mentioned pharmaceutical preparation comprises an anti-CLDN18.2 antibody at a concentration of about 20 to 40 mg / ml. In some embodiments, the above-mentioned pharmaceutical preparation comprises an acetate buffer or a histidine buffer at a concentration of about 10 to 30 mg / ml.

[0118] In some embodiments, the pharmaceutical preparation of the present application comprises an anti-CLDN18.2 antibody (e.g., a specific anti-CLDN18.2 antibody provided herein) at a concentration of about 1 to 200 mg / ml, an acetate buffer or histidine buffer at a concentration of about 5 to 100 mmol / L, and sucrose, trehalose, or sorbitol at a concentration of about 1 to 20% (w / v), wherein the pH of the pharmaceutical preparation is 4.5 to 6.0. In some embodiments, the above-mentioned pharmaceutical preparation comprises an anti-CLDN18.2 antibody at a concentration of about 20 to 40 mg / ml. In some embodiments, the above-mentioned pharmaceutical preparation comprises an acetate buffer or histidine buffer at a concentration of about 10 to 30 mg / ml. In some embodiments, the above-mentioned pharmaceutical preparation comprises sucrose or trehalose at a concentration of about 5 to 10% (w / v). In some embodiments, the above-mentioned pharmaceutical preparation comprises sorbitol at a concentration of about 2 to 8% (w / v).

[0119] In some embodiments, the pharmaceutical preparation of the present application comprises an anti-CLDN18.2 antibody (e.g., a specific anti-CLDN18.2 antibody provided herein) at a concentration of about 1 to 200 mg / ml, an acetate buffer or a histidine buffer at a concentration of about 5 to 100 mmol / L, sucrose, trehalose or sorbitol at a concentration of about 1 to 20% (w / v), and polysorbate 80 or poloxamer 188 at a concentration of about 0.005% (w / v) to 0.4% (w / v), wherein the pH of the pharmaceutical preparation is 4.5 to 6.0. In some embodiments, the above-mentioned pharmaceutical preparation comprises an anti-CLDN18.2 antibody at a concentration of about 20 to 40 mg / ml. In some embodiments, the above-mentioned pharmaceutical preparation comprises an acetate buffer or a histidine buffer at a concentration of about 10 to 30 mg / ml. In some embodiments, the pharmaceutical preparation comprises sucrose or trehalose at a concentration of about 5-10% (w / v), or sorbitol at a concentration of about 2-8% (w / v). In some embodiments, the pharmaceutical preparation 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).

[0120] In some embodiments, the pharmaceutical preparation of the present application comprises an anti-CLDN18.2 antibody (e.g., a specific anti-CLDN18.2 antibody provided herein) at a concentration of about 1 to 200 mg / ml, an acetate buffer or histidine buffer at a concentration of about 5 to 100 mmol / L, sucrose, trehalose or sorbitol at a concentration of about 1 to 20% (w / v), polysorbate 80 or poloxamer 188 at a concentration of about 0.005% (w / v) to 0.4% (w / v), and a chelating agent at a concentration of about 30 to 350 μmol / L, wherein the pH of the pharmaceutical preparation is 4.5 to 6.0. In some embodiments, the above-mentioned pharmaceutical preparation comprises an anti-CLDN18.2 antibody at a concentration of about 20 to 40 mg / ml. In some embodiments, the above-mentioned pharmaceutical preparation comprises an acetate buffer or histidine buffer at a concentration of about 10 to 30 mg / ml. In some embodiments, the above-mentioned pharmaceutical preparation contains sucrose or trehalose at a concentration of about 5 to 10% (w / v), or sorbitol at a concentration of about 2 to 8% (w / v). In some embodiments, the above-mentioned pharmaceutical preparation contains 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 above-mentioned pharmaceutical preparation contains a chelating agent (e.g., EDTA, EDTA-2Na) at a concentration of about 40 to 60 μmol / L. In some other embodiments, the pharmaceutical preparation of the present application contains almost no chelating agent.

[0121] In some embodiments, the pharmaceutical formulation of the present application comprises an anti-CLDN18.2 antibody at a concentration of about 20-40 mg / ml, an acetate buffer at a concentration of about 10-30 mmol / L, sucrose or trehalose at a concentration of about 5-10% (w / v), and polysorbate 80 at a concentration of about 0.01% (w / v) to 0.2% (w / v), wherein the pH of the pharmaceutical formulation is about 4.5-6.0 or about 5.0-5.5.

[0122] In some embodiments, the pharmaceutical formulation of the present application comprises an anti-CLDN18.2 antibody at a concentration of about 30 mg / ml, an acetate buffer at a concentration of about 20 mM, sucrose at a concentration of about 9% (w / v), polysorbate 80 at a concentration of about 0.05% (w / v), and a pH of about 5.3.

[0123] In some embodiments, the pharmaceutical preparation of the present application comprises an anti-CLDN18.2 antibody at a concentration of about 20-40 mg / ml, an acetate buffer at a concentration of about 10-30 mmol / L, sucrose or trehalose at a concentration of about 5-10% (w / v), polysorbate 80 at a concentration of about 0.01% (w / v) to 0.2% (w / v), and EDTA or EDTA-2Na at a concentration of about 40-60 μmol / L, wherein the pH of the pharmaceutical preparation is about 4.5-6.0 or about 5.0-5.5.

[0124] In some embodiments, the pharmaceutical formulation of the present application comprises an anti-CLDN18.2 antibody at a concentration of about 30 mg / ml, an acetate buffer at a concentration of about 20 mM, sucrose at a concentration of about 9% (w / v), polysorbate 80 at a concentration of about 0.05% (w / v), and EDTA or EDTA-2Na at a concentration of about 50 μmol / L, and the pH value is about 5.3.

[0125] In some embodiments, the pharmaceutical formulation of the present application comprises an anti-CLDN18.2 antibody at a concentration of about 20-40 mg / ml, an acetate buffer at a concentration of about 10-30 mmol / L, sorbitol at a concentration of about 2-8% (w / v), and polysorbate 80 at a concentration of about 0.01% (w / v) to 0.1% (w / v), wherein the pH of the pharmaceutical formulation is about 4.5-6.0 or about 5.0-5.5.

[0126] In some embodiments, the pharmaceutical preparation of the present application comprises an anti-CLDN18.2 antibody at a concentration of about 20-40 mg / ml, an acetate buffer at a concentration of about 10-30 mmol / L, sorbitol at a concentration of about 2-8% (w / v), polysorbate 80 at a concentration of about 0.01% (w / v) to 0.1% (w / v), and 40-60 μmol / L EDTA, wherein the pH of the pharmaceutical preparation is about 4.5-6.0 or about 5.0-5.5.

[0127] In some embodiments, the pharmaceutical formulation of the present application comprises an anti-CLDN18.2 antibody at a concentration of about 20-40 mg / ml, a histidine buffer at a concentration of about 10-30 mmol / L, sucrose at a concentration of about 6-12% (w / v), and poloxamer 188 at a concentration of about 0.05% (w / v) to 0.2% (w / v), wherein the pH of the pharmaceutical formulation is about 4.5-6.0 or about 5.0-5.5.

[0128] In some embodiments, the pharmaceutical formulation of the present application comprises an anti-CLDN18.2 antibody at a concentration of about 20-40 mg / ml, a histidine buffer at a concentration of about 10-30 mmol / L, a sorbitol at a concentration of about 2-8% (w / v), and a poloxamer 188 at a concentration of about 0.05% (w / v) to 0.2% (w / v), wherein the pH of the pharmaceutical formulation is about 4.5-6.0 or about 5.0-5.5.

[0129] In some embodiments, the pharmaceutical formulation of the present application comprises an anti-CLDN18.2 antibody at a concentration of about 20-40 mg / ml, a histidine buffer at a concentration of about 10-30 mmol / L, sucrose at a concentration of about 6-12% (w / v), and polysorbate 80 at a concentration of about 0.01% (w / v) to 0.1% (w / v), wherein the pH of the pharmaceutical formulation is about 4.5-6.0 or about 5.0-5.5.

[0130] In some embodiments, the pharmaceutical formulation of the present application comprises an anti-CLDN18.2 antibody at a concentration of about 20-40 mg / ml, a histidine buffer at a concentration of about 10-30 mmol / L, sorbitol at a concentration of about 2-8% (w / v), and polysorbate 80 at a concentration of about 0.01% (w / v) to 0.1% (w / v), wherein the pH of the pharmaceutical formulation is about 4.5-6.0 or about 5.0-5.5.

[0131] In some embodiments, the pharmaceutical preparation of the present application comprises an anti-CLDN18.2 antibody at a concentration of about 20-40 mg / ml, a histidine buffer at a concentration of about 10-30 mmol / L, sucrose or trehalose at a concentration of about 6-12% (w / v), poloxamer 188 at a concentration of about 0.05% (w / v) to 0.2% (w / v), and 40-60 μmol / L EDTA, wherein the pH of the pharmaceutical preparation is about 4.5-6.0 or about 5.0-5.5.

[0132] The chemical degradation of antibody molecules, or the aggregation to form polymers, or the deglycosylation, glycosylation modification, oxidation or other structural modifications of antibody molecules that may reduce at least one functional activity of monomeric proteins, etc., may cause instability of antibody preparations. For pharmaceutical preparations containing anti-CLDN18.2 antibodies, during the storage of the pharmaceutical preparations, the anti-CLDN18.2 antibodies may undergo chemical degradation, resulting in a decrease in the concentration of the antibodies; the anti-CLDN18.2 antibodies may also aggregate to form polymers that are sometimes insoluble in the form of multimeric 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 polymer antibodies will result in a decrease in the purity of monomeric antibodies. Moreover, due to the formation of insoluble polymers, the turbidity of the pharmaceutical preparation may increase.

[0133] In some embodiments, the pharmaceutical preparations comprising anti-CLDN18.2 antibodies of the present application can maintain stability after long-term storage, high temperature and / or multiple freeze-thaw cycles, wherein the physical and / or chemical stability and / or functional activity of the anti-CLDN18.2 antibodies remain relatively constant over time. In some embodiments, antibody protein concentration, protein purity, protein activity, pH value of the preparation, osmotic pressure of the preparation, appearance of the preparation, insoluble particles in the preparation, etc. can be used as markers of the stability of the pharmaceutical preparation. There are a variety of analytical techniques for determining protein stability in the art, which are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee, ed., Marcel Dekker Inc., New York, New York Press (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993).

[0134] In some embodiments, the stability of the pharmaceutical formulation can be measured under selected conditions and time 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 measurement, protein concentration (Protein Conc) measurement, and visual inspection (Visual Inspection).

[0135] As shown in the examples of the present application, the pharmaceutical preparation comprising the anti-CLDN18.2 antibody of the present application shows high stability in visual inspection, for example, high stability under long-term storage, high stability at high temperature (for example, 40° C.), and high stability after freezing and thawing.

[0136] In some embodiments, a stable pharmaceutical preparation refers to a pharmaceutical preparation in which the protein particle size does not change significantly during storage in a DLS test, and the KD is positive, that is, the force between the monoclonal antibody molecules is a repulsive force, and the monoclonal antibody molecules do not aggregate. As shown in the examples of the present application, the pharmaceutical preparation comprising an anti-CLDN18.2 antibody of the present application shows high stability in a DLS test, for example, high stability under long-term storage, high stability at high temperature (e.g., 40° C.), and high stability after freeze-thaw.

[0137] In some embodiments, a stable pharmaceutical preparation refers to a pharmaceutical preparation that has only a small amount of protein degradation during storage in the SEC test, and the rate of increase of polymers and oligomers is slow. As shown in the examples of the present application, the pharmaceutical preparation comprising anti-CLDN18.2 antibodies of the present application shows high stability in the SEC test, for example, high stability under long-term storage, high stability at high temperature (e.g., 40° C.), and high stability after freeze-thaw.

[0138] In some embodiments, a stable pharmaceutical preparation refers to a pharmaceutical preparation in which the charge heterogeneity of the pharmaceutical preparation does not change significantly during storage, and the degree of separation of the acidic peak and the basic peak does not change significantly in the CEX test. As shown in the examples of the present application, the pharmaceutical preparation comprising the anti-CLDN18.2 antibody of the present application shows high stability in the CEX test, for example, high stability under long-term storage, high stability at high temperature (e.g., 40° C.), and high stability after freeze-thaw.

[0139] In some embodiments, a stable pharmaceutical preparation refers to a pharmaceutical preparation that has only a small amount of protein degradation during storage in the NR CE-SDS test, and the rate of increase of polymers and oligomers is slow. As shown in the examples of the present application, the pharmaceutical preparation comprising the anti-CLDN18.2 antibody of the present application shows high stability in the NR CE-SDS test, for example, high stability under long-term storage, high stability at high temperature (e.g., 40° C.), and high stability after freeze-thaw.

[0140] In some embodiments, a stable pharmaceutical preparation means that when the pharmaceutical preparation is visually inspected, no significant change in appearance of the pharmaceutical preparation is observed during storage, and the pharmaceutical preparation still appears as a colorless clear liquid.

[0141] In some embodiments, a stable pharmaceutical formulation means that when the pharmaceutical formulation is tested for protein concentration, the change in protein concentration in the pharmaceutical formulation does not exceed + / -20%, does not exceed + / -19%, does not exceed + / -18%, does not exceed + / -17%, does not exceed + / -16%, does not exceed + / -15%, does not exceed + / -14%, does not exceed + / -13%, does not exceed + / -12%, does not exceed + / -11%, does not exceed + / -10%, does not exceed + / -9%, does not exceed + / -8%, does not exceed + / -7%, does not exceed + / -6%, does not exceed + / -5%, does not exceed + / -4%, does not exceed + / -3%, does not exceed + / -2%, does not exceed + / -1%, or does not exceed + / -0.5%, wherein the protein concentration can be determined by UV-visible spectrophotometry according to the General Rule 0401 of the Chinese Pharmacopoeia 2010 edition, Part III.

[0142] Preparation

[0143] In another aspect, the present application provides a method for preparing a pharmaceutical preparation, comprising:

[0144] 1) providing a formulation medium and an anti-CLDN18.2 antibody stock solution, wherein the formulation medium comprises a buffer and a stabilizer, and optionally a surfactant;

[0145] 2) Using the formulation solvent to replace the anti-CLDN18.2 antibody stock solution, thereby obtaining a pharmaceutical preparation as described herein. In some embodiments, the solution replacement is buffer replacement, such as buffer replacement by dialysis. In some embodiments, the solution replacement is filtration. In some embodiments, the filtration is ultrafiltration, diafiltration, gel filtration, and / or other filtration methods known to those skilled in the art.

[0146] The present application also provides a method for preparing a pharmaceutical preparation, which comprises:

[0147] 1) providing a formulation medium and an anti-CLDN18.2 antibody stock solution, wherein the formulation medium comprises a buffer and a stabilizer, and wherein the anti-CLDN18.2 antibody stock solution contains a chelating agent (e.g., EDTA, EDTA-2Na);

[0148] 2) using the preparation solvent to replace the anti-CLDN18.2 antibody stock solution, wherein the product after the replacement contains almost no chelating agent;

[0149] 3) adding a surfactant to the product after the liquid exchange (i.e., the preparation medium containing the anti-CLDN18.2 antibody) to obtain the pharmaceutical preparation as described herein.

[0150] In some embodiments, the surfactant comprises polysorbate 80.

[0151] In some embodiments, the buffer in the formulation vehicle is an 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).

[0152] In some embodiments, the fluid replacement is buffer replacement. In some embodiments, the anti-CLDN18.2 antibody stock solution is dialyzed using the formulation medium. For example, a certain volume of sample can be placed in a dialysis bag (e.g., Snake The dialysis bag is sealed and placed in a target buffer with a volume of ≧100 times the volume, and stirred continuously to promote substitution. The dialysis is performed for an appropriate number of times (e.g., 3 times), for an appropriate time (e.g., 4 h, 4 h, and overnight), and is completed at an appropriate stirring speed (e.g., 150 rpm).

[0153] In some embodiments, the liquid exchange is a filtration liquid exchange. In some embodiments, the filtration is ultrafiltration, diafiltration, gel filtration, and / or other filtration methods known to those skilled in the art.

[0154] As used herein, the term "ultrafiltration" refers to a process in which a mixture (eg, an anti-CLDN18.2 antibody stock solution) moves through a membrane (eg, an ultrafiltration membrane) under pressure-driven conditions, for example, and different substances (eg, solvents and solutes) are separated by differences in the rates at which they filter through the membrane in response to a given pressure driving force.

[0155] As used herein, the term "diafiltration" refers to the process of separating a mixture (e.g., an anti-CLDN18.2 antibody stock solution) according to the molecular size of each component thereof using, for example, a permeable membrane filter. Typically, the protein stock solution is diafiltered through a membrane that retains the protein and allows buffer exchange, whereby, over time, the protein-containing stock solution is replaced by a new buffer.

[0156] As used herein, the term "gel filtration" refers to the process of separating smaller molecules using a gel by repelling molecules (e.g., proteins) that are larger than the resin pores, causing them to pass through a solid phase faster than smaller molecules that diffuse into the resin pores and are therefore retained and move more slowly through the solid phase.

[0157] In another aspect, the present application provides a method for preparing a pharmaceutical preparation, comprising: mixing a high-concentration anti-CLDN18.2 antibody stock solution with a high-concentration formulation excipient (such as a buffer and a stabilizer, and optionally a surfactant), and then diluting the mixture to a target concentration.

[0158] application

[0159] In another aspect, the present application also provides a method for treating a disease in a subject, comprising administering a therapeutically effective amount of the pharmaceutical preparation of the present application to the subject, wherein the subject has or may have a disease that needs to be treated with an antibody against CLDN18.2.

[0160] As used herein, the term "treat" refers to reducing or ameliorating the severity and / or duration of a disorder or one or more symptoms thereof, inhibiting or preventing the progression of a disorder, causing regression of a disorder, inhibiting or preventing the recurrence, development, onset or progression of one or more symptoms associated with a disorder. Subjects in need of treatment include those already suffering from the disease.

[0161] The term "therapeutically effective amount" refers to the minimum concentration required to achieve a measurable improvement or prevention of a particular condition.

[0162] The pharmaceutical preparation of the present application can be used to treat diseases associated with CLDN18.2, including chronic and acute diseases. Diseases associated with CLDN18.2 include cancer, etc. In some embodiments, the CLDN18.2-related disease is a cancer expressing CLDN18.2. Examples of cancers expressing CLDN18.2 include, but are not limited to, 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 / hepatoma), squamous cell carcinoma, peritoneal cancer, brain tumor (e.g., glioblastoma / multiforme glioblastoma (GBM), non-glioblastoma brain tumor or meningioma), glioma (e.g., ependymoma, astrocytoma, anaplastic astrocytoma, oligodendroglioma or mixed glioma, such as oligoastrocytoma), cervical cancer, ovarian cancer, liver cancer (e.g., hepatoblastoma, hepatocellular carcinoma / hepatoma, liver cancer), bladder cancer (e.g., urothelial carcinoma), breast cancer, colon cancer, colorectal cancer, rectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer (e.g., renal rhabdoid tumor), prostate cancer, vulvar cancer, penis cancer, anal cancer (e.g., anal squamous cell carcinoma), thyroid cancer, head and neck cancer (e.g., nasopharyngeal cancer), skin cancer (e.g., melanoma or squamous cell carcinoma), osteosarcoma, Ewing's sarcoma, chondrosarcoma, soft tissue sarcomas (e.g., rhabdomyosarcoma, fibrosarcoma, Kaposi's sarcoma), carcinoid tumor, eye cancer (e.g., retinoblastoma), mesothelioma , lymphocytic / lymphoblastic leukemias (e.g., acute lymphocytic / lymphoblastic leukemia (ALL) of the T-cell lineage and B-cell precursor lineage, chronic lymphoblastic / lymphocytic leukemia (CLL), acute myeloid / myeloblastic leukemia (AML), including mast cell leukemia, chronic myeloid / myelocytic / myeloblastic leukemia (CML), hairy cell leukemia (HCL), Hodgkin's disease, non-Hodgkin's lymphoma, chronic myelomonocytic leukemia (CMML), follicular lymphoma (FL), diffuse large B-cell lymphoma (DL The following are examples of cancers that may be treated with chemotherapy: leukemia, leukemia, lymphoma of the ovary, cerebral infarction, mycosis fungoides, mycosis fungoides, mycobacterial infarction, myeloma, myelodysplastic syndrome, myelodysplastic syndrome, myelosuppressive disease, myelofibrosis, myelodysplastic syndrome ...elodysplastic syndrome, myelofibrosis, myelodysplastic syndrome, myelodysplastic syndrome, myelodysplastic syndrome, myelodysplastic syndrome, myelodysplastic syndrome, myelodysplastic syndrome, myelodysplastic syndrome, myelodysplastic syndrome, myelodysplastic syndrome, myelodysplastic syndrome, myelodysplastic syndrome, myelodysplastic syndrome, myelodysplastic syndrome, myelodysplastic syndrome, myelodysplastic syndrome, myelodysplastic syndrome, myelodysplastic syndrome, myelodysplastic syndrome, myelodysplastic syndrome, myelodysplastic syndrome, myelodysplastic syndrome, myelodysplastic syndrome, myelodysplastic syndrome, myelodysplastic syndrome, myelodysplastic

[0163] The pharmaceutical preparation of the present application can be administered to a subject by any suitable route. For example, the pharmaceutical preparation can be administered to a subject by intravenous injection.

[0164] In yet another aspect, the present application provides use of a pharmaceutical preparation in the preparation of a medicament for preventing and / or treating a CLDN18.2-related disease.

[0165] Example

[0166] Overview of experimental methods:

[0167] In the following examples, anti-CLDN18.2 antibodies were subjected to formulation screening tests. The anti-CLDN18.2 antibodies used in the following examples contain a heavy chain variable region as shown in SEQ ID NO: 7 and a light chain variable region as shown in SEQ ID NO: 8. The anti-CLDN18.2 antibodies used in the following examples contain a heavy chain as shown in SEQ ID NO: 9 and a light chain as shown in SEQ ID NO: 10.

[0168] The anti-CLDN18.2 antibody formulations tested in the following examples of this application were prepared as follows:

[0169] 1. Prepare the target formulation buffer: The preparation of the buffer uses specific acidic and alkaline ion pairs. Accurately weigh the excipients of the acidic and alkaline ion pairs, add about 60% of the volume of the target buffer to Milli-Q water, mix well, and measure the pH of the solution. If the pH value deviates from the target value, the corresponding ion pair needs to be used to adjust the pH. Then dilute the solution with Milli-Q water to the target weight or target volume. Finally, measure the conductivity, osmotic pressure and pH of the solution for verification.

[0170] 2. Prepare samples (dialysis or direct dilution):

[0171] a) Using dialysis to replace the buffer containing the anti-CLDN18.2 antibody (i.e., stock solution, also referred to as DS in the following examples) into the target formulation buffer. Specifically, a certain volume of DS sample is placed in Snake The dialysis bag was sealed and placed in ≥100 times the volume of the target buffer, with continuous stirring to promote replacement. The dialysis was performed three times, with durations of 4 h, 4 h and overnight, respectively, and the stirring speed was 150 rpm.

[0172] b) Direct dilution method: Add high-concentration excipients and surfactant stock solutions to high-concentration DS, and then dilute DS to the target concentration using the target buffer system.

[0173] The following examples of the present application analyze the anti-CLDN18.2 antibody formulations tested using the following analytical methods:

[0174] 1. Dynamic light scattering (DLS): The protein particle size and distribution were measured by dynamic light scattering (DLS) with the following parameters: acquisition time 5 s, 20 acquisitions per measurement, and measurement temperature 25 °C.

[0175] 2. Size Exclusion Chromatography (SEC): Protein aggregation was determined by SEC using an Agilent 1260 system and a TSKgelG3000SWXL column (300×7.8 mm, 5 μm). The mobile phase was 50 mM sodium phosphate buffer, 300 mM NaCl, pH 6.8±0.1. The flow rate was 1.0 mL / min. The sample was diluted to 10 mg / mL, the detection volume was 10 μL, and the detection wavelength was 280 nm.

[0176] 3. Cation Exchange Chromatography (CEX): Charge heterogeneity of proteins was analyzed by CEX using a Thermo Propac Elite WCX-10 4 mm ×

[0177] 150mm 5um column determination. Dilute the sample to 2.00mg / mL with a mixed solution of mobile phase A and B.

[0178] 4. Non-reducing capillary electrophoresis (NR CE-SDS): Protein fragmentation was determined by CE-SDS (NR) method. The standard or test sample was diluted to 4 mg / mL with phosphate-citrate buffer, and then 25 μL was vortexed with 75 μL SDS sample buffer and 5 μL NEM (100 mM N-ethylmaleimide) for denaturation. The denatured sample was centrifuged, incubated at 70 ± 2 ° C for 10 ± 2 min, cooled at room temperature, and then centrifuged again. Separation was performed on PA800 plus using SDS separation gel kit and uncoated fused silica capillaries.

[0179] 5.pH: By installing The Seven Compact pH meter with ExpertPro electrodes measures sample pH.

[0180] 6. Protein Conc: Protein concentration was determined by absorbance at 280 nm using a Nano Drop 2000 spectrophotometer. The extinction coefficient (E1%) used throughout the study was 1.511 L / g-cm. Each sample was measured twice at a loading volume of 2.0 μL. The average concentration was reported.

[0181] 7. Visual Inspection: Check the appearance of the sample against a black background using a YB-2 clarity tester. Report transparency and color.

[0182] The sources of the reagents used in this experiment are as follows, and other reagents are conventional reagents in the field.

[0183] Reagent name supplier Part Number Citric acid monohydrate Merck 137003.1000 Trisodium Citrate Dihydrate Merck 1.06446.1000 Histidine Merck 1.04352.1000 Histidine hydrochloride monohydrate Merck 1.04354.0500 Sodium acetate trihydrate Merck 1.37012.1000 glacial acetic acid JT Baker UN2789 Polysorbate 80 NOF Polysorbate80(HX2) sucrose Pfanstiehl S-124-2-MC Arginine hydrochloride Merck 1.01587.1000 Sodium chloride Merck 1.16224.5000 Trehalose dihydrate Pfanstiehl T-104-4 Sorbitol MERCK 1.11597.9028 Poloxamer 188 MERCK 1.37065.1000 PVDF membrane Merck Millipore 33mm,0.22μm PVDF membrane Merck Millipore 13mm,0.22μm PES membrane Merck Millipore 33mm,0.22μm

[0184] Example 1: pH screening experiment

[0185] 1. Experimental Design

[0186] The anti-CLDN18.2 antibody protein was subjected to a pH screening test for the formulation. The anti-CLDN18.2 antibody protein was replaced into the formulation buffer shown in Table 1 by dialysis, and diluted with the corresponding formulation buffer to adjust it to 30.0 mg / mL. Finally, the sample was filtered through a 0.22 μm PVDF membrane and dispensed into a 2 mL vial packaging container system using a pipette. The samples were incubated under the different conditions listed in Table 1 and tested.

[0187] Table 1. pH screening experiment design

[0188]

[0189]

[0190] 2. Results Analysis

[0191] (1) At T0, the appearance of preparations F7, F8 and F9 was colorless and slightly opalescent liquid, and the other preparations were colorless and clear liquid. After incubation at 40°C for 14 days, no obvious changes were observed in the appearance of all preparations except F7, F8 and F9.

[0192] Table 2. pH screening test appearance, pH and concentration results_40℃

[0193]

[0194]

[0195] (2) At T0, the KD of preparations F7, F8 and F9 is negative, indicating that the interaction between the monoclonal antibody molecules is attractive, and the KD of the other preparations is positive, indicating that the interaction between the monoclonal antibody molecules is repulsive. At T0, the radius of the monoclonal antibody molecules in preparations F7, F8 and F9 is significantly larger than that in other preparations, so F1, F2, F3, F4, F5 and F6 meet the requirements in KD and T0 particle size.

[0196] Table 3. pH screening experiment DLS results_40℃

[0197]

[0198] (3) SEC test was performed on F1, F2, F3, F4, F5, and F6. As shown in Table 4, after incubation at 40°C for 14 days, F1, F2, F3, F4, F5, and F6 showed a small amount of main peak (MP) degradation to form fragments (LMW) and a slower increase rate of high polymers (HMW), which met the acceptance criteria under the test conditions. All preparations met the stability requirements.

[0199] Table 4. SEC results of pH screening experiment_40℃

[0200]

[0201]

[0202] (4) CEX. After incubation at 40°C for 14 days, all formulations showed major peak degradation and formation of an acidic peak, meeting the acceptance criteria under this test condition. There were no significant differences among the formulations.

[0203] Table 5. pH screening experiment CEX results_40℃

[0204]

[0205] (5) NR CE-SDS: After incubation at 40°C for 14 days, all formulations showed a small amount of main peak degradation and fragmentation, meeting the acceptance criteria under this test condition.

[0206] Table 6. pH screening experiment NR CE-SDS results_40℃

[0207]

[0208] In summary, after incubation at 40°C, the citric acid system was excluded based on the DLS and appearance results because the intermolecular force of the monoclonal antibody is attractive and the larger molecular radius of the monoclonal antibody indicates that the monoclonal antibody molecules have the risk of aggregation during long-term storage. The acetic acid system and the histidine system (i.e., F1, F2, F3, F4, F5, and F6) are relatively stable and can meet the stability requirements of protein preparations.

[0209] Example 2: Surfactant screening experiment-1

[0210] 1. Experimental Design

[0211] The anti-CLDN18.2 antibody protein was subjected to a surfactant screening test. The anti-CLDN18.2 antibody protein was filtered through a 0.22 μm PVDF membrane, sucrose solution and polysorbate 80 solution were added, and then diluted with 20 mM acetate buffer pH 5.5 and milli-Q water to replace the following formulations (F10, F11, F12 and F13). Acetic acid and sucrose were used as buffers and stabilizers. 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 PVDF membrane and dispensed into a 2 mL vial packaging container system using a pipette, and then the stability studies listed in Table 7 were performed. The samples were tested after incubation.

[0212] Table 7. Surfactant screening experimental design

[0213]

[0214] 2. Results Analysis

[0215] The data at 40°C (Table 8) and shaking (Table 9) showed that polysorbate 80 had a significant protective effect on the stability of the monoclonal antibody molecule in the concentration range of 0.01% (w / v)-0.1% (w / v). All formulations met the test requirements. In subsequent experiments, a concentration of 0.05% (w / v) polysorbate 80 was selected for further evaluation.

[0216] Table 8. Results of surfactant screening experiment_40℃

[0217]

[0218]

[0219] Table 9. Results of surfactant screening experiment_Shaking

[0220]

[0221] Example 3. Surfactant screening experiment-2

[0222] 1. Experimental Design

[0223] The anti-CLDN18.2 antibody protein was subjected to a surfactant screening test. The anti-CLDN18.2 antibody protein was filtered through a 0.22 μm PES membrane, sucrose solution and poloxamer 188 solution were added, and then diluted with 20 mM histidine buffer pH 5.5 and replaced with the following formulation. Histidine and sucrose were selected as buffers and stabilizers. 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 and subjected to the stability study listed in Table 10. The sample was tested after incubation.

[0224] Table 10. Surfactant screening experimental design

[0225]

[0226]

[0227] 2. Results Analysis

[0228] From the SEC, CE-SDS and CEX data, no significant differences were observed among formulations F14, F15 and F16 ( Figure 1-3 ). Therefore, 0.05%-0.20% (W / V) of Poloxamer 188 surfactant also meets the prescription requirements.

[0229] Example 4. Excipient screening experiment

[0230] 1. Experimental Design

[0231] The anti-CLDN18.2 antibody protein was subjected to an excipient screening test. The anti-CLDN18.2 antibody protein was filtered through a 0.22 μm PVDF membrane, added to the excipient solution and polysorbate 80 solution to replace the following formulation, and then diluted with 20 mM acetic acid-sodium acetate buffer pH 5.5 and water. The final concentration of the CLDN18.2 antibody protein was 30.0 mg / mL. Finally, the sample was filtered through a 0.22 μm PVDF membrane and dispensed into a 2 mL vial packaging container system using a pipette, and then the stability study listed in Table 11 was performed.

[0232] Table 11. Experimental design for excipient screening

[0233]

[0234]

[0235] 2. Results Analysis

[0236] (1) Freeze-thaw test

[0237] The results are shown in Table 12. At T0, the appearance of preparations F17 and F20 was colorless and slightly opalescent liquid, while preparations F18 and F19 were both colorless and clear liquid. After 5 cycles of freeze-thaw, no significant changes were observed in the protein concentration and appearance of preparations F18 and F19. At T0, the KD of preparations F17 and F20 was negative, indicating that the force between monoclonal antibody molecules was attractive, and the KD of preparations F18 and F19 was positive, indicating that the force between monoclonal antibody molecules was repulsive. At T0, the radius of the monoclonal antibody molecules in preparations F17 and F20 was significantly larger than that in other preparations. After 5 cycles of freeze-thaw, the radius of the monoclonal antibody molecules in preparations F18 and F19 remained basically unchanged and showed a unimodal distribution; no significant differences and changes were observed in the main peaks of SEC, NR CE-SDS and CEX; no significant differences and changes were observed in the sub-visible particles. Therefore, F18 and F19 meet the experimental requirements.

[0238] Table 12. Results of excipient screening experiment_freeze-thaw

[0239]

[0240] (2) High temperature test

[0241] The results are shown in Table 13. After incubation at 40°C for 7 and 14 days, no significant changes were observed in the protein concentration, appearance and particle size of F18 and F19. A small amount of degradation occurred in the main peaks of SEC, NR-CE-SDS and CEX, but all met the acceptable standards, and there was no significant difference between the formulations.

[0242] Table 13. Results of excipient screening experiment_40℃

[0243]

[0244] (3) Summary

[0245] Based on the DLS and appearance results, the excipients sodium chloride and arginine hydrochloride were excluded because the addition of these excipients changed the interaction between monoclonal antibody molecules from repulsive force to attractive force, resulting in the risk of aggregation of molecules during long-term storage, which is not conducive to the long-term storage of the preparation. The results of the 40°C and freeze-thaw studies showed that the protective effects of sucrose and trehalose on the stability of monoclonal antibody molecules are basically the same, and both can meet the needs of developing stable preparations.

[0246] Example 5: Excipient screening experiment-2

[0247] 1. Experimental Design

[0248] The anti-CLDN18.2 antibody protein was subjected to an excipient screening test. The CLDN18.2 antibody protein was filtered through a 0.22 μm PES membrane, added to the excipient solution and polysorbate 80 solution to replace the following formulation, and then diluted with 20 mM histidine buffer pH 5.5. The final concentration of the CLDN18.2 antibody protein was 30.0 mg / mL. Finally, the sample was filtered through a 0.22 μm PES membrane and subjected to the stability study listed in Table 14.

[0249] Table 14. Excipient screening experiment-2

[0250]

[0251] 2. Results Analysis

[0252] From the stirring experiment and the results of SEC, CEX, and CE-SDS, it can be seen that both F21 and F22 meet the requirements ( Figure 4-6 , Table 15).

[0253] Table 15. Results of auxiliary material screening experiment-2_Stirring

[0254]

[0255] Therefore, sucrose and sorbitol both meet the requirements.

[0256] Example 6: Formulation Confirmation Experiment

[0257] 1. Experimental Design

[0258] Based on the above experiments, two prescriptions that meet the requirements are selected to confirm the optimal prescription.

[0259] The surfactant and excipient solutions were added to the buffer system with protein and the protein concentration was adjusted to 30.0 mg / mL to prepare the following formulation. The samples were filtered through a 0.22 μm PVDF membrane and dispensed into 2 mL vial packaging container system and 5 mL polycarbonate bottles using a pipette and then subjected to stability studies.

[0260] Table 16. Formulation confirmation study design

[0261]

[0262] 2. Results Analysis

[0263] In the accelerated test (Table 17), the protein purity (SEC and NR CE-SDS) was slightly degraded in both the acetate buffer system (F23) and the histidine buffer system (F22), and the degradation degree of the acetate buffer system (F23) was smaller; the CEX purity did not change significantly in the acetate buffer system, while the CEX purity in the histidine buffer system decreased significantly. In the long-term test (Table 18) and the low-temperature test (Table 19), the acetate buffer system (F23) and the histidine buffer system (F22) did not change significantly in appearance, pH, concentration, DLS, purity (SEC, NR CE-SDS and CEX). Considering that the conditions of the accelerated test can better simulate the protein degradation trend under long-term storage conditions, the acetate buffer system (F23) that is more stable in the accelerated test was selected as the final prescription, namely: 30.0 mg / mL anti-CLDN18.2 antibody protein, 20 mM acetic acid / sodium acetate, 9% (w / v) sucrose, 0.05% (w / v) polysorbate 80, pH 5.3.

[0264] Table 17. Accelerated test_25℃

[0265]

[0266] Table 18. Formulation confirmation test results_5℃

[0267]

[0268]

[0269] Table 19. Formulation confirmation test results_-20℃

[0270]

[0271] Example 7: Investigating the protective effect of EDTA-Na2

[0272] 1. Experimental Design

[0273] The following samples were prepared as shown in the table below, and the protective effect of disodium edetate on preventing polysorbate 80 (PS80) degradation and protein quality was investigated for F23, F24 (EDTA was added to the stock solution, and after mixing and shaking the stock solution, EDTA was removed by dialysis and prepared into the final formulation), and F25. The preparation method of F24 is as follows: EDTA was added to the antibody stock solution, and then replaced with a formulation solvent containing a buffer and a stabilizer, thereby achieving the effect of simultaneously removing EDTA and filtering and replacing the solution, and finally a surfactant was added to prepare the final formulation.

[0274] Table 20. Experimental design of edetate disodium protection study

[0275]

[0276]

[0277] 2. Results Analysis

[0278] Under the forced experimental conditions of 40℃, Figure 7 It can be seen that adding EDTA during downstream purification (F24) and adding EDTA in the preparation (F25) can effectively prevent PS80 degradation. Figure 8 It can be seen that the degradation of the main protein peak can be reduced at F24 and F25.

Claims

1. A pharmaceutical preparation, 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 preparation is 4.5 to 6.

0. 2 . The pharmaceutical preparation according to claim 1 , wherein the concentration of the buffer in the pharmaceutical preparation is 5 mM to 50 mM, or 10 mM to 30 mM.

3. The pharmaceutical preparation according to claim 1 or 2, further comprising a stabilizer.

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

5. The pharmaceutical preparation according to claim 3 or 4, wherein the stabilizer is selected from the group consisting of sucrose, trehalose and sorbitol.

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

7. A pharmaceutical formulation according to any preceding claim, further comprising a surfactant.

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

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

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

11. A pharmaceutical formulation according to any preceding claim, further comprising a chelating agent. 12 . The pharmaceutical preparation according to claim 11 , wherein the concentration of the chelating agent in the pharmaceutical preparation is 30 μM-350 μM, or 40 μM-60 μM.

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

14. The pharmaceutical formulation according to any one of the preceding claims, wherein the concentration of the anti-CLDN18.2 antibody in the pharmaceutical formulation is 1 mg / ml to 200 mg / ml. 15 . The pharmaceutical preparation according to claim 14 , wherein the concentration of the anti-CLDN18.2 antibody in the pharmaceutical preparation is 20 mg / ml to 40 mg / ml.

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

6.

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

8.

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

8.

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

10.

20. The pharmaceutical formulation according to any preceding claim, 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 4.5-6.

0.

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

22. The pharmaceutical formulation according to claim 21, wherein 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)-0.1% (w / v), and the pH value is about 5.0-5.

5.

23. The pharmaceutical formulation of claim 22, wherein 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.

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

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

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

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

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) - 0.1% (w / v).

30. The pharmaceutical formulation according to any one of claims 26 to 28, wherein 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).

31. A method for preparing a pharmaceutical preparation, the method comprising: 1) providing a formulation medium and an anti-CLDN18.2 antibody stock solution, wherein the formulation medium comprises a buffer and a stabilizer, and optionally a surfactant; 2) using the preparation solvent to replace the anti-CLDN18.2 antibody stock solution, thereby obtaining the pharmaceutical preparation according to any one of claims 1 to 30.

32. The method according to claim 31, wherein the anti-CLDN18.2 antibody stock solution contains a chelating agent (eg, EDTA), the formulation medium does not contain a chelating agent, and the pharmaceutical formulation is made to contain almost no chelating agent by filtration.

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

34. The method of claim 31, wherein the formulation vehicle comprises a buffer, a stabilizer, and a chelating agent (eg, EDTA).

35. The method according to claim 33 or 34, wherein a surfactant is added after the anti-CLDN18.2 antibody stock solution is replaced with the formulation solvent, thereby obtaining the pharmaceutical preparation according to any one of claims 1 to 30.

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

38. The pharmaceutical preparation according to claim 37, wherein the CLDN18.2-related disease is selected from gastric cancer, gastroesophageal junction adenocarcinoma, lung cancer, bronchial cancer, bone cancer, hepatobiliary cancer, 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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