A liquid pharmaceutical composition comprising an anti-CLDN18.2 antibody

Abstract

The application provides a liquid pharmaceutical composition containing an anti-CLDN18.2 antibody, comprising: a therapeutically effective amount of an anti-CLDN18.2 antibody, a surfactant, an oligosaccharide, water and a buffer, and the pH of the preparation is 5.2-6.2. The liquid pharmaceutical composition of the application has simple components, good stability, is suitable for storage and transportation, can reduce the cost of packaging materials, transportation and storage and the like, and meanwhile, the biological activity of the antibody is maintained.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedicine, in particular to a pharmaceutical composition containing an anti-CLDN18.2 antibody. BACKGROUND

[0002] Claudins are a family of sealing proteins that regulate paracellular ion channels. Certain members of the sealing protein family are differentially expressed in malignant tumors. Claudin 18.2 is a highly selective gastric family antigen that is specifically expressed in short-term differentiated gastric epithelial cells, and has limited accessibility to antibody drugs.

[0003] Claudin 18.2 is continuously present in the process of malignant metastasis, and therefore is often displayed on the surface of human gastric cancer cells. Claudin 18.2 has recently been found as a target for antibodies to treat gastric cancer and esophageal cancer, and it is also a target for developing antibody drugs for pancreatic cancer. WO2020 / 018852 has disclosed a novel antibody that binds to Claudin 18.2.

[0004] The development of biotechnology has enabled the production of a series of proteins for pharmaceutical applications through recombinant DNA technology in the past two decades. Protein drugs such as monoclonal antibodies can be used for tumor treatment, for example, for specific immunotherapy or tumor vaccination. Therapeutic proteins are larger and more complex in structure than conventional organic and inorganic active ingredients, they have complex three-dimensional structures and many functional groups, and the three-dimensional structure and functional groups affect the biological activity of the protein or cause other undesirable effects. During preparation, storage and transportation, protein drugs are exposed to many external factors that can weaken the stability of the protein active ingredient. In order to maintain the biological activity of the protein, the formulation must maintain the conformational integrity of at least one core sequence of the amino acids of the protein, while protecting the multiple functional groups of the protein from degradation. Therefore, it is necessary to take some measures to improve the stability of the protein, for example, by adding certain pharmaceutically acceptable excipients.

[0005] Although many formulations of therapeutic proteins are disclosed in the prior art, due to the specific physicochemical properties and degradation reactions of different proteins, there are certain limitations in developing existing protein formulations for new protein active ingredients. Therefore, for a new antibody against CLDN18.2, it is still a serious challenge for those skilled in the art to develop a pharmaceutical formulation with high stability and appropriate components. SUMMARY

[0006] The purpose of the present application is to provide a formulation containing a new antibody against CLDN18.2 with high stability and appropriate components.

[0007] The first aspect of the present application provides a liquid pharmaceutical composition containing an anti-CLDN18.2 antibody, which comprises:

[0008] a therapeutically effective amount of an anti-CLDN18.2 antibody;

[0009] a surfactant;

[0010] an oligosaccharide;

[0011] water; and

[0012] a buffer;

[0013] optionally containing a pH adjuster, wherein the pH of the pharmaceutical composition is 5.2-6.2.

[0014] Preferably, the anti-CLDN18.2 antibody is a monoclonal antibody.

[0015] Preferably, the monoclonal antibody is a humanized antibody.

[0016] Preferably, the monoclonal antibody binds to human CLDN18.2 protein.

[0017] Preferably, the surfactant is polysorbate 20 or polysorbate 80.

[0018] Preferably, the oligosaccharide is sucrose or trehalose.

[0019] Preferably, the buffer is phosphate, histidine-acetate or acetate.

[0020] Preferably, the water in the pharmaceutical composition is water for injection.

[0021] Preferably, the pharmaceutical composition contains a pH adjuster.

[0022] More preferably, the pH adjuster is glacial acetic acid.

[0023] Further, the content of the anti-CLDN18.2 antibody is 20-30 mg / mL.

[0024] Further, the content of the surfactant is 0.02wt%-0.08wt%.

[0025] Further, the content of the oligosaccharide is 6wt%-8wt%.

[0026] Further, the concentration of the buffer is 20-50 mmol / L.

[0027] More further, the pharmaceutical composition comprises:

[0028] 25-30 mg / mL of the anti-CLDN18.2 antibody;

[0029] 0.02-0.06 wt% of polysorbate 80;

[0030] 6-8 wt% of sucrose; and

[0031] 20-30 mmol / L of histidine-acetate;

[0032] wherein the pH of the formulation is 5.5-5.8.

[0033] Further, the pharmaceutical composition comprises:

[0034] 25 mg / mL of the anti-CLDN18.2 antibody;

[0035] 0.04 wt% of polysorbate 80;

[0036] 7 wt% of sucrose; and

[0037] 20 mmol / L of histidine-acetate; wherein the pH of the formulation is 5.8.

[0038] Preferably, the liquid pharmaceutical composition can also be prepared into a lyophilized formulation.

[0039] The second aspect of the present application also provides a method for preparing the pharmaceutical composition of the first aspect, which comprises the steps of preparing the anti-CLDN18.2 antibody, exchanging the antibody into other components of the liquid pharmaceutical composition, and then filtering sterilization and aseptic filling.

[0040] The third aspect of the present application also provides a lyophilized formulation prepared by lyophilizing the liquid pharmaceutical composition of the first aspect.

[0041] Further, the method for preparing the lyophilized formulation comprises the steps of pre-freezing, vacuumizing, primary drying, and secondary drying.

[0042] Further, the temperature increasing rate of the primary drying is 0.2℃ / min.

[0043] Further, the temperature increasing rate of the secondary drying is 0.1℃ / min.

[0044] The fourth aspect of the present application provides the use of the composition of the first aspect in the preparation of a medicament for treating cancer.

[0045] Preferably, the cancer is gastric cancer, esophageal cancer, pancreatic cancer, or liver cancer.

[0046] Advantages:

[0047] The present application provides an excellent liquid pharmaceutical composition containing an anti-CLDN18.2 antibody, the liquid pharmaceutical composition of the present application has simple components, good stability, is suitable for storage and transportation, can reduce the cost of packaging materials, transportation and storage, etc., while maintaining the biological activity of the antibody.

[0048] The anti-CLDN18.2 antibody liquid pharmaceutical composition of the present application shows good stability in the shaking / vibration, 25±2℃ acceleration test, and 5±3℃ long-term storage stability test, and has good application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 DSF research result curve chart for the first round of prescription screening, wherein 1-7 represent prescriptions A1-A7 respectively;

[0050] Figure 2 DLS research result curve chart for the first round of prescription screening, wherein 1-7 represent prescriptions A1-A7 respectively;

[0051] Figure 3 DSF research result curve chart for the second round of prescription screening, wherein 1-6 represent prescriptions B1-B6 respectively;

[0052] Figure 4 DLS research result curve chart for the second round of prescription screening, wherein 1-6 represent prescriptions B1-B6 respectively;

[0053] Figure 5 DSF research result curve chart for the third round of prescription screening, wherein 1-6 represent prescriptions C1-C6 respectively;

[0054] Figure 6 DLS research result curve chart for the third round of prescription screening, wherein 1-6 represent prescriptions C1-C6 respectively. DETAILED DESCRIPTION

[0055] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not used to limit the scope of the present application. The experimental methods in the following examples without specific conditions are usually carried out according to the conventional conditions, or according to the conditions recommended by the manufacturers.

[0056] TERMS

[0057] An antibody, as used herein, refers to a polypeptide comprising a framework region of an immunoglobulin gene or fragments thereof that specifically binds or recognizes an antigen. Immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin isotypes IgG, IgM, IgA, IgD and IgE, respectively. Typically, the antigen binding domain of an antibody is most decisive in terms of specificity and affinity of binding.

[0058] A typical immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one light (about 25 kD) and one heavy chain (50-70 kD). The N-terminus of each chain is a variable region of about 100-110 or more amino acids that is primarily responsible for antigen recognition. The variable light (VL) and variable heavy (VH) chains refer to these light and heavy chains, respectively.

[0059] Antibodies exist, for example, as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various proteolytic enzymes. Thus, for example, pepsin digests an antibody at the sites of the disulfide bonds linking light chains to VHand at the sites of the disulfide bonds linking CH1 to CH2. This produces two identical Fab' fragments, each with a single antigen binding site, and a residual Fc'2 piece. The F(ab)'2 fragment also can be produced by the partial reduction of an intact immunoglobulin, but the disulfide bonds linking the CH1 portion of one of the light chains to the CH1 portion of the other are not reduced. The F(ab)'2 has two antigen binding sites. Pepsin digestion of an F(ab)'2 fragment results in the cleavage of the disulfide bond of each Fab fragment, yielding an Fab' monomer. Each Fab' monomer has the same structure as that of the original two disulfide bond F(ab)'2 piece, except that it has only a single antigen binding site. Unlike any of the above, the single chain variable fragment (scFv) is a noncovalent heterodimer of two polypeptide chains, each of which has VHand VLdomains joined by a short linker peptide of ten to about 25 amino acids (Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); McCafferty et al., Nature 348:552-554 (1990); and Orlandi et al., Proc. Natl. Acad. Sci. USA 86:3833-3837 (1989)). The scFv can be produced by recombinant DNA techniques, or it can be selected from a phage display library (McCafferty et al., Nature 348:552-554 (1990)).

[0060] "Antibody stability" refers to an antibody that retains substantially its physical stability and / or chemical stability and / or biological activity after storage. The storage period is generally selected based on the intended shelf life of the formulation. Various analytical techniques for measuring antibody stability are well known in the art.

[0061] Generally, the stability can be determined at a selected temperature for a selected time, and the injectable formulation is stable for at least 3 months, at least 6 months, preferably 1 year, more preferably 2 years at 2-8°C.

[0062] An antibody retains its physical stability in a formulation if there is essentially no evidence of substantial aggregation, precipitation, and / or denaturation as determined by visual inspection for color and / or clarity, or by UV light scattering, or by size exclusion chromatography.

[0063] An antibody retains its chemical stability in a formulation if the chemical stability is such that the antibody is considered to still retain the biological activity as defined below at a particular time. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the antibody.

[0064] An antibody retains its biological activity in a formulation if the antibody in the formulation has the biological activity for its intended use. For example, an antibody is considered to retain its biological activity if the biological activity of the antibody in the formulation is within about 70% to 130% (within the error of the assay) of the biological activity exhibited at the time the formulation was prepared (e.g., as determined by an antigen binding assay).

[0065] For stability of a formulation, the stability of a liquid formulation can be assessed qualitatively and / or quantitatively in a number of different ways, including assessing dimer, multimer, and / or aggregate formation (e.g., using size exclusion chromatography (SEC), matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), analytical ultracentrifugation, light scattering (photon correlation spectroscopy, dynamic light scattering (DLS), static light scattering, multi-angle laser light scattering (MALLS)), flow-based microscopy imaging, electronic impedance (coulter) counting, light obscuration or other liquid particle counting systems, by measuring turbidity, and / or by visual inspection); assessing charge heterogeneity by using cation exchange chromatography (CEX), isoelectric focusing (IEF) (e.g., capillary technology (cIEF)), or capillary zone electrophoresis; amino terminal or carboxy terminal sequence analysis; mass spectrometry analysis; SDS-PAGE or SEC analysis to compare fragmented, intact, and multimeric (i.e., dimeric, trimeric, etc.) antibodies; peptide mapping (e.g., trypsin or LYS-C) analysis; assessing biological activity or antigen binding function of the antibody; etc. Instability can include any one or more of the following: aggregation (e.g., non-covalent soluble aggregation, covalent soluble aggregation (e.g., disulfide rearrangement / heterogeneity), insoluble aggregation), deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), truncation / hydrolysis / fragmentation (e.g., hinge region fragmentation), succinimide formation, N-terminal extension, C-terminal processing, glycosylation differences, etc.

[0066] An "anti-CLDN18.2 antibody" refers to an antibody that binds to a CLDN18.2 protein with sufficient affinity and specificity. The antibody affinity can be determined, for example, by surface plasmon resonance-based assays; enzyme-linked immunosorbent assays (ELISA) and competition assays (e.g., RIA). The anti-CLDN18.2 antibody of the present application is preferably Zolbetuximab, molecule M5 disclosed in WO2020 / 018852. CLDN18.2 can also be written as CLDN18_2 in the present application.

[0067] A "therapeutically effective amount" or "therapeutically effective dose" is any amount of an agent that, when used alone or in combination with another therapeutic agent, protects a subject against the onset of a disease or promotes the regression of a disease (as evidenced by a decrease in the severity of symptoms of the disease, an increase in the frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease). The ability of a therapeutic agent to promote the regression of a disease can be assessed using various methods known to those skilled in the art, for example, assessment in human subjects during clinical trials, assessment in animal model systems predictive of efficacy in humans, or assessment by determining the activity of the agent in in vitro assays.

[0068] An "injection formulation" refers to a preparation which is adapted to release the active ingredient in a form that is effective for the biological activity and that is free of components that are otherwise unacceptable, i.e., that are unacceptable to a subject to which the formulation will be administered.

[0069] An "oligosaccharide" refers to a saccharide material formed by the linkage of 2 to 20 monosaccharides through glycosidic bonds. Preferably, in the present application, it is a disaccharide, including but not limited to sucrose, lactose, maltose, trehalose.

[0070] A "buffer" refers to a buffering agent that resists changes in pH through the action of its acid-base conjugate components. Examples of buffers that control the pH in an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0071] A "histidine salt buffer" is a buffer that contains a histidine ion. Examples of histidine salt buffers include histidine-hydrochloric acid, histidine-acetic acid, histidine-phosphoric acid, histidine-sulfuric acid, and the like, wherein the histidine-acetic acid buffer is prepared from histidine and acetic acid, and the histidine-hydrochloric acid buffer is prepared from histidine and hydrochloric acid.

[0072] A "lyophilized formulation" means a formulation or pharmaceutical composition in the form of a liquid or solution obtained after a vacuum freeze-drying step of a liquid or solution formulation.

[0073] The freeze-drying disclosed herein includes pre-freezing, vacuuming, and drying, wherein drying further includes primary drying and secondary drying. The purpose of pre-freezing is to freeze the product and obtain a crystalline solid. The pre-freezing temperature and the pre-freezing speed are two important process parameters, and the pre-freezing speed can be set at 1 °C / min. Primary drying, also known as main drying, is the main stage of freeze-drying of the sample. The purpose is to remove water in the product while maintaining the shape of the product and minimizing damage to the product. If the temperature and vacuum degree of primary drying are not properly selected, it will cause the product to collapse. Both higher temperature and vacuum degree will accelerate the freeze-drying efficiency, but at the same time, it will also increase the risk of product collapse. The temperature of the primary drying disclosed herein can be the temperature conventional in the art. The size and type of the formulation, the container containing the sample (e.g., a glass vial), and the volume of the liquid determine the time required for primary drying, which can range from several hours to several days. Secondary drying is the main step to remove bound water in the product by drawing an extreme vacuum and increasing the temperature. The secondary drying time is determined by the desired residual moisture level in the product, and it usually takes at least about 5 hours. The time of freeze-drying is related to the freezer, the freeze-dried formulation dose, and the container of the freeze-dried drug. The adjustment of such time is well known to those skilled in the art.

[0074] The freeze-dried agent described in the present application should be reconstituted using an aqueous reconstitution component before being administered to a patient. This step allows the antibodies and other components in the freeze-dried agent to be re-dissolved to obtain a solution suitable for injection into a patient.

[0075] A typical reconstitution component for freeze-dried antibodies includes sterile water or a buffer, optionally containing a preservative. If the freeze-dried agent includes a buffer, the reconstitution component can further include a buffer (which can be the same or different from the buffer of the freeze-dried agent), or it can also not include a buffer (e.g., WFI (water for injection), or physiological saline).

[0076] Also provided herein is the use of an injection preparation containing an anti-CLDN18.2 antibody in the preparation of a medicament for preventing / treating cancer, neurodegenerative disease, or infectious disease.

[0077] “Prevention” refers to all initiatives to control or delay the occurrence of a disease.

[0078] “Treatment” refers to all initiatives to alleviate, improve, or relieve the symptoms of a disease. In the present specification, “treatment” refers to alleviating, improving, or relieving the symptoms of cancer, neurodegeneration, or infectious disease by using the antibody of the present application.

[0079] A "subject" refers to those who are suspected of having or diagnosed with cancer, neurodegenerative disease or infectious disease. However, any subject treated with the injection preparation disclosed in the present application is included, but not limited thereto. The injection preparation disclosed in the present application comprises an anti-CLDN18.2 antibody, which is administered to a subject who is suspected of having cancer, neurodegenerative disease or infectious disease.

[0080] The cancer described herein is gastric cancer, esophageal cancer, pancreatic cancer or liver cancer.

[0081] The prescription design of the present application is mainly based on the excipients available for antibody drug preparations, combined with the physicochemical characteristics of anti-CLDN18.2 antibody molecules, etc., to preliminarily determine the prescription composition. With the help of various analysis and evaluation means, four rounds of preparation prescription screening were carried out, and the screening process was as follows: the first round of screening determined the suitable solution buffer system (histidine-acetate), and the second round of screening was carried out based on this buffer system, and the suitable oligosaccharide (sucrose or trehalose) and surfactant (polysorbate 80) were screened out. The third and fourth rounds of screening were based on the screening results of the previous two rounds (histidine-acetate, sucrose or trehalose, polysorbate 80), combined with stability research comparison and osmotic pressure screening results, to determine the preparation prescription.

[0082] Preparation of a liquid pharmaceutical composition containing an anti-CLDN18.2 antibody of Example 1

[0083] The sequences of the heavy and light chains of the anti-CLDN18.2 antibody have been disclosed in WO2020 / 018852, and the molecule M5 was selected to construct CHO cells expressing the anti-CLDN18.2 antibody. After suspension culture, the supernatant was taken, and the antibody solution was obtained after three-step chromatography separation and purification. Then, the antibody was exchanged into other components of the liquid pharmaceutical composition by dialysis to prepare a stock solution, which was then frozen. Subsequently, the antibody stock solution was thawed, double-filtered, and aseptically filled to prepare the final liquid pharmaceutical composition.

[0084] CHO cells expressing anti-CLDN18.2 antibody (with known amino acid sequence) can be constructed according to conventional molecular biology in the art. For example, CN110862454A Example 1 discloses the construction of CHO cells expressing IMAB362 (an anti-CLDN18.2 antibody in clinical research, generic name Zolbetuximab).

[0085] Example 2 First round of preparation prescription screening (sugar, different pH and buffer system selection)

[0086] According to the antibody drug preparation prescription and biological product available excipients, combined with the properties of anti-CLDN18.2 antibody molecules and the initial forced degradation experiment data, 7 prescriptions were initially selected. Then the differential scanning fluorescence (DSF, which measures the thermal stability parameters of proteins) and dynamic light scattering (DLS, which measures the hydrodynamic size distribution of proteins) were used to test the 7 prescription samples.

[0087] Table 1 First round of prescription screening

[0088]

[0089] The results of DSF showed that the anti-CLDN18.2 antibody protein in prescription A3 had the smallest change in intrinsic fluorescence, indicating the highest thermal stability. The results of DLS showed that the anti-CLDN18.2 antibody protein molecules in the 7 prescriptions had similar size distribution. Prescriptions A3 and A4 had smaller hydrodynamic diameters, so they were relatively more stable.

[0090] According to the results of DSF and DLS, prescription A3 showed good thermal stability, and the combination of histidine-acetate buffer system at pH 5.8 and sugar was initially selected, and further research will be carried out on this basis.

[0091] Example 3 Second round of prescription screening (selection of sugar and surfactant, determination of buffer system)

[0092] According to the results of the first round of screening, the histidine-acetate buffer system at pH 5.8 was selected as the basis for the second round of screening, and the histidine-hydrochloride buffer system at pH 5.8 was set as the control. The second round of screening experiment mainly compared the effects of different sugars (sucrose vs. trehalose) and different surfactants (polysorbate 20 vs. polysorbate 80) in the prescription on protein stability, and 6 prescriptions in the table were designed.

[0093] Table 2 Second round of prescription screening

[0094]

[0095] The results of DSF showed that prescriptions B3 and B4 had the smallest change in fluorescence of anti-CLDN18.2 antibody, and the highest thermal stability. The results of DLS showed that prescriptions B2-B4 were relatively stable, while prescriptions B1, B5 and B6 showed new aggregates.

[0096] According to the results of DSF and DLS studies, Formulations B3 and B4 performed better, and 20 mmol / L Histidine-Acetate, 0.04 wt% polysorbate 80, 6 wt% trehalose or 5 wt% sucrose were determined as the base, further combined with other conditions for stability study.

[0097] Example 4 Third round of formulation screening (sugar and pH determination)

[0098] According to the results of the first round of Example 1 and the second round of Example 2, 20 mmol / L Histidine-Acetate, 0.04 wt% polysorbate 80, 6 wt% trehalose or 5 wt% sucrose were determined as the more suitable formulation for the anti-CLDN18.2 antibody. The third round of experiment was to further compare the sugar (sucrose and trehalose) and the pH value selection, and 6 formulations in the table were designed for the third round of screening.

[0099] Table 3 Third round of formulation screening

[0100]

[0101] From the results of DSF studies, there was no significant difference among the 6 formulations. The results of DLS studies showed that Formulation C5 had a smaller hydrodynamic diameter, and thus was relatively more stable.

[0102] Example 5 Preliminary stability study at high temperature 40°C

[0103] Preliminary stability study at high temperature 40°C was performed on the samples of Formulations C1-C6 in Example 4, focusing on SEC-HPLC, WCX-HPLC and CE-SDS purity.

[0104] SEC-HPLC: The SEC-HPLC purity change of each formulation at high temperature 40°C from 0 days to 4 weeks was investigated. It was found that the monomer content of Formulation C3 significantly decreased at 4 weeks, and there was no significant difference among the other formulations.

[0105] Table 4 SEC-HPLC purity change at high temperature 40°C (0 days to 4 weeks)

[0106]

[0107] Reduced CE-SDS: The reduced CE-SDS purity change of each formulation at high temperature 40°C from 0 days to 4 weeks was investigated. There was no significant difference among the formulations.

[0108] Table 5 Reduced CE-SDS purity change at high temperature 40°C (0 days to 4 weeks)

[0109]

[0110] Non-reducing CE-SDS: The non-reducing CE-SDS purity change of each formulation under high temperature 40℃ condition from 0 day to 4 weeks was investigated. It was found that the non-glycosylated protein content of formulation C6 increased significantly, and there was no significant difference in other formulations.

[0111] Table 6 Non-reducing CE-SDS purity change of each formulation under high temperature 40℃ condition (0 day to 4 weeks)

[0112]

[0113] WCX-HPLC: The WCX-HPLC purity change of each formulation under high temperature 40℃ condition from 0 day to 4 weeks was investigated. There was no significant difference in each formulation.

[0114] Table 7 WCX-HPLC purity change of each formulation under high temperature 40℃ condition (0 day to 4 weeks)

[0115]

[0116]

[0117] The results of the study showed that formulations C1, C2, C4 and C5 were superior to formulations C3 and C6, and formulations C1, C2, C4 and C5 would be further compared and optimized as candidate formulations.

[0118] Example 6 Selection of sucrose and trehalose

[0119] Under frozen glass state, trehalose is more likely to separate from the protein, thereby losing the protective effect of the protein. Since the antibody stock of the product needs to be stored at -80±10℃, in order to effectively protect the protein, sucrose is more optimal. Formulations C4 and C5 are superior to formulations C1 and C2, so formulations C4 and C5 will be further compared and optimized as candidate formulations.

[0120] Example 7 Selection of different pH

[0121] Based on the results of the first round of screening in Example 2 and the DLS study, it was found that the thermal stability of the protein solution at pH 5.8 was relatively robust, so the preferred formulation of the liquid pharmaceutical composition containing the anti-CLDN18.2 antibody was initially formulated as formulation C5 (20 mmol / L histidine-acetate, 5 wt% sucrose, 0.04 wt% polysorbate 80, pH 5.8).

[0122] Example 8 Fourth round of formulation screening (amount of sucrose)

[0123] The osmotic pressure of normal human blood ranges from 285 to 310 mOsmol / kg. To ensure that the osmotic pressure level of the anti-CLDN18.2 antibody-containing injection solution (diluted for infusion with 0.9% sodium chloride solution) is equivalent to that of human blood during clinical use, a small sample of prescription C5 in the third round of screening was subjected to osmotic pressure detection.

[0124] Table 8 Osmotic pressure detection results

[0125]

[0126] The results of the study showed that the osmotic pressure of the formulation solution containing 5 wt% sucrose was low. To further increase the osmotic pressure of the formulation solution, the osmotic pressure of the formulation solution containing 6 wt%, 7 wt%, 8 wt%, and 10 wt% sucrose was detected, respectively. The results showed that increasing the sucrose concentration in the formulation prescription also increased the osmotic pressure of the formulation solution.

[0127] Table 9 Osmotic pressure detection results

[0128]

[0129]

[0130] During clinical use of the anti-CLDN18.2 antibody-containing injection solution, 0.9% sodium chloride solution was used for dilution and infusion. The formulation solution containing 6 wt% and 7 wt% sucrose was diluted with 0.9% sodium chloride solution to 1 mg / ml and 10 mg / ml, respectively, and subjected to osmotic pressure detection.

[0131] Table 10 Osmotic pressure detection results

[0132]

[0133] The results showed that when the sucrose content in the prescription was increased to 6 wt% to 8 wt%, the final osmotic pressure value of the liquid for intravenous administration was close to the osmotic pressure molar concentration of human blood. Subsequently, the above-mentioned prescriptions were further investigated in combination with stability studies.

[0134] Example 9 Stability test

[0135] Test sample information: The anti-CLDN18.2 antibody-containing injection stability test sample was derived from Jiangsu Aoxikang Pharmaceutical Co., Ltd., and the detailed information is shown in the following table.

[0136] Table 11 Stability test sample information

[0137]

[0138]

[0139] (1) Impact factor test (high temperature)

[0140] The stability of the sample under high temperature (40 ± 2°C) was investigated, focusing on SEC-HPLC, WCX-HPLC and CE-SDS purity. SEC-HPLC: The SEC-HPLC purity change of each formulation sample under high temperature (40 ± 2°C) from 0 days to 4 weeks was investigated.

[0141] Table 12 High temperature (40 ± 2°C), upright investigation results (sample 3-1)

[0142]

[0143] Table 13 High temperature (40 ± 2°C), inverted investigation results (sample 3-1)

[0144]

[0145]

[0146] Table 14 High temperature (40 ± 2°C), upright investigation results (sample 3-2)

[0147]

[0148]

[0149] Table 15 High temperature (40 ± 2°C), inverted investigation results (sample 3-2)

[0150]

[0151] Table 16 High temperature (40 ± 2°C), upright investigation results (sample 3-3)

[0152]

[0153]

[0154] Table 17 High temperature (40 ± 2°C), inverted investigation results (sample 3-3)

[0155]

[0156]

[0157] The three groups of samples investigated in this example were investigated at 40±2°C (upright / inverted) for 4 weeks, and the SEC-HPLC purity, non-reduced CE-SDS purity, WCX-HPLC purity and polysorbate 80 content all showed a clear downward trend. Among them, the main peak purity of WCX-HPLC and the content of polysorbate 80 were lower than the quality standard at the second week, so the product should not be exposed to 40°C for more than 1 week.

[0158] (2) Influence factor test (light)

[0159] The stability of the samples under light (4500±500lx / 25±2°C) was investigated, with a focus on SEC-HPLC, WCX-HPLC and CE-SDS purity.

[0160] Table 18 Light (4500±500lx / 25±2°C), horizontal investigation results (sample 3-1)

[0161]

[0162] Table 19 Light (4500±500lx / 25±2°C), horizontal investigation results (sample 3-2)

[0163]

[0164]

[0165] Table 20 Light (4500±500lx / 25±2°C), horizontal investigation results (sample 3-3)

[0166]

[0167] The three groups of samples investigated in this example were investigated at 4500±500lx / 25±2°C (horizontal) for 5 days, and the non-reduced CE-SDS purity and WCX-HPLC purity showed a downward trend. Among them, the WCX-HPLC purity decreased significantly, and the main peak purity of 2 groups of samples was lower than the quality standard at the third day, and the main peak purity of 1 group of samples was lower than the quality standard at the fifth day, so the product should not be exposed to light for more than 1 day.

[0168] (3) Influence factor test (transport simulation)

[0169] During the storage, transportation and clinical use of injection preparations, shaking will occur, leading to protein denaturation and inactivation. One of the important indicators for investigating injection preparations is the stability of the investigation period under shaking conditions. In this example, the samples were shaken (150rpm) at 5±3°C for 1 week to evaluate the performance of different formulations.

[0170] Table 21 Transportation simulation (5±3°C / 150rpm), horizontal (sample 3-1)

[0171]

[0172] Table 22 Transportation simulation (5±3°C / 150rpm), horizontal (sample 3-2)

[0173]

[0174]

[0175] Table 23 Transportation simulation (5±3°C / 150rpm), horizontal (sample 3-3)

[0176]

[0177] The 3 groups of samples investigated in this example were investigated at 5±3°C / 150rpm (horizontal) for 1 week, and there were no significant changes in the results of each investigation, so the product can be transported at 5±3°C for 1 week.

[0178] Example 10 Accelerated test

[0179] In this example, the accelerated stability, i.e. the stability at a temperature higher than the storage condition (the storage temperature is 2-8°C), was investigated. The temperature investigated was 25±2°C. The accelerated stability is helpful to distinguish different formulations, and can also be used to estimate the stability of the product under long-term storage conditions. In addition, the room temperature stability for a certain period of time is helpful for future clinical conditions, reducing the requirement for cold chain, etc. The samples placed at 25±2°C were stored for 0 days, 1 month, 2 months, 3 months and 6 months, and then sampled, to detect the changes in the content of multimers and fragments, the changes in insoluble particles and the changes in thermal stability at different time points, so as to investigate the performance of different formulations under this condition.

[0180] Table 24 Accelerated test of finished product (25±2°C / 60±%RH), upright investigation results (sample 3-1)

[0181]

[0182]

[0183] Table 25 Accelerated test of finished product (25±2°C / 60±%RH), inverted investigation results (sample 3-1)

[0184]

[0185]

[0186] Table 26 Accelerated test of finished product (25±2℃ / 60±%RH), upright investigation results (sample 3-2)

[0187]

[0188]

[0189] Table 27 Accelerated test of finished product (25±2℃ / 60±%RH), inverted investigation results (sample 3-2)

[0190]

[0191]

[0192] Table 28 Accelerated test of finished product (25±2℃ / 60±%RH), upright investigation results (sample 3-3)

[0193]

[0194]

[0195] Table 29 Accelerated test of finished product (25±2℃ / 60±%RH), inverted investigation results (sample 3-3)

[0196]

[0197]

[0198] The 3 groups of samples investigated in this example were investigated under accelerated conditions of 25±2℃ (upright / inverted) for 6 months, and the number of insoluble particles ≥10μm showed a trend of increase compared with before placement, SEC-HPLC and non-reducing CE-SDS purity showed a downward trend, WCX-HPLC main peak purity and polysorbate 80 content decreased significantly, and at the 3rd month, they were lower than the quality standard, so the product should not be stored at 25±2℃ for more than 2 months.

[0199] Example 11 Long-term test

[0200] The proposed long-term storage temperature of the anti-CLDN18.2 antibody injection solution is 2-8℃. In this example, the stability of samples with different formulations under long-term storage conditions was investigated. Samples were taken on the 0th day, 6th month, 12th month and 24th month of upright or inverted storage of each sample, and the changes in multimer and fragment content, insoluble particle changes and thermal stability at different time points were detected to test the performance of samples with different formulations under the conditions.

[0201] Table 30 Long-term test of finished product (5±3℃), upright investigation results (sample 3-1)

[0202]

[0203]

[0204] Table 31 Long-term test of finished product (5±3℃), inverted observation results (sample 3-1)

[0205]

[0206] Table 32 Long-term test of finished product (5±3℃), upright observation results (sample 3-2)

[0207]

[0208] Table 33 Long-term test of finished product (5±3℃), inverted observation results (sample 3-2)

[0209]

[0210]

[0211] Table 34 Long-term test of finished product (5±3℃), upright observation results (sample 3-3)

[0212]

[0213]

[0214] Table 35 Long-term test of finished product (5±3℃), inverted observation results (sample 3-3)

[0215]

[0216]

[0217] The 3 groups of samples investigated in this example were investigated for 24 months under the condition of long-term 5±3℃ (upright / inverted), and the appearance, clarity, pH, visible foreign matter, osmolality, protein concentration, bacterial endotoxin, and relative biological activity before and after storage all met the requirements. The SEC-HPLC purity, reduced CE-SDS purity, non-reduced CE-SDS purity, WCX-HPLC purity, and polysorbate 80 content were not significantly changed compared with before storage. The number of insoluble particles ≥10μm increased compared with before storage, but all met the quality standards. It was shown that the above three groups of anti-CLDN18.2 antibody pharmaceutical compositions had long-term stability under the storage condition of 5±3℃.

[0218] Freeze-drying method of the preparation of Example 12

[0219] (1) Plate cooling: open the circulating pump, compressor, and condenser valve, and turn on the electric heating, and adjust the temperature of the heat conducting oil to pre-cool the baffle temperature to 4 DEG C;

[0220] (2) Pre-freezing: after the product is put into the box, close the box door, close the electric heating, open the condenser valve, open the plate cooling valve, adjust the heat conducting oil to make the product temperature reach-45 DEG C (about 1 DEG C / min), and keep for more than 2 hours;

[0221] (3) Cold trap: switch the compressor to "cold trap" refrigeration;

[0222] (4) Vacuumizing: when the "cold trap" temperature is reduced to below-45 DEG C, open the vacuum pump and the small butterfly valve, and when the "cold trap" vacuum degree of the rear box reaches about 100 mTorr (13.33 pa), open the middle baffle valve;

[0223] (5) Primary drying: set the temperature of the heat conducting oil to-35 DEG C within 1.5-2 hours, and keep for 24-30 hours;

[0224] (6) Desorption drying: set the temperature of the heat conducting oil to 20 DEG C within 9 hours, and the vacuum to 50 mTorr (6.67 pa), and keep for 15-20 hours or more.

[0225] In the primary drying of step (5), the temperature rising speed can be set to 0.2 DEG C / min; and in the desorption drying of step (6), the temperature rising speed can be set to 0.1 DEG C / min.

[0226] The freeze-drying protection process freezes the water contained in the product at low temperature, and then dries it in a vacuum environment, so that the water is directly sublimated into water vapor from the solid state and removed from the product, so that the product is dried. Active. This method effectively prevents changes in the physical and chemical properties of the product, causes less damage to the structure and characteristics of biological tissues and cells, quickly puts them into a dormant state, and effectively protects the stability of many heat-sensitive pharmaceutical biological products.

[0227] The liquid pharmaceutical composition of the present application has simple components and good stability, is suitable for storage and transportation, can reduce the cost of packaging materials, transportation and storage, etc., while maintaining the biological activity of the antibody.

[0228] The liquid pharmaceutical composition containing the anti-CLDN18.2 antibody of the present application shows good stability in the shaking / vibration, 25±2 DEG C accelerated test, and 5±3 DEG C long-term storage stability test, and has good application prospect.

[0229] It should be understood that although certain aspects of this specification are highlighted by reference to specific embodiments, those skilled in the art will readily understand that these disclosed embodiments are merely illustrative of the principles of the subject matter disclosed herein. Therefore, it should be understood that the disclosed subject matter is by no means limited to the specific compounds, compositions, products, instruments, methods, procedures, and / or reagents described herein, unless explicitly stated otherwise. Furthermore, it should be recognized that certain variations, modifications, substitutions, alterations, additions, deletions, and sub-combinations of those common techniques in the art can be derived from the teachings herein without departing from the spirit of this specification. Therefore, the dependent claims and claims set forth below are to be construed as including all variations, modifications, substitutions, alterations, additions, deletions, and sub-combinations within their true subject matter and scope.

[0230] Certain embodiments of the invention have been described herein, including the inventors' known best mode for carrying out the invention. Of course, variations of the described embodiments will be apparent to those skilled in the art upon reading the foregoing description. The inventors intend that those skilled in the art will appropriately adopt these variations, and that the embodiments of the invention are not limited to the specific descriptions herein. Therefore, the invention includes all modifications and equivalent substitutions permitted by applicable law to the content of the claims. Moreover, any combination of all possible changes to the foregoing embodiments is included in the invention unless otherwise stated herein or clearly contradicted by the context.

[0231] Finally, the terminology used herein is for describing particular embodiments only and does not limit the scope of the invention, which is defined only by the claims. Therefore, the invention is not limited to the details shown and described.

Claims

1. A liquid pharmaceutical composition comprising an anti-CLDN18.2 antibody, characterized in that, The pharmaceutical composition comprises: an anti-CLDN18.2 antibody, a surfactant, an oligosaccharide, water, a buffer, a pH regulator; the anti-CLDN18.2 antibody is molecule M5 in WO2020 / 018852, and the content of the anti-CLDN18.2 antibody is 20-30 mg / mL; the surfactant is polysorbate 80, and the content of the surfactant is 0.02wt%-0.06wt%; the oligosaccharide is sucrose, and the content of the oligosaccharide is 6wt%-8wt%; the buffer is histidine-acetate, and the concentration of the buffer is 20-30 mmol / L; the pH regulator is glacial acetic acid, and the pH of the pharmaceutical composition is 5.5-5.

8.

2. The pharmaceutical composition of claim 1, wherein, The pharmaceutical composition comprises: 25-30 mg / mL of an anti-CLDN18.2 antibody.

3. The pharmaceutical composition of claim 2, wherein The pharmaceutical composition comprises: 25 mg / mL of an anti-CLDN18.2 antibody; 0.04wt% of polysorbate 80; 7wt% of sucrose; and 20 mmol / L of histidine-acetate; wherein the pH of the pharmaceutical composition is 5.

8.

4. Process for the preparation of a pharmaceutical composition as claimed in any one of claims 1 to 3, characterized in that, The method comprises the steps of preparing an anti-CLDN18.2 antibody, replacing the antibody with other components of the liquid pharmaceutical composition, sterilizing filtration, and aseptic filling.

5. A lyophilized formulation characterized in that The liquid pharmaceutical composition of any one of claims 1-3 is freeze-dried.

6. Use of the liquid pharmaceutical composition of any one of claims 1-3 in the preparation of a medicament for treating cancer; the cancer is gastric cancer or pancreatic cancer.

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