Pharmaceutical composition containing anti-CD19 antibody and application thereof

Abstract

The invention relates to a pharmaceutical composition of an anti-CD19 monoclonal antibody and application of the pharmaceutical composition. The freeze-dried preparation of the anti-CD19 monoclonal antibody comprises an anti-CD19 antibody irnerizumab, a first protective agent, a second protective agent and a surfactant. The pharmaceutical composition is used for treating immune globulin G4 related diseases, neuromyelitis optica spectrum disorders and other diseases.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical preparations, and particularly relates to a pharmaceutical composition containing an anti-CD19 monoclonal antibody and uses thereof. Background Art

[0002] CD19 is a glycoprotein expressed on the surface of B cells and belongs to the immunoglobulin (Ig) superfamily. It is closely involved in B cell activation, proliferation, and differentiation. It acts as a co-receptor in B cell activation and signaling, regulating B cell activation and proliferation, participating in B cell signaling, and mediating T cell cytotoxicity against target cells. CD19 expression begins early in B cell development and is downregulated during B cell differentiation into plasma cells. Furthermore, CD19 is a crucial component of the BCR complex, forming a complex with CD21 and CD81 that lowers the threshold for BCR-mediated B cell activation. CD21 provides a bridge to surface immunoglobulins, CD81 regulates CD19 expression, and CD19 plays a primary role in signaling. CD19 regulates B cell development, proliferation, and differentiation in both BCR-dependent and -independent ways. CD19 can also form heterodimers or complexes with other membrane proteins (such as CD21 and CD81) to amplify BCR signals. The intracellular domain of CD19 physically binds to a series of tyrosine kinases (such as Lyn and Fyn), triggering downstream signaling pathways such as the Src kinase family. Furthermore, CD19 plays a role in the interaction between B cells and T cells, participating in the regulation of immune responses.

[0003] CD19 is widely expressed in almost all B-cell malignancies, including chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), and non-Hodgkin's lymphoma. Therefore, CD19 has become a specific molecular target for the treatment of B-cell malignancies and an important diagnostic marker for these diseases. Furthermore, CD19 also plays a role in certain autoimmune diseases, making it a popular target in the development of tumor immunotherapy and autoimmune disease drugs.

[0004] WO2008 / 031056 discloses inelizumab, an anti-CD19 monoclonal antibody. It acts by targeting CD19 antigen-mediated B cell depletion, depleting multiple B cell subsets, including plasmablasts. Currently, inelizumab injection has been approved in China, the United States, and other countries for the treatment of neuromyelitis optica spectrum disorder (NMOSD). Several other indications related to tumor immunity and autoimmune diseases, such as myasthenia gravis, systemic sclerosis, systemic lupus erythematosus, and diffuse large B-cell lymphoma, are in clinical trials. However, liquid formulations of monoclonal antibodies often face stability challenges, with a short shelf life and the potential loss of biological activity during storage due to chemical and physical instabilities. Therefore, there is an urgent need to develop formulations with higher stability. Summary of the Invention

[0005] The present invention provides a lyophilized formulation of inerizumab, obtained by vacuum freeze-drying a liquid formulation. The lyophilized formulation further comprises a protective agent and a surfactant, wherein the protective agent is selected from sucrose and xylitol, and the surfactant is poloxamer. The weight ratio of inerizumab to protective agent is 1-4:12, the weight ratio of sucrose to xylitol is 2:1, and the weight ratio of poloxamer to sucrose to xylitol is 1:160-200:80-100. This pharmaceutical composition has excellent stability.

[0006] The above-mentioned inelizumab and its preparation method are described in WO2008 / 031056, the entire contents of which are incorporated into this application by reference. The relevant sequences are shown in Tables A and B.

[0007] Table A. Inerizumab CDR and variable region sequence information

[0008]

[0009] Note: CDR sequences were defined according to the Kabat definition scheme.

[0010] Table B. Inerizumab full-length sequence information

[0011]

[0012] In certain preferred embodiments of the present invention, the weight ratio of poloxamer, sucrose and xylitol is 1:200:100.

[0013] In certain embodiments of the invention, the weight of inerizumab is 100 mg.

[0014] In certain embodiments of the present invention, the lyophilized formulation further comprises a citrate buffer, and the citrate buffer is citric acid-sodium citrate.

[0015] In preferred certain embodiments of the application, the poloxamer is poloxamer 188.

[0016] In preferred certain embodiments of the application, the concentration of inebilizumab in the liquid formulation is 10-40 mg / mL, for example 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL.

[0017] In preferred certain embodiments of the application, the concentration of inebilizumab in the liquid formulation is 20 mg / mL.

[0018] In certain embodiments of the application, the concentration of sucrose in the liquid formulation is 50-100 mg / mL, for example 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL.

[0019] In preferred certain embodiments of the application, the concentration of sucrose in the liquid formulation is 50-80 mg / mL, for example 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL.

[0020] In preferred certain embodiments of the application, the concentration of sucrose in the liquid formulation is 80 mg / mL.

[0021] In certain embodiments of the application, the concentration of xylitol in the liquid formulation is 20-50 mg / mL, for example 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL.

[0022] In preferred certain embodiments of the application, the concentration of xylitol in the liquid formulation is 20-40 mg / mL, for example 20 mg / mL, 30 mg / mL, 40 mg / mL.

[0023] In preferred certain embodiments of the application, the concentration of xylitol in the liquid formulation is 40 mg / mL.

[0024] In certain embodiments of the application, the weight ratio of sucrose to xylitol is 1-5: 1.

[0025] In preferred certain embodiments of the application, the weight ratio of sucrose to xylitol is 1-4: 1.

[0026] In preferred certain embodiments of the application, the weight ratio of sucrose to xylitol is 2: 1.

[0027] In certain embodiments of the present invention, the concentration of poloxamer in the liquid preparation is 0.3-0.5 mg / mL, for example, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL.

[0028] In certain preferred embodiments of the present invention, the concentration of poloxamer in the liquid preparation is 0.4-0.5 mg / mL, for example, 0.4 mg / mL, 0.5 mg / mL.

[0029] In certain preferred embodiments of the present invention, the concentration of poloxamer in the liquid preparation is 0.4 mg / mL.

[0030] In certain preferred embodiments of the present invention, the concentration of the citric acid-sodium citrate buffer in the liquid preparation is 20 mM.

[0031] In certain embodiments of the present invention, the pH of the liquid preparation is 5.5-6.0.

[0032] In certain preferred embodiments of the present invention, the pH of the liquid preparation is 5.5.

[0033] In certain embodiments of the present invention, the concentration of inelizumab in the liquid preparation is 10-40 mg / mL, the concentration of sucrose is 50-100 mg / mL, the concentration of xylitol is 20-50 mg / mL, the concentration of poloxamer 188 is 0.3-0.5 mg / mL, the concentration of citric acid-sodium citrate buffer is 20 mM, and the pH is 5.5-6.0.

[0034] In certain preferred embodiments of the present invention, the concentration of inelizumab in the liquid preparation is 10-40 mg / mL, the concentration of sucrose is 50-80 mg / mL, the concentration of xylitol is 20-40 mg / mL, the concentration of poloxamer 188 is 0.4-0.5 mg / mL, the concentration of citric acid-sodium citrate buffer is 20 mM, and the pH is 5.5-6.0.

[0035] In certain preferred embodiments of the present invention, the concentration of inelizumab in the liquid preparation is 10-40 mg / mL, the concentration of sucrose is 80 mg / mL, the concentration of xylitol is 40 mg / mL, the concentration of poloxamer 188 is 0.4-0.5 mg / mL, the concentration of citric acid-sodium citrate buffer is 20 mM, and the pH is 5.5.

[0036] In certain preferred embodiments of the present invention, the concentration of inelizumab in the liquid preparation is 20 mg / mL, the concentration of sucrose is 80 mg / mL, the concentration of xylitol is 40 mg / mL, the concentration of poloxamer 188 is 0.4 mg / mL, the concentration of citric acid-sodium citrate buffer is 20 mM, and the pH is 5.5.

[0037] Another object of the present invention is to provide the use of the pharmaceutical composition in the preparation of drugs for treating immunoglobulin G4-related diseases, neuromyelitis optica spectrum disorders, myasthenia gravis, systemic scleroderma, systemic lupus erythematosus, diffuse large B-cell lymphoma and related diseases.

[0038] Another object of the present invention is to provide a freeze-drying process:

[0039] Pre-freezing stage: maintain at 5℃ for 1 hour; maintain at -45℃ for 2 hours.

[0040] Primary drying: -25℃ for 50 hours.

[0041] Secondary drying: 25°C, vacuum degree 10Pa for 2 hours; then 25°C, vacuum degree 1Pa for 10 hours.

[0042] By selecting specific protective agents and surfactants and optimizing their combined weight ratio, the pharmaceutical composition prepared in this invention exhibits excellent stability. Specifically, under forced degradation conditions such as 40°C for one, three, and six months, the sample's quality indicators remained unchanged compared to the zero-day results, demonstrating high stability. Furthermore, the prepared product exhibits a good morphology, resists collapse, and is easily reconstituted. The process is robust, with strong production adaptability, making it suitable for large-scale industrial production.

[0043] Detailed Description of the Invention

[0044] Unless otherwise stated, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art. Specifically, the terms used in the specification and claims have the following meanings.

[0045] The three letter and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0046] The term "antibody" as used herein is used in the broadest sense and encompasses various antibody structures, including but not limited to full-length antibodies and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. Typically, a natural intact antibody consists of a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains connected by interchain disulfide bonds.

[0047] "Buffer" refers to a buffer that tolerates changes in pH through the action of its acid-base conjugate components. Examples of buffers that control pH within an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0048] A "histidine salt buffer" is a buffer containing histidine ions. Examples of histidine salt buffers include histidine hydrochloride, histidine acetate, histidine phosphate, histidine sulfate, and the like. Histidine acetate buffer is preferred. Histidine acetate buffer is a mixture of histidine and acetic acid, while histidine hydrochloride buffer is a mixture of histidine and hydrochloric acid.

[0049] "Citrate buffer" is a buffer comprising citrate ions. Examples of citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-sodium bicarbonate, and the like. A preferred citrate buffer is citric acid-sodium citrate.

[0050] A "succinate buffer" is a buffer comprising succinate ions. Examples of succinate buffers include succinic acid-sodium succinate, succinic acid-potassium succinate, succinic acid-calcium succinate, and the like. A preferred succinate buffer is succinic acid-sodium succinate. For example, the succinic acid-sodium succinate can be prepared from succinic acid and sodium hydroxide, or from succinic acid and sodium succinate.

[0051] "Phosphate buffer" is a buffer containing phosphate ions. Examples of phosphate buffers include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, disodium hydrogen phosphate-citric acid, and the like. A preferred phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate.

[0052] "Poloxamer" is a block copolymer of ethylene oxide and propylene oxide that is water-soluble and used as a surfactant in pharmaceutical formulations. Examples of poloxamers include poloxamer 188 (P188).

[0053] A "pharmaceutical composition" refers to a mixture containing one or more antibodies described herein, or their physiologically / pharmaceutically acceptable salts or prodrugs, together with other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to maintain the stability of the active ingredient of the antibody, facilitate administration to an organism, and promote absorption of the active ingredient, thereby exerting its biological activity.

[0054] In the present invention, "pharmaceutical composition" and "preparation" are not mutually exclusive.

[0055] Unless otherwise specified, the solvent in the solution form of the pharmaceutical composition of the present invention is water.

[0056] "Lyophilized formulation" refers to a pharmaceutical composition in liquid or solution form or a formulation or pharmaceutical composition obtained after a liquid or solution formulation has been subjected to a vacuum freeze-drying step.

[0057] As used herein, the terms "about" and "approximately" refer to values ​​that are within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which value depends in part on how it is measured or determined (i.e., the limits of the measurement system). For example, in each practice in the art, "about" can mean within 1 or more than 1 standard deviation. Alternatively, "about" or "substantially comprising" can mean a range of up to 20%. In addition, particularly for biological systems or processes, the terms can mean up to an order of magnitude or up to 5 times the value. Unless otherwise indicated, when a specific value appears in the application and claims, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for that specific value.

[0058] The pharmaceutical compositions of the present invention can achieve a stable effect: the antibody-drug conjugate therein substantially retains its physical stability and / or chemical stability and / or biological activity after storage. Preferably, the pharmaceutical composition substantially retains its physical and chemical stability as well as its biological activity after storage. The storage period is generally selected based on the intended shelf life of the pharmaceutical composition. Currently, there are various analytical techniques for measuring protein stability after storage at a selected temperature for a selected period of time.

[0059] A stable formulation is one in which no significant changes are observed when stored at refrigerated temperatures (2-8°C) for at least one month, preferably six months, more preferably one year, and even more preferably up to two years. Additionally, stable liquid formulations include those that exhibit the desired characteristics after storage at 25°C for periods of time, including one month, three months, or six months. Typical examples of stability include: typically no more than about 10%, preferably no more than about 5%, of antibody monomers aggregate or degrade as measured by SEC-HPLC. Visually, the formulation is a pale yellow, nearly colorless, clear liquid, or colorless, or clear to slightly opalescent. The concentration, pH, and osmolality of the formulation vary by no more than ±10%. A decrease of no more than about 10%, preferably no more than about 5%, is typically observed. Aggregates typically form no more than about 10%, preferably no more than about 5%.

[0060] The antibody "retains its physical stability" in the pharmaceutical formulation if the antibody drug conjugate shows no significant increase in aggregation, precipitation, and / or denaturation as measured by visual inspection of color and / or clarity, or by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS). Changes in protein conformation can be assessed by fluorescence spectroscopy (which determines protein tertiary structure) and by FTIR spectroscopy (which determines protein secondary structure).

[0061] An antibody "retains its chemical stability" in a pharmaceutical formulation if it does not show significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that often change the chemical structure of a protein include hydrolysis or truncation (assessed by methods such as size exclusion chromatography and CE-SDS), oxidation (assessed by methods such as peptide mapping coupled to mass spectrometry or MALDI / TOF / MS), deamidation (assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartate measurement), and isomerization (assessed by measuring isoaspartate content, peptide mapping, etc.).

[0062] An antibody "retains its biological activity" in a pharmaceutical formulation if the biological activity of the antibody at a given time is within a predetermined range of the biological activity exhibited when the pharmaceutical formulation is prepared.

[0063] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable region of the specified sequence may but need not be present.

[0064] "Substituted" means that one or more hydrogen atoms, preferably up to five, and more preferably one to three, hydrogen atoms in a group are independently replaced by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine (either experimentally or theoretically) which substitutions are possible or impossible without undue effort. For example, an amino or hydroxyl group with a free hydrogen may be unstable when combined with a carbon atom with an unsaturated (e.g., olefinic) bond.

[0065] The preparation of conventional pharmaceutical compositions can be found in the Chinese Pharmacopoeia.

[0066] The term "carrier" as used in the context of the present disclosure for a pharmaceutical means a system that modifies the way a pharmaceutical enters the body and its distribution in the body, controls the rate of release of the pharmaceutical and delivers the pharmaceutical to a target organ. Pharmaceutical carrier release and targeting systems can reduce degradation and loss of the pharmaceutical, reduce side effects, and increase bioavailability. As a result of their unique amphiphilic structure, high molecular weight surfactants, which can be used as carriers, can self-assemble to form various forms of aggregates, such as micelles, microemulsions, gels, liquid crystals, vesicles, and the like. These aggregates have the ability to encapsulate drug molecules and at the same time have good membrane permeability, and can be used as excellent pharmaceutical carriers.

[0067] "Administering" and "treatment" when applied to an animal, human, test subject, cell, tissue, organ or biological fluid means the contact of an exogenous pharmaceutical, therapeutic agent, diagnostic agent or composition with the animal, human, test subject, cell, tissue, organ or biological fluid. "Administering" and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Treatment of a cell includes contact of the agent with the cell, as well as contact of the agent with a fluid that is in contact with the cell. "Administering" and "treatment" also mean treatment of a cell in vitro and ex vivo by an agent, diagnostic, binding composition or by another cell. "Treatment" when applied to a human, veterinary or research test subject means therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

[0068] "Treatment" means the administration of an internal or external therapeutic agent, such as a composition comprising any of the binding compounds of the present disclosure, to a patient having one or more symptoms of a disease, where the therapeutic agent is known to have a therapeutic effect on those symptoms. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of a disease in a patient or population being treated, to induce regression of such symptoms or to inhibit the progression of such symptoms to any clinically measurable extent. The amount of a therapeutic agent effective to alleviate any particular symptom of a disease (also referred to as a "therapeutically effective amount") can vary according to factors such as the disease state, age, and weight of the patient, and the ability of the pharmaceutical to elicit a desired effect in the patient. Whether a disease symptom has been alleviated can be assessed by any clinical test method typically used by a physician or other professional health care provider to assess the severity or progression of the symptom. While embodiments of the present disclosure (e.g., a method of treatment or article of manufacture) can not be effective in alleviating every symptom of a disease of interest, it is determined that a statistically significant number of patients should have alleviation of the disease symptom of interest according to any statistical test method known in the art, such as the Student t-test, the Chi-square test, the U-test according to Mann and Whitney, the Kruskal-Wallis test (H-test), the Jonckheere-Terpstra test, and the Wilcoxon test.

[0069] An "effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also means an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

[0070] "Displacement" refers to the replacement of the solvent system in which the antibody protein is dissolved. For example, a high-salt or hypertonic solvent system containing the antibody protein is replaced by a physical manipulation using a buffer system that stabilizes the formulation, thereby stabilizing the antibody protein in the formulation. Such physical manipulations include, but are not limited to, ultrafiltration, dialysis, or centrifugation followed by reconstitution. DETAILED DESCRIPTION

[0071] The present invention is further described below with reference to the following examples, but these examples are not intended to limit the scope of the invention. Experimental methods in the examples herein where specific conditions are not specified generally follow conventional conditions, such as those in the Cold Spring Harbor Laboratory's "Antibody Techniques Laboratory Manual" and "Molecular Cloning Manual," or according to the conditions recommended by the raw material or product manufacturer. Reagents whose sources are not specified are commercially available conventional reagents.

[0072] The equipment used in the test and the calculation method of the results are as follows:

[0073] 1) SEC size exclusion chromatography:

[0074] An analytical method that separates solutes based on the relative relationship between the pore size of the gel and the coil size of the polymer sample molecules.

[0075] SEC% (SEC monomer content percentage) = A monomer / A total * 100% (A monomer is the peak area of ​​the main peak monomer in the sample, and A total is the sum of all peak areas).

[0076] SEC instrument: Agilent 1260; column: waters, XBrige BEH200Å SEC (300×7.8mm3.5μm).

[0077] 2) CE capillary gel electrophoresis:

[0078] A method of electrophoresis in which gel is transferred to a capillary tube as a supporting medium and samples are separated according to their molecular weight at a certain voltage.

[0079] Reduction CE purity percentage = A main peak / A total * 100% (A main peak is the peak area of ​​the light chain main peak + the heavy chain main peak in the sample, and A total is the sum of all peak areas.

[0080] CE measurement instrument: Beckman model plus800.

[0081] 3) Turbidity measurement:

[0082] The degree of obstruction to light passing through the water layer indicates the water's ability to scatter and absorb light. This is related not only to the suspended matter content but also to the particle composition, size, shape, and surface reflectivity. By comparing the absorbance values ​​of the same protein sample at the same concentration and wavelength (near-UV and visible wavelength regions), higher absorbance values ​​indicate greater turbidity and a more pronounced tendency for protein molecules in the sample to aggregate. The measurement instrument is a Molecular Devices M5 multi-function microplate reader. Equal volumes of sample are added to a 96-well plate and the absorbance values ​​are read.

[0083] Example 1: Buffer system and pH screening

[0084] An anti-CD19 monoclonal antibody formulation containing 100 mg / mL sucrose, 0.2 mg / mL polysorbate 80, and 20 mg / mL inerizumab was prepared using the following buffer:

[0085] 1) 20 mM citric acid-sodium citrate, pH 5.5;

[0086] 2) 20 mM citric acid-sodium citrate, pH 6.0;

[0087] 3) 20 mM citric acid-sodium citrate, pH 6.5;

[0088] 4) 20 mM histidine-hydrochloride, pH 5.5;

[0089] 5) 20 mM histidine-hydrochloride, pH 6.0;

[0090] 6) 20 mM succinic acid-sodium succinate, pH 5.5;

[0091] 7) 20 mM succinic acid-sodium succinate, pH 6.0;

[0092] The prepared formulation was filtered, filled, stoppered, and capped. The stability of the sample was evaluated under forced degradation conditions (40°C M1, i.e., 1 month at 40°C; 300 rpm shaking D10, i.e., 10 days). The stability of the formulation was assessed using appearance, SEC, and reduced CE-SDS. The experimental results are shown in Table 1.

[0093] Table 1 pH and buffer system screening results

[0094]

[0095] Note: D0 means the start of the experiment.

[0096] The results showed that the monoclonal antibody preparation was most stable when citric acid-sodium citrate was used as the buffer system and the pH was 5.5-6.0.

[0097] Example 2: Screening of protective agent types

[0098] Anti-CD19 monoclonal antibody preparations containing 0.2 mg / mL polysorbate 80, 20 mg / mL inerizumab, and different types of protective agents were prepared using a 20 mM citric acid-sodium citrate, pH 5.5 buffer system. The protective agents are as follows:

[0099] 1) 100 mg / mL sucrose;

[0100] 2) 100 mg / mL trehalose;

[0101] 3) 100 mg / mL mannitol;

[0102] 4) 50 mg / mL sucrose + 50 mg / mL lactose;

[0103] 5) 50 mg / mL sucrose + 50 mg / mL fructose;

[0104] 6) 50 mg / mL sucrose + 50 mg / mL xylitol;

[0105] The prepared formulation was filtered, filled, stoppered, and capped. The stability of the sample was evaluated under forced degradation conditions (shaking D10, i.e., 10 days of shaking; FT 5Cycle, i.e., five freeze-thaw cycles at -35°C to -2°C to 8°C; and 40°C M1, i.e., one month at 40°C). The stability of the formulation was assessed using appearance, SEC, and reduced CE-SDS. The experimental results are shown in Table 2.

[0106] Table 2 Screening results of protective agent types

[0107]

[0108] Note: D0 means the start of the experiment.

[0109] The results showed that the monoclonal antibody preparation was more stable when sucrose-lactose, sucrose-fructose or sucrose-xylitol were used as protective agents.

[0110] Example 3: Screening of protective agent dosage

[0111] Anti-CD19 monoclonal antibody preparations containing 0.2 mg / mL polysorbate 80, 20 mg / mL inerizumab, and different types and amounts of protective agents were prepared using a 20 mM citric acid-sodium citrate, pH 5.5 buffer system. The protective agents were as follows:

[0112] 1) 50 mg / mL sucrose + 50 mg / mL lactose;

[0113] 2) 60 mg / mL sucrose + 40 mg / mL lactose;

[0114] 3) 50 mg / mL sucrose + 50 mg / mL fructose;

[0115] 4) 60 mg / mL sucrose + 40 mg / mL fructose;

[0116] 5) 50 mg / mL sucrose + 50 mg / mL xylitol;

[0117] 6) 60 mg / mL sucrose + 40 mg / mL xylitol;

[0118] 7) 80 mg / mL sucrose + 20 mg / mL xylitol;

[0119] 8) 80 mg / mL sucrose + 40 mg / mL xylitol;

[0120] The prepared preparation is filtered, filled, semi-stoppered, and freeze-dried. The freeze-drying procedure is shown in Table 3:

[0121] Table 3 Freeze-drying procedure

[0122]

[0123] The stability of the samples was investigated under forced degradation conditions (40°C M1, i.e., 1 month at 40°C). Appearance, reconstitution rate, SEC, and reduced CE-SDS were used as evaluation indicators to assess the stability of the formulation. The experimental results are shown in Table 4.

[0124] Table 4 Screening results of protective agent dosage

[0125]

[0126] The results showed that when sucrose-xylitol was used as a protective agent at a ratio of 2:1, the monoclonal antibody preparation was more stable.

[0127] Example 4: Surfactant screening

[0128] Anti-CD19 monoclonal antibody preparations containing 80 mg / mL sucrose, 40 mg / mL xylitol, 20 mg / mL inerizumab, and different types and amounts of surfactants were prepared using a 20 mM citric acid-sodium citrate, pH 5.5 buffer system. The surfactants were as follows:

[0129] 1) 0.2 mg / mL polysorbate 80;

[0130] 2) 0.2 mg / mL polysorbate 20;

[0131] 3) 0.3 mg / mL poloxamer 188;

[0132] 4) 0.4 mg / mL poloxamer 188;

[0133] 5) 0.5 mg / mL poloxamer 188;

[0134] The prepared formulation was filtered, filled, partially stoppered, and lyophilized. The lyophilization procedure is shown in Table 3. The stability of the sample was evaluated under forced degradation conditions (40°C M1, i.e., 1 month at 40°C). Appearance, reconstitution rate, SEC, and reduced CE-SDS were used as evaluation criteria to assess formulation stability. The experimental results are shown in Table 5.

[0135] Table 5 Surfactant screening results

[0136]

[0137] The results showed that the monoclonal antibody preparation was more stable when poloxamer 188 was used as a surfactant.

[0138] Example 5: Protein concentration screening

[0139] Anti-CD19 monoclonal antibody preparations containing 80 mg / mL sucrose, 40 mg / mL xylitol, 0.4 mg / mL poloxamer 188, and different protein concentrations were prepared using a 20 mM citric acid-sodium citrate, pH 5.5 buffer system as follows:

[0140] 1) 10 mg / mL inelizumab;

[0141] 2) 20 mg / mL inelizumab;

[0142] 3) 30 mg / mL inelizumab;

[0143] 4) 40 mg / mL inelizumab;

[0144] The prepared formulation was filtered, filled, partially stoppered, and lyophilized. The lyophilization procedure is shown in Table 3. The stability of the samples was evaluated under forced degradation conditions (40°C M1, i.e., 40°C high temperature exposure for one month; 40°C M3, i.e., 40°C high temperature exposure for three months; and 40°C M6, i.e., 40°C high temperature exposure for six months). The stability of the formulation was assessed using appearance, reconstitution rate, SEC, and reduced CE-SDS. The results are shown in Table 5.

[0145] Table 6 Protein concentration screening results

[0146]

[0147] The results show that under forced degradation conditions, there is no significant difference in appearance and purity of inebilizumab preparations of different concentrations; and the stability is excellent, and the purity indicators decrease by not more than 1% under the condition of 40℃ for 6 months.

Claims

1. A lyophilized formulation of inelizumab, obtained by vacuum freeze-drying a liquid formulation, the lyophilized formulation further comprising a protective agent and a surfactant, wherein the protective agent is selected from sucrose and xylitol, the surfactant is poloxamer, the weight ratio of inelizumab to the protective agent is 1-4:12, the weight ratio of sucrose to xylitol is 2:1, and the weight ratio of poloxamer, sucrose, and xylitol is 1:160-200:80-100.

2. The freeze-dried preparation according to claim 1, characterized in that The weight ratio of the poloxamer, sucrose and xylitol is 1:200:

100.

3. The freeze-dried preparation according to claim 1, characterized in that The invention also comprises a citrate buffer, wherein the citrate buffer is a citric acid-sodium citrate buffer.

4. The freeze-dried preparation according to claim 3, characterized in that The poloxamer is poloxamer 188.

5. The freeze-dried preparation according to claim 1, characterized in that The weight of inerizumab is 100 mg.

6. The freeze-dried preparation according to claim 4, characterized in that The liquid preparation has an inelizumab concentration of 10-40 mg / mL, a sucrose concentration of 50-100 mg / mL, a xylitol concentration of 20-50 mg / mL, a poloxamer 188 concentration of 0.3-0.5 mg / mL, a citric acid-sodium citrate buffer concentration of 20 mM, and a pH of 5.5-6.

0.

7. The freeze-dried preparation according to claim 4, characterized in that The liquid preparation has an inelizumab concentration of 10-40 mg / mL, a sucrose concentration of 50-80 mg / mL, a xylitol concentration of 20-40 mg / mL, a poloxamer 188 concentration of 0.4-0.5 mg / mL, a citric acid-sodium citrate buffer concentration of 20 mM, and a pH of 5.5-6.

0.

8. The freeze-dried preparation according to claim 4, characterized in that The liquid preparation has an inelizumab concentration of 10-40 mg / mL, a sucrose concentration of 80 mg / mL, a xylitol concentration of 40 mg / mL, a poloxamer 188 concentration of 0.4-0.5 mg / mL, a citric acid-sodium citrate buffer concentration of 20 mM, and a pH of 5.

5.

9. The freeze-dried preparation according to claim 8, characterized in that The liquid preparation has a concentration of inelizumab of 20 mg / mL, a concentration of sucrose of 80 mg / mL, a concentration of xylitol of 40 mg / mL, a concentration of poloxamer 188 of 0.4 mg / mL, a concentration of citric acid-sodium citrate buffer of 20 mM, and a pH of 5.

5.

10. Use of the lyophilized preparation according to any one of claims 1 to 8 in the preparation of a medicament for treating immunoglobulin G4-related diseases, neuromyelitis optica spectrum disorder, myasthenia gravis, systemic scleroderma, systemic lupus erythematosus, diffuse large B-cell lymphoma and related diseases.

Images