A pharmaceutical composition containing an anti-cd19 antibody and uses thereof

Abstract

The present application relates to a kind of pharmaceutical composition of anti-CD19 monoclonal antibody and its use. Specifically related to a kind of freeze-dried preparation of anti-CD19 monoclonal antibody, including anti-CD19 antibody inali Zhu single antibody, first protective agent, second protective agent and surfactant. The pharmaceutical composition is used to treat immunoglobulin G4 related diseases, opticospinal cord spectrum disorder and the like diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmaceutical preparations, and particularly relates to a pharmaceutical composition containing an anti-CD19 monoclonal antibody and use thereof. BACKGROUND

[0002] CD19 is a glycoprotein expressed on the surface of B cells, belonging to the immunoglobulin (Ig) superfamily, which is closely related to the activation, proliferation and differentiation of B cells. It plays a synergistic receptor role in B cell activation and signal transmission, regulates B cell activation and proliferation, participates in the signal transduction function of B cells, and mediates T cell killing of target cells. CD19 is expressed in the early stage of B cell development and down-regulated when B cells differentiate into plasma cells. Secondly, CD19 is an important part of the BCR complex, which forms a BCR complex with CD21 and CD81. This complex reduces the threshold of B cell activation mediated by BCR. Among them, CD21 provides a bridge between surface immunoglobulin, CD81 regulates the expression of CD19, and CD19 plays a major role in signal transduction. CD19 regulates the development, proliferation and differentiation of B cells in a BCR-dependent and independent manner. CD19 can also form a heterodimer or a complex with other membrane proteins (such as CD21, CD81) to amplify the BCR signal. The intracellular domain of CD19 is physically combined with a series of tyrosine kinases (such as Lyn, Fyn), triggering downstream signaling pathways such as Src kinase family. In addition, CD19 plays a role in the interaction between B cells and T cells, and is involved in the regulation of immune response.

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

[0004] WO2008 / 031056 discloses an anti-CD19 monoclonal antibody inebilizumab which exerts its effect by targeting the B-cell depletion mechanism mediated by CD19 antigen, and can clear various B-cell subpopulations including plasmablasts. At present, inebilizumab injection has been approved in China, the United States and other countries for the treatment of neuromyelitis optica spectrum disorder (NMOSD), and still has a number of indications related to tumor immunity and autoimmune diseases such as myasthenia gravis, systemic scleroderma, systemic lupus erythematosus, diffuse large B-cell lymphoma, etc. in the clinical trial stage. However, the liquid preparation of the monoclonal antibody often faces the challenge of stability, and has a short shelf life, and the biological activity of the antibody can be lost due to chemical and physical instability during storage. Therefore, it is urgent to develop a preparation product with higher stability. SUMMARY

[0005] The purpose of the present application is to provide an inebilizumab freeze-dried preparation obtained by vacuum freeze-drying from a liquid preparation, the freeze-dried preparation 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 inebilizumab 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 to xylitol is 1:160-200:80-100. The pharmaceutical composition has excellent stability.

[0006] The above-mentioned inebilizumab and the preparation method thereof are described in WO2008 / 031056, the entire contents of which are incorporated herein by reference. The relevant sequences are specifically shown in Tables A and B.

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

[0008]

[0009] Note: The CDR sequence is defined according to the Kabat definition scheme.

[0010] Table B. Full-length sequence information of inebilizumab

[0011]

[0012] In some embodiments of the present application, the weight ratio of poloxamer, sucrose to xylitol is 1:200:100.

[0013] In some embodiments of the present application, the weight of inebilizumab is 100 mg.

[0014] In some embodiments of the present application, the freeze-dried preparation 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 application, the concentration of poloxamer in the liquid formulation 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 application, the concentration of poloxamer in the liquid formulation is 0.4-0.5 mg / mL, for example 0.4 mg / mL, 0.5 mg / mL.

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

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

[0031] In certain embodiments of the application, the pH of the liquid formulation is 5.5-6.0.

[0032] In certain preferred embodiments of the application, the pH of the liquid formulation is 5.5.

[0033] In certain embodiments of the application, the concentration of inebilizumab in the liquid formulation 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 application, the concentration of inebilizumab in the liquid formulation 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 application, the concentration of inebilizumab in the liquid formulation 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 preferred certain embodiments of the present application, the concentration of inebilizumab in the liquid formulation 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 application is to provide the use of the pharmaceutical composition in the preparation of a medicament for treating immunoglobulin G4-related disease, neuromyelitis optica spectrum disorder, myasthenia gravis, systemic scleroderma, systemic lupus erythematosus, diffuse large B-cell lymphoma and related diseases.

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

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

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

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

[0042] The present application screens specific protective agents and surfactants, and optimizes the weight ratio of their combination, so that the prepared pharmaceutical composition has excellent stability. In particular, under the forced degradation conditions of 40℃ high temperature for 1 month, 3 months and 6 months, compared with the results of 0 days, the quality indicators of the sample do not change significantly, and the stability is high. And the prepared product has good morphology, no collapse, easy to reconstitute, stable process, strong production compliance, and is easy for industrialized mass production.

[0043] Detailed description of the invention

[0044] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art in the field of the application, and in particular, the terms used in the specification and claims have their normal meaning to one of ordinary skill in the art. Unless otherwise required by context, singular terms shall include pluralities and vice versa.

[0045] The three-letter and one-letter codes for amino acids used herein are as described in J. Biol. Chem, 243, p 3558 (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) so long as they exhibit the desired antigen-binding activity. Typically, a native, intact antibody is a four polypeptide chain structure consisting of two identical heavy chains and two identical light chains connected by interchain disulfide bonds.

[0047] "Buffer" means a buffering agent that resists pH change by action of its acid-base conjugate components. Examples of buffers that control pH in an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0048] "Histidine salt buffer" is a buffer that includes histidine ion. Examples of histidine salt buffers include histidine-hydrochloric acid, histidine-acetic acid, histidine-phosphoric acid, histidine-sulfuric acid, and the like, preferably histidine-acetic acid buffer, and histidine-hydrochloric acid buffer.

[0049] "Citrate buffer" is a buffer that includes citrate ion. Examples of citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-sodium bicarbonate, and the like. Preferred citrate buffer is citric acid-sodium citrate.

[0050] "Succinate buffer" is a buffer that includes succinate ion. Examples of succinate buffers include succinic acid-sodium succinate, succinic acid-potassium succinate, succinic acid-calcium succinate, and the like. Preferred succinate buffer is succinic acid-sodium succinate. Exemplarily, 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 that includes phosphate ion. 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. Preferred phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate.

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

[0053] "Pharmaceutical composition" means a mixture of one or more antibodies described herein or physiologically / pharmaceutically acceptable salts or prodrugs thereof with other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to maintain the stability, facilitate administration to an organism, and promote absorption of the active ingredient into the body to exert biological activity.

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

[0055] The solution form of the pharmaceutical composition described in the present application, if not otherwise specified, the solvent therein is water.

[0056] "Lyophilized formulation" means a formulation or pharmaceutical composition obtained after a liquid or solution form of the pharmaceutical composition or liquid or solution formulation is subjected to a vacuum freeze-drying step.

[0057] The term "about", "approximately" as used herein means values that are within an acceptable error range for the specific value being sought to be measured or determined, such range as would be understood by one of ordinary skill in the art to be within the scope of the values the specific value is intended to be measured or determined. For example, "about" can mean within 1 or more than 1 standard deviation, per each implementation of the disclosure. Alternatively, "about" or "approximately" can mean ranges within 20% and / or 10%, of a given value. Furthermore, the term "about" can mean a range of up to one order of magnitude or up to 5-fold of a value, especially with respect to biological systems or processes. Unless otherwise stated, the meaning of "about" or "comprising substantially" should be assumed to be within an acceptable error range of the specific value stated in the application and claims, when that specific value occurs.

[0058] The pharmaceutical composition described in the present application is capable of achieving 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 and its biological activity after storage. The storage period is generally selected based on the intended shelf life of the pharmaceutical composition. There are various analytical techniques to measure protein stability, which can measure the 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 upon storage at refrigerated temperatures (2-8°C) for at least 1 month, preferably 6 months, more preferably 1 year, and even more preferably up to 2 years. In addition, a stable liquid formulation includes a liquid formulation that exhibits the desired characteristics upon storage at a temperature including 25°C for a period of time including 1 month, 3 months, 6 months. A typical example of stability is that no more than about 10%, preferably no more than about 5% of the antibody monomer aggregates or degrades as measured by SEC-HPLC. The formulation is a pale yellow to nearly colorless clear liquid or colorless, or clear to slightly opalescent by visual analysis. The concentration, pH, and osmolality of the formulation have no more than a ±10% change. A decrease of no more than about 10%, preferably no more than about 5% is typically observed. No more than about 10%, preferably no more than about 5% aggregation is typically formed.

[0060] An antibody "retains its physical stability" in a pharmaceutical formulation if, after visual inspection for color and / or clarity, or by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS), the antibody drug conjugate shows no significant increase in aggregation, precipitation, and / or denaturation. 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 the antibody shows no significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that frequently alter the chemical structure of proteins include hydrolysis or truncation (assessed by methods such as size exclusion chromatography and CE-SDS), oxidation (assessed by methods such as peptide mapping in conjunction with 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 at the time of preparation of the pharmaceutical formulation.

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

[0064] "Substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1-3, of a group are independently of one another replaced with a corresponding number of substituents. It goes without saying that substituents are only in their possible chemical positions, which can or cannot be possible, as determined (experimentally or theoretically) by one of skill in the art without undue effort. For example, an amino or hydroxyl group with a free hydrogen can not be stable when bound to a carbon atom with an unsaturated (e.g., olefinic) bond.

[0065] Preparation of conventional pharmaceutical compositions is described 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" includes an amount sufficient to effect an improvement or prevention of a symptom or condition of a medical disease. An effective amount also means an amount sufficient to allow or facilitate diagnosis. An effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the overall health status of the patient, the method route and dose of administration, and the severity of side effects. An effective amount can be the maximum dose or administration regimen that avoids significant side effects or toxic effects.

[0070] "Displacement" refers to the displacement of a solvent system in which an antibody protein is dissolved, for example, a high salt or high osmotic solvent system containing an antibody protein is displaced by a physical manipulation using a buffer system of a stable formulation, so that the antibody protein exists in the stable formulation. The physical manipulation includes, but is not limited to, ultrafiltration, dialysis, or resuspension after centrifugation. DETAILED DESCRIPTION

[0071] The present application is further described in connection with the following examples, which are not intended to limit the scope of the application. The experimental methods in the examples of the present application, unless otherwise specified, are generally performed according to conventional conditions, for example, as described in Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, or Molecular Cloning: A Laboratory Manual, or as suggested by the manufacturer of the materials or products. Reagents, unless otherwise specified, are commercially available and are conventional reagents.

[0072] The apparatus used in the detection process and the result calculation method are as follows:

[0073] 1) SEC molecular exclusion chromatography:

[0074] An analytical method for separating solutes according to the relative relationship between the pore size of the gel pores and the size of the polymer sample molecules.

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

[0076] SEC determination instrument: Agilent 1260; column: waters, XBrige BEH200 Å SEC (300x7.8mm 3.5μm).

[0077] 2) CE capillary gel electrophoresis:

[0078] A method of electrophoresis in which the gel is moved into a capillary as a support medium, and the sample molecules are separated according to their molecular weights under a certain voltage.

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

[0080] Instrument for CE assay: Beckman Model plus800.

[0081] 3) Turbidity assay:

[0082] The degree of hindrance of light passing through the water layer represents the ability of the water layer to scatter and absorb light, which is not only related to the content of suspended matter, but also to the particle composition, size, shape and reflection performance of its surface. By comparing the same protein sample with the same concentration and the same wavelength (near ultraviolet and visible light wavelength region) absorption value, the greater the absorption value, the greater the turbidity, and the more obvious the aggregation tendency of the protein molecules in the sample. The instrument for determination is multifunctional enzyme marker (Molecular Devices M5), and the absorbance value of the same volume of sample is read in a 96-well plate.

[0083] Example 1: Buffer system and pH screening

[0084] Using the following buffers, an anti-CD19 monoclonal antibody formulation containing 100 mg / mL sucrose, 0.2 mg / mL polysorbate 80, 20 mg / mL inebilizumab was prepared, wherein the buffers are as follows:

[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, plugged, and capped. The stability of the sample under forced degradation conditions (40°C M1, i.e. 40°C high temperature condition for 1 month; 300 rpm shaking D10, i.e. shaking for 10 days) was investigated, and the appearance, SEC and reduced CE-SDS were used as evaluation indexes to investigate the stability of the formulation. The experimental results are shown in Table 1.

[0093] Table 1: Results of pH and buffer system screening

[0094]

[0095] Note: D0 represents the beginning of the experiment.

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

[0097] Example 2: Screening of the type of protective agent

[0098] Using a 20 mM citric acid-sodium citrate, pH 5.5 buffer system, an anti-CD19 monoclonal antibody preparation containing 0.2 mg / mL polysorbate 80, 20 mg / mL inebilizumab and different types of protective agents was prepared, wherein the protective agents were 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 preparation was filtered, filled, plugged and capped. The stability of the sample under forced degradation conditions (shaking D10, i.e. shaking for 10 days; FT 5 Cycle, i.e. -35°C-2-8°C freeze-thaw 5 cycles; 40°C M1, i.e. 40°C high temperature for 1 month) was investigated, and the appearance, SEC and reduced CE-SDS were used as evaluation indexes to investigate the stability of the preparation. The experimental results are shown in Table 2.

[0106] Table 2: Results of screening of the type of protective agent

[0107]

[0108] Note: D0 represents the time when the experiment starts.

[0109] The results show that the monoclonal antibody preparation has higher stability when sucrose-lactose, sucrose-fructose or sucrose-xylitol is used as the protective agent.

[0110] Example 3: Screening of the amount of protective agent

[0111] Using a 20 mM citric acid-sodium citrate, pH 5.5 buffer system, an anti-CD19 monoclonal antibody preparation containing 0.2 mg / mL polysorbate 80, 20 mg / mL inebilizumab and different types and amounts of protective agents was prepared, wherein 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 finished formulation was filtered, filled, half-closed, and lyophilized. The lyophilization procedure is shown in Table 3:

[0121] Table 3 Lyophilization procedure

[0122]

[0123] The stability of the samples under forced degradation conditions (40°C M1, i.e., 40°C high-temperature conditions for 1 month) was investigated, and the appearance, reconstitution speed, SEC, and reduced CE-SDS were used as evaluation indexes to investigate the stability of the formulation. The experimental results are shown in Table 4.

[0124] Table 4 Results of protective agent dosage screening

[0125]

[0126] The results show that when sucrose-xylitol is used as a protective agent and the ratio is 2:1, the stability of the monoclonal antibody formulation is higher.

[0127] Example 4: Surfactant screening

[0128] Using a 20 mM citric acid-sodium citrate, pH 5.5 buffer system, an anti-CD19 monoclonal antibody formulation containing 80 mg / mL sucrose, 40 mg / mL xylitol, 20 mg / mL inebilizumab, and different types and amounts of surfactants was prepared, wherein 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 formulations were filtered, filled, semi-closed, and lyophilized. The lyophilization procedure is shown in Table 3. The stability of the samples under forced degradation conditions (40°C M1, i.e., 40°C high-temperature condition for 1 month) was investigated, and the appearance, reconstitution speed, SEC, and reduced CE-SDS were used as evaluation indexes to investigate the stability of the formulations. The experimental results are shown in Table 5.

[0135] Table 5. Results of surfactant screening

[0136]

[0137] The results show that the stability of the monoclonal antibody formulation is higher when poloxamer 188 is used as a surfactant.

[0138] Example 5: Protein concentration screening

[0139] Using a 20 mM citric acid-sodium citrate, pH 5.5 buffer system, an anti-CD19 monoclonal antibody formulation containing 80 mg / mL sucrose, 40 mg / mL xylitol, 0.4 mg / mL poloxamer 188, and different concentrations of protein was prepared as follows:

[0140] 1) 10 mg / mL inebilizumab;

[0141] 2) 20 mg / mL inebilizumab;

[0142] 3) 30 mg / mL inebilizumab;

[0143] 4) 40 mg / mL inebilizumab;

[0144] The prepared formulations were filtered, filled, semi-closed, and lyophilized. The lyophilization procedure is shown in Table 3. The stability of the samples under forced degradation conditions (40°C M1, i.e., 40°C high-temperature condition for 1 month; 40°C M3, i.e., 40°C high-temperature condition for 3 months; 40°C M6, i.e., 40°C high-temperature condition for 6 months) was investigated, and the appearance, reconstitution speed, SEC, and reduced CE-SDS were used as evaluation indexes to investigate the stability of the formulations. The experimental results are shown in Table 5.

[0145] Table 6. Results of protein concentration screening

[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 inhaled zanolimab, which is obtained by vacuum freeze-drying a liquid formulation, the lyophilized formulation further comprising a protective agent, a surfactant and a citrate buffer, wherein the protective agent is selected from sucrose and xylitol, the surfactant is poloxamer 188, and the citrate buffer is a citric acid-sodium citrate buffer; the concentration of inhaled zanolimab in the liquid formulation 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 the citric acid-sodium citrate buffer is 20 mM, and the pH is 5.

5.

2. The lyophilized formulation of claim 1, wherein, The weight of inhaled zanolimab is 100 mg.

3. Use of the lyophilized formulation according to claim 1 or 2 for the manufacture of a medicament for the treatment of an immunoglobulin G4-related disease.

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