Stable formulation of Anti-human il-4ra monoclonal antibody

Abstract

The present application provides a stable formulation of an anti-human IL-4Rα monoclonal antibody. Particularly, the present application provides a pharmaceutical composition, which comprises: an anti-human IL-4Rα monoclonal antibody or an antigen-binding fragment thereof, a buffer agent, a stabilizer, and a surfactant. The concentration of the anti-human IL-4Rα monoclonal antibody or the antigen-binding fragment thereof is 7-200 mg / mL. The buffer agent is a histidine hydrochloride buffer solution, the concentration thereof is 15-25 mM, and the pH value thereof is 5.0-6.5. The stabilizer is selected from one or more of sucrose, trehalose, sorbitol, and arginine hydrochloride, and the total concentration thereof in the pharmaceutical composition is 50-400 mM. The surfactant is selected from polysorbate 80 and polysorbate 20, and the concentration thereof in the pharmaceutical composition is 0.001-0.1%. The antibody formulation of the present application can be used for stably storing the anti-human IL-4Rα monoclonal antibody for clinical treatment.

Description

A stable anti-human IL-4Rα monoclonal antibody formulation Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically to a stable anti-human IL-4Rα monoclonal antibody preparation. Background Technology

[0002] Asthma is one of the most common respiratory diseases. It typically manifests as airway inflammation, bronchial hyperresponsiveness, and structural changes in the bronchial walls (airway remodeling). Currently, there is no effective cure for asthma; treatment primarily involves progressive therapy to control symptoms and reduce treatment risks. Inhaled corticosteroids are the standard treatment for moderate asthma; severe cases may require the addition of long-acting beta-blockers. For the most severe cases, additional controller medications are needed. Currently, 5%-10% of asthma patients are incurable. As a chronic disease that significantly impacts quality of life, asthma urgently requires safe and effective treatments.

[0003] When the body is stimulated by antigens, antigen-specific lymphocytes recognize the antigens and undergo activation, proliferation, and differentiation responses, ultimately clearing the invading antigens. T cells and B cells are the main effector cells. T cells exert their immune effect by directly killing target cells and secreting different types of cytokines to amplify and enhance the immune response against different types of antigens. Recent studies have shown that Th2 cytokines, including interleukins (IL)-4, IL-5, and IL-13, are the main mediators of the pathological mechanisms of allergic asthma. Abnormally high expression of Th2 cytokines has been found in the bronchi in allergic asthma, and it has been confirmed that Th2 cytokines mediate the occurrence and development of inflammatory responses and promote pathological changes in the respiratory tract. These cytokines promote the activation of inflammatory cells, including eosinophils and mast cells, and their chemotaxis toward inflammatory sites. IL-4 and IL-13 target B cells, causing them to convert their secreted antibodies from IgM to IgE. Simultaneously, they induce bronchial remodeling through goblet cell proliferation, conversion of bronchial fibroblasts into myofibroblasts, collagen deposition, and respiratory smooth muscle cell proliferation. Both IL-4 and IL-13 can activate corresponding signaling pathways by binding to interleukin-4 receptor α (IL-4Rα). Therefore, developing IL-4Rα-targeting antibodies could simultaneously block the pathological responses of IL-4 and IL-13, potentially leading to the treatment of IL-4Rα-related diseases, including asthma.

[0004] Antibody drugs are highly specific, have low off-target effects, and typically possess a long half-life, allowing for extended dosing cycles and offering significant advantages in the treatment of chronic diseases. As biological macromolecules, antibodies have highly complex structures. Therefore, during production, expressed antibody molecules undergo various post-translational modifications and degradation reactions, such as N-terminal cyclization, glycosylation, deamidation, isomerization, oxidation, fragmentation, and disulfide bond mismatches. Furthermore, various environmental factors (such as temperature, high pressure, physical / mechanical stress, and organic solvents) can lead to antibody denaturation (e.g., aggregation, dissociation, and adsorption onto container surfaces). The heterogeneity of monoclonal antibodies can affect their spatial conformation and even alter their biological activity, and they are prone to self-aggregation. These quality attributes can impact the safety and efficacy of the final product; therefore, controlling the accuracy and consistency of product quality is crucial.

[0005] Improving the physicochemical stability of anti-human IL-4Rα monoclonal antibody preparations, enhancing product quality uniformity and consistency, extending product shelf life, and improving their stability in clinical use are urgent issues to be addressed. Summary of the Invention

[0006] The present invention provides a pharmaceutical composition comprising: an anti-human IL-4Rα monoclonal antibody or its antigen-binding fragment, a buffer, a stabilizer, and a surfactant.

[0007] In some embodiments, the amino acid sequences of the anti-human IL-4Rα monoclonal antibody HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are as shown in SEQ ID NO: 1-6, representing the anti-human IL-4Rα monoclonal antibody or its antigen-binding fragment.

[0008] In some embodiments, the amino acid sequence of the heavy chain variable region of the anti-human IL-4Rα monoclonal antibody is shown in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8.

[0009] In some embodiments, the amino acid sequence of the heavy chain of the anti-human IL-4Rα monoclonal antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain is shown in SEQ ID NO: 10.

[0010] In some embodiments, the concentration of the anti-human IL-4Rα monoclonal antibody or its antigen-binding fragment is as described in any of the embodiments herein.

[0011] In some embodiments, the buffer solution is selected from acetate buffer, citrate buffer, histidine buffer, and phosphate buffer.

[0012] In some embodiments, the pH of the buffer solution is 4.5–7.5, 5.0–6.5, 5.0–6.0, or 5.5 ± 0.3.

[0013] In some embodiments, the concentration of the buffer solution in the pharmaceutical composition is as described in any of the embodiments herein.

[0014] In some embodiments, the stabilizer is selected from one or more of sucrose, trehalose, sorbitol, arginine hydrochloride, and sodium chloride.

[0015] In some embodiments, the concentration of the stabilizer is as described in any of the embodiments herein.

[0016] In some embodiments, the surfactant is selected from polysorbate 80 and polysorbate 20.

[0017] In some embodiments, the surfactant is as described in any of the embodiments herein.

[0018] The present invention also provides a formulation comprising, or consisting of, the pharmaceutical composition described in any embodiment herein. Detailed Implementation

[0019] To maintain the stability of anti-human IL-4Rα monoclonal antibodies and ensure their stable preservation for clinical use, the inventors, through extensive research, developed a stable pharmaceutical composition for anti-human IL-4Rα monoclonal antibodies by selecting appropriate buffer systems, optimizing stabilizers, and adding surfactants. This pharmaceutical composition significantly inhibits the formation of acid peaks, dimers, polymers, degradation products, and insoluble particles during repeated freeze-thaw cycles, long-term storage, and temperature changes. This completes the present invention.

[0020] The term "approximately" in this article refers to the permissible error range of national metrological standards.

[0021] The pharmaceutical composition of the present invention contains: an anti-human IL-4Rα monoclonal antibody, a buffer solution, a stabilizer, and a surfactant. Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0022] Antibody

[0023] In this article, "antibody" refers to an immunoglobulin molecule comprising four polypeptide chains: two heavy chains (H) and two light chains (L) linked by disulfide bonds, as well as its multimers (e.g., IgM). Each heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region contains one domain (CL1). The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with conserved regions called framework regions (FRs).

[0024] An "antigen-binding fragment" of an antibody refers to a portion or segment of the complete antibody molecule responsible for binding to an antigen. Antigen-binding fragments of antibodies can be prepared from complete antibody molecules using any suitable standard technique, including proteolytic digestion or recombinant genetic engineering. Non-limiting examples of antigen-binding fragments include: Fab fragments; F(ab′)2 fragments; Fd fragments; Fv fragments; single-chain Fv (scFv) molecules; single-domain antibodies; dAb fragments; and minimal recognition units (e.g., isolated CDRs) consisting of amino acid residues mimicking the hypervariable region of an antibody.

[0025] In this article, the terms “heavy chain variable region (VH)” and “light chain variable region (VL)” refer to the variable heavy chain and light chain regions of a single antibody, respectively, which include FR1, 2, 3 and 4 and CDR 1, 2 and 3.

[0026] It is well known to those skilled in the art that complementarity-determining regions (CDRs, typically CDR1, CDR2, and CDR3) are the regions in the variable region that have the greatest impact on antibody affinity and specificity. There are two common ways to define the CDR sequence for VH or VL: the Kabat definition and the Chothia definition, for example, see Kabat et al., “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273: 927-948 (1997); and Martin et al., P. oc. Natl. Acad. Sci. USA 86: 9268-9272 (1989). For a given antibody's variable region sequence, the CDR sequence in the VH and VL sequences can be determined according to either the Kabat or Chothia definition. In the embodiments of this application, the Kabat definition of the CDR sequence is used. In this paper, the CDR1, CDR2 and CDR3 of the heavy chain variable region are abbreviated as HCDR1, HCDR2 and HCDR3, respectively; the CDR1, CDR2 and CDR3 of the light chain variable region are abbreviated as LCDR1, LCDR2 and LCDR3, respectively.

[0027] In this document, the anti-human IL-4Rα monoclonal antibody can be any anti-human IL-4Rα monoclonal antibody well known in the art, preferably an anti-human IL-4Rα monoclonal antibody or its antigen-binding fragment with HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 1–6, respectively. In some embodiments, the amino acid sequence of the heavy chain variable region of the anti-human IL-4Rα monoclonal antibody described herein is shown in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8. In some embodiments, the amino acid sequence of the heavy chain of the anti-human IL-4Rα monoclonal antibody described herein is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain is shown in SEQ ID NO: 10.

[0028] The sequences are shown below:

[0029] HCDR1 (SEQ ID NO: 1)

[0030] HCDR2 (SEQ ID NO: 2)

[0031] HCDR3 (SEQ ID NO: 3)

[0032] LCDR1 (SEQ ID NO: 4)

[0033] LCDR2 (SEQ ID NO: 5)

[0034] LCDR3 (SEQ ID NO: 6)

[0035] VH(SEQ ID NO: 7)

[0036] VL (SEQ ID NO: 8)

[0037] Heavy chain (SEQ ID NO: 9)

[0038] Light chain (SEQ ID NO: 10)

[0039] The anti-human IL-4Rα monoclonal antibody or its antigen-binding fragment described herein can be prepared using methods well known in the art. For example, the anti-human IL-4Rα monoclonal antibody described herein can be expressed in CHO cells by genetic engineering and then purified through a series of standard chromatographic steps.

[0040] In the pharmaceutical compositions described herein, the antibody concentration can be 7–200 mg / mL, such as within the ranges of 10–200 mg / mL, 15–200 mg / mL, 15–180 mg / mL, or 15–160 mg / mL. In some embodiments, the antibody concentration in the pharmaceutical composition can be 20±5 mg / mL, 50±5 mg / mL, 80±5 mg / mL, 100±10 mg / mL, 130±10 mg / mL, 150±10 mg / mL, 170±10 mg / mL, or 180±10 mg / mL. In some embodiments, the concentration of the antibody in the pharmaceutical composition may be about 20 mg / mL, about 50 mg / mL, about 80 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, about 150 mg / mL, about 160 mg / mL, about 170 mg / mL, or about 180 mg / mL.

[0041] buffer solution

[0042] In the pharmaceutical composition described herein, the buffer solution may be selected from acetate buffer, citrate buffer, histidine buffer, and phosphate buffer to provide the pharmaceutical composition with a pH of 4.5 to 7.5, preferably 5.0 to 6.5, more preferably 5.0 to 6.0, and even more preferably 5.5 ± 0.3.

[0043] In this document, "acetate buffer" refers to a buffer solution containing acetate ions. Examples of acetate buffer solutions include acetate-sodium acetate buffer, acetate-potassium acetate buffer, acetate-calcium acetate buffer, and acetate-magnesium acetate buffer. The preferred acetate buffer solution is acetate-sodium acetate buffer.

[0044] In this document, "citrate buffer" refers to a buffer solution containing citrate ions. Examples of citrate buffer solutions include citrate-sodium citrate buffer, citrate-potassium citrate buffer, citrate-calcium citrate buffer, citrate-magnesium citrate buffer, etc. The preferred citrate buffer solution is citrate-sodium citrate buffer.

[0045] In this document, "histidine buffer" refers to a buffer solution containing histidine ions. Histidine buffer may contain histidine and / or histidine salts. Histidine salts include, but are not limited to, histidine hydrochloride, histidine acetate, histidine phosphate, and histidine sulfate. An exemplary histidine buffer is histidine hydrochloride buffer.

[0046] In this document, "phosphate buffer" refers to a buffer solution containing phosphate ions. Examples of phosphate buffer solutions include disodium hydrogen phosphate-sodium dihydrogen phosphate buffer and dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer. A preferred phosphate buffer solution is disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

[0047] In this document, the concentration of the buffer solution in the pharmaceutical composition may be in the range of 5-60 mM. It should be understood that the concentration of the buffer solution described herein refers to the final concentration of the buffering component (such as acetic acid and / or its salts, citric acid and / or its salts, histidine and / or its salts, phosphate and / or its salts) in the pharmaceutical composition. In some embodiments, the concentration of the buffer solution in the pharmaceutical composition may be 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, or within a range defined by any two of the above values, such as 10-60 mM, 10-50 mM, 10-40 mM, 10-30 mM, 15-25 mM, etc. In one embodiment, the concentration of the buffer solution in the pharmaceutical composition is 20 ± 3 mM.

[0048] In some embodiments, the buffer solution in the pharmaceutical composition described herein is an acetate-sodium acetate buffer solution with a concentration of 15–25 mM and a pH of 4.5–5.5.

[0049] In some embodiments, the buffer solution in the pharmaceutical composition described herein is a citrate-sodium citrate buffer solution with a concentration of 15–25 mM and a pH of 4.5–5.5.

[0050] In some embodiments, the buffer solution in the pharmaceutical composition described herein is a histidine hydrochloride buffer with a concentration of 15–25 mM and a pH of 5.0–6.5, preferably 5.0–6.0. ​​In some embodiments, the histidine hydrochloride buffer has a concentration of 20 ± 3 mM and a pH of 5.5 ± 0.3. In some embodiments, the buffer solution in the pharmaceutical composition described herein is a histidine hydrochloride buffer containing 3.0–4.0 mg / mL, preferably 3.1–3.5 mg / mL, of histidine hydrochloride and 1.0–1.5 mg / mL, preferably 1.2–1.4 mg / mL, of histidine. In some embodiments, the buffer solution in the pharmaceutical composition of the present invention is a histidine hydrochloride buffer containing 3.1–3.5 mg / mL, of histidine hydrochloride and 1.2–1.4 mg / mL of histidine, providing a pH of 5.5 ± 0.3 for the pharmaceutical composition.

[0051] In some embodiments, the buffer solution in the pharmaceutical composition described herein is a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with a concentration of 15–25 mM and a pH of 6.0–7.5, preferably 6.5–7.5.

[0052] stabilizer

[0053] In this article, "stabilizer" refers to a pharmaceutically acceptable excipient that protects the active pharmaceutical ingredient from chemical and / or physical degradation during manufacturing, storage, and application. Stabilizers include, but are not limited to, sugars, amino acids, salts, and polyols such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, trehalose, arginine or its salts (such as arginine hydrochloride), glycine, alanine, betaine, leucine, lysine, glutamic acid, L-methionine, aspartic acid, proline, 4-hydroxyproline, sarcosine, γ-aminobutyric acid, alanine, and octopine.

[0054] The stabilizers used herein may be selected from one or more of sucrose, trehalose, sorbitol, arginine hydrochloride, and sodium chloride. In the pharmaceutical compositions described herein, the total concentration of the stabilizer may be 50–400 mM, such as 50–300 mM, 70–300 mM, 100–300 mM, 150–300 mM, or 200–280 mM. In some embodiments, the total concentration of the stabilizer in the pharmaceutical compositions described herein may be 100 mM, 130 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, or 300 mM, or within any two of the above values.

[0055] In some embodiments, the pharmaceutical composition described herein contains sucrose as a stabilizer, preferably at a concentration of 200–300 mM, more preferably 200–250 mM.

[0056] In some embodiments, the pharmaceutical composition described herein contains trehalose as a stabilizer, preferably at a concentration of 200–300 mM, more preferably 200–250 mM.

[0057] In some embodiments, the pharmaceutical composition described herein contains sorbitol as a stabilizer, preferably at a concentration of 150–400 mM, more preferably 180–300 mM or 180–230 mM. In some embodiments, the pharmaceutical composition described herein contains sorbitol as a stabilizer, at a concentration of 200 ± 10 mM, more preferably 200 ± 5 mM, and even more preferably 200 ± 3 mM.

[0058] In some embodiments, the pharmaceutical composition described herein contains arginine hydrochloride as a stabilizer, preferably at a concentration of 100–200 mM, more preferably 120–160 mM.

[0059] In some embodiments, the pharmaceutical composition described herein contains sucrose and arginine hydrochloride as stabilizers, preferably with a sucrose concentration of 100–150 mM and an arginine hydrochloride concentration of 50–100 mM.

[0060] In some embodiments, the pharmaceutical composition described herein contains sucrose and sodium chloride as stabilizers, preferably 100-200 mM, more preferably 130-170 mM, and the concentration of sodium chloride is 20-60 mM, more preferably 30-50 mM.

[0061] In some embodiments, the pharmaceutical composition described herein contains sorbitol and arginine hydrochloride as stabilizers, preferably sorbitol at a concentration of 100-250 mM, more preferably 130-230 mM, and arginine hydrochloride at a concentration of 20-100 mM, preferably 40-80 mM.

[0062] In some embodiments, the pharmaceutical composition described herein also contains an appropriate amount of L-methionine. Preferably, the concentration of L-methionine in the pharmaceutical composition is 0.5–1.5 mg / mL, more preferably 0.6–1.2 mg / mL, and even more preferably 0.8 ± 0.05 mg / mL.

[0063] In some embodiments, the pharmaceutical compositions described herein do not contain any of sodium chloride, sodium gluconate, and sodium lactate.

[0064] surfactants

[0065] In this document, "surfactant" includes agents that protect proteins, such as antibodies, from air / solution interface-induced stress and solution / surface-induced stress to reduce antibody aggregation or minimize particulate formation in formulations. Exemplary surfactants include, but are not limited to, nonionic surfactants. Exemplary surfactants include, but are not limited to, polyoxyethylene sorbitan fatty acid esters (such as polysorbate 20 and polysorbate 80), polyethylene-polypropylene copolymers, polyethylene-polypropylene glycol, polyoxyethylene-stearate, polyoxyethylene alkyl ethers, such as polyoxyethylene monolauryl ether, alkylphenyl polyoxyethylene ether (Triton-X), polyoxyethylene-polyoxypropylene copolymer (pluronic), and sodium dodecyl sulfate (SDS).

[0066] In some embodiments, the surfactant included in the pharmaceutical compositions described herein may be selected from polysorbate 80 and polysorbate 20. Preferred surfactant is polysorbate 80. The concentration of the surfactant in the pharmaceutical compositions of the present invention, on a w / v basis, is 0.001–0.1%, preferably 0.005–0.05%, more preferably 0.01–0.05%, and even more preferably 0.02–0.04%. As a non-limiting example, the concentration of the surfactant in the pharmaceutical compositions of the present invention is about 0.02%, about 0.03%, or about 0.04%.

[0067] In some embodiments, the pharmaceutical composition described herein contains polysorbate 80 at a concentration of 0.08–0.15 mg / mL, preferably 0.10 ± 0.02 mg / mL.

[0068] Pharmaceutical Composition

[0069] The pharmaceutical compositions described herein contain the antibodies, buffer solutions, stabilizers, and surfactants described in any of the preceding embodiments. The types and concentrations of the antibodies, buffer solutions, stabilizers, and surfactants in the pharmaceutical compositions are as described in any of the preceding embodiments or combinations thereof. Preferably, the pH value of the pharmaceutical compositions described herein is 4.5 to 7.5, more preferably 5.0 to 6.5, more preferably 5.0 to 6.0, even more preferably 5.5 ± 0.3, such as about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, or about 5.8. It should be understood that water is generally used to prepare the pharmaceutical compositions of the present invention; therefore, in addition to the antibodies, buffer components, stabilizers, and surfactants described herein, the pharmaceutical compositions of this application typically also contain water.

[0070] In some embodiments, the pharmaceutical composition of the present invention comprises:

[0071] (1) 100-200 mg / mL of antibody, 15-25 mM histidine hydrochloride buffer, 200-250 mM sucrose, 0.01-0.03% polysorbate 80, and the pH of the drug composition is 5.5±0.3.

[0072] (2) 100-200 mg / mL of antibody, 15-25 mM histidine hydrochloride buffer, 200-250 mM trehalose, 0.01-0.03% polysorbate 80, and the pH of the drug composition is 5.5±0.3.

[0073] (3) 100-200 mg / mL of antibody, 15-25 mM histidine hydrochloride buffer, 180-250 mM sorbitol, 0.01-0.03% polysorbate 80, and the pH of the drug composition is 5.5±0.3.

[0074] (4) 100-200 mg / mL of antibody, 15-25 mM of histidine hydrochloride buffer, 100-180 mM of arginine hydrochloride, 0.01-0.03% of polysorbate 80, and the pH of the drug composition is 5.5±0.3.

[0075] (5) 100-200 mg / mL of antibody, 15-25 mM histidine hydrochloride buffer, 80-150 mM sucrose and 40-100 mM arginine hydrochloride, 0.01-0.03% polysorbate 80, and the pH of the drug composition is 5.5±0.3.

[0076] (6) 100-200 mg / mL of antibody, 15-25 mM histidine hydrochloride buffer, 100-200 mM sucrose and 20-60 mM sodium chloride, 0.01-0.03% polysorbate 80, and the pH of the drug composition is 5.5±0.3.

[0077] (7) 100-200 mg / mL of antibody, 15-25 mM histidine hydrochloride buffer, 240-300 mM sorbitol, 0.01-0.10% polysorbate 20, and the pH of the drug composition is 5.5 ± 0.3.

[0078] (8) 100–200 mg / mL antibody, 15–25 mM histidine hydrochloride buffer, 240–300 mM sorbitol, 0.005–0.05% polysorbate 80, and the pH of the drug composition is 5.5 ± 0.3; or

[0079] (9) 100-200 mg / mL of antibody, 15-25 mM of histidine hydrochloride buffer, 240-300 mM of sorbitol, 0.01-0.03% of polysorbate 80, 0.6-1.0 mg / mL of L-methionine, and the pH of the drug composition is 5.5±0.3.

[0080] In some embodiments, the pharmaceutical composition of the present invention comprises:

[0081] (1) 150±10 mg / mL antibody, 20±3 mM histidine hydrochloride buffer, 200-250 mM sucrose, 0.01-0.03% polysorbate 80, and the pH of the drug composition is 5.5±0.3.

[0082] (2) 150±10 mg / mL antibody, 20±3 mM histidine hydrochloride buffer, 200-250 mM trehalose, 0.01-0.03% polysorbate 80, and the pH of the drug composition is 5.5±0.3.

[0083] (3) 150±10 mg / mL antibody, 20±3 mM histidine hydrochloride buffer, 200±10 mM sorbitol, 0.01~0.03% polysorbate 80, and the pH of the drug composition is 5.5±0.3.

[0084] (4) 150±10 mg / mL antibody, 20±3 mM histidine hydrochloride buffer, 100-180 mM arginine hydrochloride, 0.01-0.03% polysorbate 80, and the pH of the drug composition is 5.5±0.3.

[0085] (5) 150±10 mg / mL antibody, 20±3 mM histidine hydrochloride buffer, 80-150 mM sucrose and 40-100 mM arginine hydrochloride, 0.01-0.03% polysorbate 80, and the pH of the drug composition is 5.5±0.3.

[0086] (6) 150±10 mg / mL antibody, 20±3 mM histidine hydrochloride buffer, 100-200 mM sucrose and 20-60 mM sodium chloride, 0.01-0.03% polysorbate 80, and the pH of the drug composition is 5.5±0.3.

[0087] (7) 150±10 mg / mL antibody, 20±3 mM histidine hydrochloride buffer, 240-300 mM sorbitol, 0.01-0.10% polysorbate 20, and the pH of the drug composition is 5.5±0.3.

[0088] (8) 150±10 mg / mL antibody, 20±3 mM histidine hydrochloride buffer, 240–300 mM sorbitol, 0.005–0.05% polysorbate 80, and the pH of the drug composition is 5.5±0.3; or

[0089] (9) 150±10 mg / mL antibody, 20±3 mM histidine hydrochloride buffer, 240-300 mM sorbitol, 0.01-0.03% polysorbate 80, 0.6-1.0 mg / mL L-methionine, and the pH of the drug composition is 5.5±0.3.

[0090] In some embodiments, the pharmaceutical composition of the present invention contains 150 mg / mL of antibody, 20 mM histidine hydrochloride buffer, 200 mM sorbitol, 0.02% polysorbate 80, and the pH of the pharmaceutical composition is 5.6 ± 0.1. In some embodiments, the pharmaceutical composition of the present invention has a pH of 5.6 ± 0.1 and consists of 150 mg / mL of antibody, 20 mM histidine hydrochloride buffer, 200 mM sorbitol, 0.02% polysorbate 80, and water.

[0091] In some embodiments, the pharmaceutical composition of the present invention contains about 150 mg / mL of antibody, about 3.35 mg / mL of histidine hydrochloride, about 0.62 mg / mL of histidine, about 36.43 mg / mL of sorbitol, and about 0.10 mg / mL of polysorbate 80.

[0092] preparation

[0093] This invention also provides a pharmaceutical preparation comprising, or being the pharmaceutical composition described in any embodiment herein, either the pharmaceutical composition itself or a preparation obtained by reconstituted the pharmaceutical composition with water for injection or physiological saline for injection. The preparation of this invention may be an injectable formulation, suitable for subcutaneous or intravenous injection.

[0094] In some embodiments, the pharmaceutical formulation of the present invention may be a lyophilized agent. It may be reconstituted using an injectable solution such as water for injection or physiological saline for injection.

[0095] Methods and uses

[0096] In some embodiments, the present invention provides a method for treating or preventing IL-4Rα-related diseases, the method comprising administering to a desired subject the pharmaceutical composition described in any embodiment of the present application.

[0097] In this article, IL-4Rα-related diseases refer to diseases in which IL-4Rα plays a role in the occurrence or development of the disease, or diseases that benefit from the blockade of IL-4 and IL-13. These diseases include, but are not limited to, atopic dermatitis (AD), asthma, chronic sinusitis with nasal polyposis (CRSwNP), eosinophilic esophagitis (EoE), nodular prurigo (PN), chronic spontaneous urticaria (CSU), eosinophilic syndrome (HES), and eosinophilic granulomatous angiopathy (EGPA).

[0098] Depending on the formulation type of the pharmaceutical composition, it can be administered in an appropriate manner, for example, by injection.

[0099] In some embodiments, the present invention also provides the use of the pharmaceutical composition described in any embodiment of this application in the preparation of a medicament for treating or preventing IL-4Rα-related diseases as described herein. In some embodiments, the present invention also provides the pharmaceutical composition described in any embodiment of this application for treating or preventing IL-4Rα-related diseases.

[0100] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0101] In the following examples, stability tests and related biological tests were performed in accordance with the specifications of the Chinese Pharmacopoeia.

[0102] SDS-PAGE (Non-reducing and Reducing): SDS-PAGE is used as an analytical technique to separate free and high molecular weight proteins from natural proteins based on their molecular weight. Non-reducing SDS-PAGE is used to assess covalent aggregates due to their high tendency to aggregate at high concentrations of large protein molecules. Reducing SDS-PAGE is used to examine protein fragmentation because of the numerous disulfide bonds between the light and heavy chains of antibody structures, including hinge regions.

[0103] The size exclusion chromatography (SEC-HPLC) method used in the following examples is as follows: The determination was carried out in accordance with Appendix IIIB of the Pharmacopoeia of the People's Republic of China (2010 edition, Part III), and the samples were detected using a hydrophilic silica size exclusion chromatography column. The sample purity was calculated using the area normalization method.

[0104] The charge variant detection (CEX-HPLC) method used in the following examples is as follows: The determination was carried out in accordance with Appendix IIIB of the Pharmacopoeia of the People's Republic of China (2010 edition, Part III), and the samples were detected using a weak cation analysis column. The acidity, baseness and main component purity of the samples were calculated by the area normalization method.

[0105] Example 1: Buffer System and pH Screening

[0106] Antibody stability studies were conducted using various buffer systems. Based on the properties of the antibody and experience, the following buffer systems were initially determined, and the specific experimental design schemes are shown in Table 1.1.

[0107] Table 1.1: Buffer System and pH Screening Range

[0108] The above-mentioned liquid formulations were incubated at 40°C for 4 weeks. Appearance, protein content, pH value, SEC-HPLC, CEX, and cIEF analyses were performed on the samples at weeks 2 and 4. The results are shown in Table 1.2.

[0109] Table 1.2: Buffer system and pH screening test results

[0110] As shown in Table 1.2, the SEC purity of His-HCl (F7-F9) and HAc-NaAc (F1-F3) formulations did not decrease significantly at 40℃; the pH value had little effect on the SEC purity of these formulations. At 40℃, the CZE and cIEF peaks of the His-HCl (F7-F9) and HAc-NaAc (F1-F3) formulations decreased the least; the alkali peak increase of the His-HCl (F7-F9) formulation was less than that of the HAc-NaAc (F1-F3) formulation. The CZE peak of formulation F7 (20mM His-HCl, pH 5.5) decreased the least.

[0111] In summary, F7 (20mM His-HCl, pH 5.5) was selected for the stability screening experiment of the excipients.

[0112] Example 2: Screening of excipient stability

[0113] The effects of different stabilizers on the stability of antibody liquid formulations were studied. The specific excipient screening and formulation design is shown in Table 2.1.

[0114] Table 2.1: Formulation Design for Excipient Stability Screening

[0115] After the above sample preparations were incubated at 40℃ for 4 weeks, the samples were analyzed for appearance, protein content, pH value, SEC-HPLC, CE_SDS_NR, and CEX. The results are shown in Table 2.2.

[0116] The results showed that formulations F1, F2, F3, and F7 were relatively clear, while F4, F5, and F6 were more opalescent; the A350 value was consistent with the degree of opalescence; there was no significant difference in protein content and SEC-HPLC purity among the formulations; only the CE_SDS_NR and CZE purity of the formulation F6-T0 sample decreased significantly.

[0117] Table 2.2: Results of excipient stability screening

[0118] After the above sample formulations were treated with shaking (Agi-D5) at 25℃, the samples were analyzed for appearance, protein content, SEC-HPLC, CE_SDS_NR, CEX, and Flowcam analysis of insoluble particles. The results are shown in Table 2.3.

[0119] As can be seen from the results in the table, formulation F7 without surfactant PS-80 showed a large number of bubbles and a slight decrease in SEC purity; the opalescence of the formulation was not significantly different from that of A350; the Flowcam results for insoluble microparticles of formulations F3 and F5 were better; there were no significant differences in protein content and purity among the formulations.

[0120] Table 2.3: Results of excipient stability screening

[0121] The above antibody formulations were subjected to repeated freeze-thaw cycles (F / T5) and 5°C strong light irradiation (SI-D11) before analysis for appearance, protein content, SEC-HPLC, CE_SDS_NR, and CZE. The results are shown in Table 2.4. The table shows that formulations F2, F3, and F5 had better appearance, while formulations F1, F4, F6, and F7 showed more particles or suspended matter; there was no significant difference in protein content and purity.

[0122] Table 2.4: Results of excipient stability screening

[0123] In summary, after 4 weeks of incubation at 40℃, formulations F1 / F2 / F3 / F7 were relatively clear; under shaking conditions at 25℃ (Agi-D5), formulations F3 and F5 showed better appearance and insoluble particulate matter; under repeated freeze-thaw cycles (F / T5) and strong light irradiation at 5℃ (SI-D11), F3 and F5 showed better appearance; there were no significant differences in protein content and purity among the formulations. Therefore, further research on the type and concentration of surfactant in sorbitol formulation F3 is needed.

[0124] Example 3: Screening of Surfactants

[0125] The effects of different surfactants on the stability of antibody liquid formulations were studied. The specific surfactant screening formulation design is shown in Table 3.1.

[0126] Table 3.1: Surfactant Screening Formulation Design

[0127] The above-mentioned formulation samples were treated with shaking (Agi-D5) at 25℃ and then analyzed for appearance, protein content, SEC-HPLC, CZE, DLS, and Flowcam insoluble particles. The results are shown in Table 3.2.

[0128] Table 3.2: Surfactant Screening Results

[0129] Experimental results show that the PS-80 formulation has a better appearance than the PS-20 formulation. High-concentration PS-20 formulations tend to produce particles; high-concentration PS-80 formulations tend to increase opalescence; samples with high PS-80 concentrations (S8, S9) showed the highest %MASS of DLS monomers at Agi-D5; there was no significant difference in insoluble microparticles among different concentrations of PS-80 formulations, all of which were lower than those of surfactant-free formulations (S1-T0); there were no significant differences in protein content, SEC, and CZE.

[0130] Table 3.1 shows the analysis and detection of appearance, protein content, SEC-HPLC, and CZE of each formulation sample under strong light irradiation (SI-D10) at 5℃. The experimental results are shown in Table 3.3. The table shows that the CZE acid peak of each formulation increased slightly; with increasing PS-80 concentration, the CZE acid peak showed a slight increasing trend; the addition of L-Met inhibited the increase of the CZE acid peak. There were no significant differences in appearance, protein content, and SEC purity. The SEC purity of the L-Met formulation was slightly higher than that of other formulations.

[0131] Table 3.3: Surfactant Screening Results

[0132] Table 3.1 shows the analysis of appearance, protein content, SEC-HPLC, CE_SDS_NR, CZE, and antibody binding activity with Human IL-4R for each group of formulation samples under 40℃-M1 conditions. The experimental results are shown in Table 3.4.

[0133] Table 3.4: Surfactant Screening Results

[0134] Experimental results showed that the PS-80 formulation had a better appearance than the PS-20 formulation. High-concentration PS-20 formulations tended to produce particles; high-concentration PS-80 formulations tended to have increased opalescence. The CZE acid peaks of all formulations increased slightly; with increasing PS-80 concentration, the CZE acid peaks showed a slight increasing trend; the addition of L-Met inhibited the increase in CZE acid peaks. There were no significant differences in protein concentration, SEC and CE_SDS_NR purity, or HumanIL-4R binding activity among the formulations. The slight increase in protein concentration may be related to sealing properties. SEC purity decreased most significantly, but there were no significant differences in SEC and CE_SDS_NR purity among the formulations (only the S2 sample showed a decrease in the main CE_SDS_NR peak). The L-Met formulation had slightly higher SEC purity than the other formulations.

[0135] In summary, the PS-80 formulation exhibits a superior appearance compared to the PS-20 formulation. At Agi and 40℃, the high-concentration PS-20 formulation tends to produce particles; at 5℃, the high-concentration PS-20 formulation tends to exhibit increased opalescence. There is no significant difference in the number of insoluble microparticles among different concentrations of PS-80 formulations, all being lower than the surfactant-free formulation (S1-T0). At SI-D10 and 40℃-M1, the CZE acid peaks of all formulations slightly increased; with increasing PS-80 concentration, the CZE acid peaks showed a slight increasing trend; at SI-D10 and 40℃-M1, the SEC purity of the L-Met formulation was slightly higher than other formulations; the addition of L-Met inhibited the increase in CZE acid peaks. There were no significant differences in protein content, CE-SDS_NR, binding assay, or the results of insoluble microparticles among the PS-80 formulations under Agi-D5 and 5℃-M1 conditions. Therefore, medium to low concentrations of PS-80 are preferred.

[0136] Example 4: Optimization of Formulation

[0137] Based on the above experimental results, the antibody liquid formulation was optimized to obtain a more stable antibody formulation. The specific optimized formulation design is shown in Table 4.1.

[0138] Table 4.1: Experimental Design for Prescription Optimization

[0139] The experimental results of the above formulation samples under shaking (Agi-D6) conditions at 25℃ are shown in Table 4.2. The data in the table show that no visible foreign matter was observed in the shaken samples of formulations O1, O8, and O9, but significant opalescence was observed; formulations O2, 4, 5, 6, 7, and 12 showed low opalescence but significant particulate matter. The protein content, SEC and CZE purity, and insoluble particulate matter results for formulations O1-10 showed no significant differences. It should be noted that the samples in this experiment were obtained by ultrafiltration concentration using separate ultrafiltration tubes, and differences in mechanical damage to the starting samples due to different excipients and concentration ratios are inevitable. With increasing protein concentration, the SEC and CZE purity of the shaken samples decreased slightly (formulations O12-14).

[0140] Table 4.2: Results of the prescription optimization experiment

[0141] The experimental results of each sample in Table 4.1 under strong light irradiation (SI-D12) at 5℃ are shown in Table 4.3. It can be seen that the protein particles in the samples irradiated by strong light of formulations O1, O4 and O7 were restored to different degrees; only the purity of SEC and CZE of the samples irradiated by light O6 and O5 decreased significantly; the insoluble particles of formulations O3, O8 and O9 were not obvious and were close to those of S1-SI-D10.

[0142] Table 4.3: Results of the prescription optimization experiment

[0143] The experimental results of each sample in Table 4.1 under the 40℃-M1 condition are shown in Table 4.4. It can be seen that the 150mg / ml formulations O1, O8, and O9 show more pronounced opalescence; there are no significant differences in protein content, SEC, CE_SDS_NR, CZE purity, and huIL-4R binding activity. With increasing protein concentration, SEC and CE purity decrease significantly (Formulations O14, O13, and O12).

[0144] Table 4.4: Results of the prescription optimization experiment

[0145] In summary, without the addition of surfactants, the O3 formulation exhibits superior appearance under strong shaking and intense light irradiation. Within the pH range of 5.0 to 6.0, there is no significant difference in the stability of sorbitol samples ranging from 200 mM to 400 mM. The addition of Arg-HCl significantly reduces the formulation viscosity; however, further increasing the protein concentration (O12) significantly increases the formulation viscosity. While the addition of Arg-HCl (formulations O8 and O9) increases the opalescence of the formulation, no obvious protein particles were observed.

Claims

1. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains an anti-human IL-4Rα monoclonal antibody or its antigen-binding fragment, a buffer, a stabilizer, and a surfactant; wherein: The concentration of the anti-human IL-4Rα monoclonal antibody or its antigen-binding fragment is 7–200 mg / mL; The buffer is histidine hydrochloride buffer with a concentration of 15–25 mM and a pH of 5.0–6.

5. The stabilizer is selected from one or more of sucrose, trehalose, sorbitol and arginine hydrochloride, and the total concentration of the stabilizer in the pharmaceutical composition is 50-400 mM; The surfactant is selected from polysorbate 80 and polysorbate 20, and the concentration of the surfactant in the pharmaceutical composition is 0.001 to 0.1 wt%.

2. The pharmaceutical composition according to claim 1, characterized in that, The amino acid sequences of the anti-human IL-4Rα monoclonal antibody HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 1 to 6, respectively.

3. The pharmaceutical composition according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the anti-human IL-4Rα monoclonal antibody is shown in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

8.

4. The pharmaceutical composition according to claim 1, characterized in that, The amino acid sequence of the heavy chain of the anti-human IL-4Rα monoclonal antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain is shown in SEQ ID NO:

10.

5. The pharmaceutical composition according to any one of claims 1 to 4, characterized in that, The concentration of the anti-human IL-4Rα monoclonal antibody or its antigen-binding fragment in the pharmaceutical composition is 130–170 mg / mL.

6. The pharmaceutical composition according to any one of claims 1 to 4, characterized in that, The concentration of the anti-human IL-4Rα monoclonal antibody or its antigen-binding fragment in the pharmaceutical composition is 20±5 mg / mL, 50±5 mg / mL, 80±5 mg / mL, 100±10 mg / mL, 130±10 mg / mL, 150±10 mg / mL, 170±10 mg / mL or 180±10 mg / mL.

7. The pharmaceutical composition according to any one of claims 1 to 4, characterized in that, The buffer solution contains 3.1–3.5 mg / mL of histidine hydrochloride and 1.2–1.4 mg / mL of histidine.

8. The pharmaceutical composition according to any one of claims 1 to 4, characterized in that: The stabilizer is sucrose, and its concentration in the pharmaceutical composition is 200–300 mM; or The stabilizer is trehalose, and its concentration in the pharmaceutical composition is 200–300 mM; or The stabilizer is sorbitol, and its concentration in the pharmaceutical composition is 180–230 mM; or The stabilizer is arginine hydrochloride, and its concentration in the pharmaceutical composition is 100–200 mM; or The stabilizer is a combination of sucrose and arginine hydrochloride, wherein in the pharmaceutical composition, the concentration of sucrose is 100-150 mM and the concentration of arginine hydrochloride is 50-100 mM; or The stabilizer is a combination of sorbitol and arginine hydrochloride, wherein the concentration of sorbitol in the pharmaceutical composition is 100-250 mM and the concentration of arginine hydrochloride is 20-100 mM.

9. The pharmaceutical composition according to any one of claims 1 to 4, characterized in that: The stabilizer is sucrose, and its concentration in the pharmaceutical composition is 200–250 mM; or The stabilizer is trehalose, and its concentration in the pharmaceutical composition is 200–250 mM; or The stabilizer is sorbitol, and its concentration in the pharmaceutical composition is 200±5 mM; or The stabilizer is arginine hydrochloride, and its concentration in the pharmaceutical composition is 120–160 mM; or The stabilizer is a combination of sorbitol and arginine hydrochloride, wherein the concentration of sorbitol in the pharmaceutical composition is 130-230 mM and the concentration of arginine hydrochloride is 40-80 mM.

10. The pharmaceutical composition according to any one of claims 1 to 4, characterized in that, The surfactant is polysorbate 80, with a concentration of 0.08–0.15 mg / mL.

11. The pharmaceutical composition of claim 10, characterized in that, The concentration of polysorbate 80 is 0.10 ± 0.02 mg / mL.

12. The pharmaceutical composition according to claim 1, characterized in that, The pharmaceutical composition has a pH of 5.5 ± 0.3 and contains or is composed of any of the components listed in (1) to (9) below: (1) 100-200 mg / mL antibody, 15-25 mM histidine hydrochloride buffer, 200-250 mM sucrose, 0.01-0.03% polysorbate 80; (2) 100-200 mg / mL antibody, 15-25 mM histidine hydrochloride buffer, 200-250 mM trehalose, 0.01-0.03% polysorbate 80; (3) 100-200 mg / mL antibody, 15-25 mM histidine hydrochloride buffer, 180-250 mM sorbitol, 0.01-0.03% polysorbate 80; (4) 100-200 mg / mL antibody, 15-25 mM histidine hydrochloride buffer, 100-180 mM arginine hydrochloride, 0.01-0.03% polysorbate 80. (5) 100-200 mg / mL antibody, 15-25 mM histidine hydrochloride buffer, 80-150 mM sucrose and 40-100 mM arginine hydrochloride, 0.01-0.03% polysorbate 80. (6) 100-200 mg / mL antibody, 15-25 mM histidine hydrochloride buffer, 100-200 mM sucrose and 20-60 mM sodium chloride, 0.01-0.03% polysorbate 80. (7) 100-200 mg / mL antibody, 15-25 mM histidine hydrochloride buffer, 240-300 mM sorbitol, 0.01-0.10% polysorbate 20; (8) 100–200 mg / mL antibody, 15–25 mM histidine hydrochloride buffer, 240–300 mM sorbitol, 0.005–0.05% polysorbate 80; or (9) 100-200 mg / mL antibody, 15-25 mM histidine hydrochloride buffer, 240-300 mM sorbitol, 0.01-0.03% polysorbate 80, and 0.6-1.0 mg / mL L-methionine.

13. The pharmaceutical composition according to claim 1, characterized in that, The pharmaceutical composition has a pH of 5.5 ± 0.3 and contains or is composed of any of the components listed in (1) to (10) below: (1) 150±10 mg / mL antibody, 20±3 mM histidine hydrochloride buffer, 200-250 mM sucrose, 0.01-0.03% polysorbate 80. (2) 150±10 mg / mL antibody, 20±3 mM histidine hydrochloride buffer, 200-250 mM trehalose, 0.01-0.03% polysorbate 80. (3) 150±10 mg / mL antibody, 20±3 mM histidine hydrochloride buffer, 200±10 mM sorbitol, 0.01~0.03% polysorbate 80. (4) 150±10 mg / mL antibody, 20±3 mM histidine hydrochloride buffer, 100-180 mM arginine hydrochloride, 0.01-0.03% polysorbate 80. (5) 150±10 mg / mL antibody, 20±3 mM histidine hydrochloride buffer, 80-150 mM sucrose and 40-100 mM arginine hydrochloride, 0.01-0.03% polysorbate 80. (6) 150±10 mg / mL antibody, 20±3 mM histidine hydrochloride buffer, 100-200 mM sucrose and 20-60 mM sodium chloride, 0.01-0.03% polysorbate 80. (7) 150±10 mg / mL antibody, 20±3 mM histidine hydrochloride buffer, 240-300 mM sorbitol, 0.01-0.10% polysorbate 20; (8) 150±10 mg / mL antibody, 20±3 mM histidine hydrochloride buffer, 240-300 mM sorbitol, 0.005-0.05% polysorbate 80; (9) 150±10 mg / mL antibody, 20±3 mM histidine hydrochloride buffer, 240–300 mM sorbitol, 0.01–0.03% polysorbate 80, 0.6–1.0 mg / mL L-methionine; or (10) 150±10 mg / mL of antibody, 3.1~3.5 mg / mL of histidine hydrochloride, 1.2~1.4 mg / mL of histidine, 200±3 mM of sorbitol, and 0.10±0.02 mg / mL of polysorbate 80.

14. A pharmaceutical preparation, characterized in that, The pharmaceutical preparation contains, or is composed of, any one of the pharmaceutical compositions according to claims 1 to 13.

15. A powder formulation obtained by drying the pharmaceutical composition according to any one of claims 1 to 13.

16. A formulation obtained by reconstituted the powder formulation of claim 15 using water for injection or physiological saline for injection.

17. Use of the pharmaceutical composition according to any one of claims 1 to 13 in the preparation of a medicament for treating or preventing IL-4Rα-related diseases.

18. The application as described in claim 17, characterized in that, The IL-4Rα-related diseases are selected from: atopic dermatitis, asthma, chronic sinusitis with nasal polyps, eosinophilic esophagitis, nodular prurigo, chronic spontaneous urticaria, eosinophilic syndrome, and eosinophilic granulomatous angiopathy.

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