Anti-il-5 antibody formulation and preparation method therefor

Abstract

Provided are an anti-IL-5 antibody lyophilized formulation and a reconstituted formulation thereof. The anti-IL-5 antibody lyophilized formulation comprises an anti-IL-5 antibody, a histidine buffer, sucrose, and polysorbate 80. The anti-IL-5 antibody lyophilized formulation has excellent stability, has a lower osmotic pressure after reconstitution, reduces injection pain for patients, and is more suitable for clinical use.

Description

Anti-IL-5 antibody preparations and their preparation methods Technical Field

[0001] This invention belongs to the field of biological agents and relates to anti-IL-5 antibody preparations. Background Technology

[0002] IL-5 plays a crucial role in many diseases and is a commonly used therapeutic target. Anti-IL-5 antibodies, such as mepolizumab, specifically bind to IL-5, blocking its binding to its receptor, and play a role in the treatment of various diseases. Protein degradation pathways can involve chemical instability (i.e., any process involving modification of proteins through bond formation or cleavage, resulting in new chemical entities) or physical instability (i.e., higher-order structural changes of proteins). Chemical instability manifests as deamidation, isomerization, hydrolysis, oxidation, fragmentation, glycan β-elimination, or disulfide exchange. Physical instability can be caused by, for example, denaturation, aggregation, precipitation, or adsorption. The four most common protein degradation pathways are protein fragmentation, aggregation, deamidation, and oxidation. The consequences of chemical or physical instability of therapeutic proteins include reduced effective dosage, decreased therapeutic safety attributed to factors such as irritation or immune reactivity, and shorter shelf life leading to more frequent manufacturing. Therefore, maintaining conformational integrity is increasingly important for antibodies to exert their full therapeutic effect.

[0003] New and more stable anti-IL-5 antibody formulations are still needed in this field. Summary of the Invention

[0004] This invention provides an anti-IL-5 antibody formulation.

[0005] On one hand, the present invention provides a lyophilized formulation of an anti-IL-5 antibody. In some embodiments, the lyophilized formulation comprises an anti-IL-5 antibody, a histidine buffer, a sugar, and polysorbate. In some embodiments, the sugar is not trehalose. In some embodiments, the sugar is sucrose. In some embodiments, the mass ratio of the anti-IL-5 antibody to the sugar is about 0.5-1.5. In some embodiments, the mass ratio of the anti-IL-5 antibody to the sugar is about 0.75-1.2. In some embodiments, the mass ratio of the anti-IL-5 antibody to the sugar is about 0.75-1. In some embodiments, the mass ratio of the anti-IL-5 antibody to the sugar is about 0.75-0.95. In some embodiments, the mass ratio of the anti-IL-5 antibody to the sugar is about 0.8-0.95. In some embodiments, the mass ratio of the anti-IL-5 antibody to the sugar is about 0.83-0.94.

[0006] In some embodiments, the lyophilized formulation comprises an anti-IL-5 antibody, a histidine buffer, sucrose, and polysorbate. In some embodiments, the mass ratio of the anti-IL-5 antibody to sucrose is about 0.5-1.5. In some embodiments, the ratio (g:mmol) of the anti-IL-5 antibody to the histidine buffer is about 3.3 g:1 mmol to 5 g:1 mmol. In some embodiments, the mass ratio of the anti-IL-5 antibody to polysorbate is about 250:1 to 1000:1.

[0007] In some embodiments, the heavy chain variable region of the anti-IL-5 antibody comprises the sequence shown in SEQ ID NO:1, and the light chain variable region comprises the sequence shown in SEQ ID NO:2. In some embodiments, the heavy chain of the anti-IL-5 antibody comprises the sequence shown in SEQ ID NO:3, and the light chain comprises the sequence shown in SEQ ID NO:4. In some embodiments, the anti-IL-5 antibody is mepolizumab (e.g., Neocate or its biosimilar).

[0008] In some embodiments, the mass ratio of the anti-IL-5 antibody to sucrose is about 0.75-1.2. In some embodiments, the mass ratio of the anti-IL-5 antibody to sucrose is about 0.75-1. In some embodiments, the mass ratio of the anti-IL-5 antibody to sucrose is about 0.75-0.95. In some embodiments, the mass ratio of the anti-IL-5 antibody to sucrose is about 0.8-0.95. In some embodiments, the mass ratio of the anti-IL-5 antibody to sucrose is about 0.83-0.94.

[0009] In some embodiments, the ratio of the anti-IL-5 antibody to the histidine buffer is approximately 3.5 g:1 mmol to 5 g:1 mmol. In some embodiments, the ratio of the anti-IL-5 antibody to the histidine buffer is approximately 3.75 g:1 mmol to 5 g:1 mmol. In some embodiments, the ratio of the anti-IL-5 antibody to the histidine buffer is approximately 3.75 g:1 mmol. In some embodiments, the ratio of the anti-IL-5 antibody to the histidine buffer is approximately 5 g:1 mmol.

[0010] In some embodiments, the mass ratio of the anti-IL-5 antibody to polysorbate is about 350:1 to 500:1. In some embodiments, the mass ratio of the anti-IL-5 antibody to polysorbate is about 370:1 to 500:1. In some embodiments, the mass ratio of the anti-IL-5 antibody to polysorbate is about 370:1. In some embodiments, the mass ratio of the anti-IL-5 antibody to polysorbate is about 375:1. In some embodiments, the mass ratio of the anti-IL-5 antibody to polysorbate is about 500:1. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80.

[0011] In some embodiments, the lyophilized formulation contains at least about 40 mg of anti-IL-5 antibody. In some embodiments, the lyophilized formulation contains at least about 100 mg of anti-IL-5 antibody.

[0012] In some embodiments, the lyophilized formulation comprises at least about 100 mg of anti-IL-5 antibody, histidine buffer, sucrose, and polysorbate, wherein the ratio of the anti-IL-5 antibody to histidine buffer is about 3.3 g:1 mmol to 5 g:1 mmol, the mass ratio of the anti-IL-5 antibody to sucrose is about 0.75-1.2, and the mass ratio of the anti-IL-5 antibody to polysorbate is about 250:1 to 1000:1.

[0013] In some embodiments, the lyophilized formulation comprises at least about 100 mg of anti-IL-5 antibody, histidine buffer, sucrose, and polysorbate, wherein the ratio of the anti-IL-5 antibody to histidine buffer is about 3.3 g:1 mmol to 5 g:1 mmol, the mass ratio of the anti-IL-5 antibody to sucrose is about 0.8-0.95, and the mass ratio of the anti-IL-5 antibody to polysorbate is about 250:1 to 1000:1.

[0014] In some embodiments, the lyophilized formulation comprises at least about 100 mg of anti-IL-5 antibody, histidine buffer, sucrose, and polysorbate, wherein the ratio of the anti-IL-5 antibody to the histidine buffer is about 3.75 g: 1 mmol, the mass ratio of the anti-IL-5 antibody to sucrose is about 0.94, and the mass ratio of the anti-IL-5 antibody to polysorbate is about 375: 1.

[0015] In some embodiments, the lyophilized formulation comprises at least about 100 mg of anti-IL-5 antibody, histidine buffer, sucrose, and polysorbate, wherein the ratio of the anti-IL-5 antibody to histidine buffer is about 5 g: 1 mmol, the mass ratio of the anti-IL-5 antibody to sucrose is about 1, and the mass ratio of the anti-IL-5 antibody to polysorbate is about 500: 1.

[0016] In some embodiments, the lyophilized formulation comprises at least about 100 mg of anti-IL-5 antibody, histidine buffer, sucrose, and polysorbate, wherein the ratio of the anti-IL-5 antibody to histidine buffer is about 5 g: 1 mmol, the mass ratio of the anti-IL-5 antibody to sucrose is about 0.83, and the mass ratio of the anti-IL-5 antibody to polysorbate is about 500:1.

[0017] In some embodiments, each lyophilized formulation containing about 100g of an anti-IL-5 antibody, such as mepolizumab, contains about 20-30 mmol of histidine buffer, about 100-133g of sucrose, and about 0.1-0.4g of polysorbate 80.

[0018] In some embodiments, each lyophilized formulation containing about 100g of an anti-IL-5 antibody, such as mepolizumab, contains about 20-30 mmol of histidine buffer, about 106-120g of sucrose, and about 0.1-0.4g of polysorbate 80.

[0019] In some embodiments, each lyophilized formulation containing about 100g of an anti-IL-5 antibody, such as mepolizumab, contains about 27 mmol of histidine buffer, about 108g of sucrose, and about 0.27g of polysorbate 80.

[0020] In some embodiments, each lyophilized formulation containing about 100g of an anti-IL-5 antibody, such as mepolizumab, contains about 26.7 mmol of histidine buffer, about 107g of sucrose, and about 0.27g of polysorbate 80.

[0021] In some embodiments, each lyophilized formulation containing about 100g of an anti-IL-5 antibody, such as mepolizumab, contains about 20 mmol of histidine buffer, about 100g of sucrose, and about 0.2g of polysorbate 80.

[0022] In some embodiments, each lyophilized formulation containing about 100g of an anti-IL-5 antibody, such as mepolizumab, contains about 20 mmol of histidine buffer, about 120g of sucrose, and about 0.2g of polysorbate 80.

[0023] In some embodiments, the lyophilized formulation does not contain phosphate or its salts. In one embodiment, the lyophilized formulation does not contain citric acid or its salts. In one embodiment, the antibody formulation does not contain acetic acid or its salts. In some embodiments, the lyophilized formulation does not contain EDTA.

[0024] In some embodiments, the lyophilized formulation is prepared by lyophilizing an aqueous solution. In some embodiments, the aqueous solution comprises an anti-IL-5 antibody such as mepolizumab, a histidine buffer, sucrose, and polysorbate. In some embodiments, the aqueous solution comprises 45-105 mg / mL of an anti-IL-5 antibody such as mepolizumab, 10-30 mM of histidine buffer, 60-200 mg / mL of sucrose, and 0.1-0.4 mg / mL of polysorbate.

[0025] In some embodiments, the pH of the aqueous solution is 5.3-6.8, or 5.7-6.3, or 5.8-6.1.

[0026] In some embodiments, the concentration of the anti-IL-5 antibody is about 45 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 75 mg / mL, about 80 mg / mL, about 90 mg / mL, about 94 mg / mL, about 96 mg / mL, about 98 mg / mL, about 99 mg / mL, about 100 mg / mL, about 101 mg / mL, about 102 mg / mL, about 103 mg / mL, about 104 mg / mL, or about 105 mg / mL, or a range (including endpoints) of any two of these values, or any value therein. In some embodiments, the concentration of the anti-IL-5 antibody is 50-100 mg / mL. In some embodiments, the concentration of the anti-IL-5 antibody is 50-75 mg / mL. In some embodiments, the concentration of the anti-IL-5 antibody is 75-100 mg / mL. In some embodiments, the concentration of the anti-IL-5 antibody is 75 mg / mL. In some embodiments, the concentration of the anti-IL-5 antibody is 100 mg / mL.

[0027] In some embodiments, the concentration of the histidine buffer is about 10 mM, about 12 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 26.7 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM, or a range (including endpoints) of any two of these values, or any value therein. In some embodiments, the concentration of the histidine buffer is 10-20 mM. In one embodiment, the concentration of the histidine buffer is about 10 mM. In one embodiment, the concentration of the histidine buffer is about 15 mM. In one embodiment, the concentration of the histidine buffer is about 20 mM.

[0028] In some embodiments, the concentration of the sucrose is about 60, 70, 75, 80, 85, 90, 95, 100, 105, 106, 107, 108, 110, 115, 120, 125, 130, 134, 135, 140, 150, 160, 170, 180, 190, or 200 mg / mL, or a range (including endpoints) of any two of these values, or any value therein. In some embodiments, the concentration of the sucrose is 60-120 mg / mL. In some embodiments, the concentration of the sucrose is 75-100 mg / mL. In some embodiments, the concentration of the sucrose is 80-100 mg / mL. In some embodiments, the concentration of the sucrose is 80-90 mg / mL. In some embodiments, the concentration of the sucrose is about 80 mg / mL.

[0029] In some embodiments, the concentration of the polysorbate is about 0.1 mg / mL, 0.15 mg / mL, 0.19 mg / mL, 0.2 mg / mL, 0.21 mg / mL, 0.22 mg / mL, 0.23 mg / mL, 0.24 mg / mL, 0.25 mg / mL, 0.26 mg / mL, 0.27 mg / mL, 0.28 mg / mL, 0.29 mg / mL, 0.3 mg / mL, 0.32 mg / mL, 0.35 mg / mL, 0.38 mg / mL, or 0.4 mg / mL, or a range (including endpoints) of any two of these values, or any value therein. In some embodiments, the concentration of the polysorbate is 0.1-0.4 mg / mL. In some embodiments, the concentration of the polysorbate is 0.1-0.3 mg / mL. In some embodiments, the polysorbate is polysorbate 80 or polysorbate 20. In some embodiments, the concentration of polysorbate 80 is 0.1-0.3 mg / mL. In some embodiments, the concentration of polysorbate 80 is about 0.1 mg / mL. In some embodiments, the concentration of polysorbate 80 is about 0.15 mg / mL. In some embodiments, the concentration of polysorbate 80 is about 0.2 mg / mL.

[0030] In some embodiments, the aqueous solution comprises: 50-100 mg / mL of an anti-IL-5 antibody, such as mepolizumab, 10-30 mM histidine buffer, 60-200 mg / mL of sucrose, and 0.1-0.4 mg / mL of polysorbate 80, with a pH of about 5.7-6.3, wherein the mass ratio of the anti-IL-5 antibody to sucrose is about 0.5-1.

[0031] In some embodiments, the aqueous solution comprises: 50-100 mg / mL of an anti-IL-5 antibody, such as mepolizumab, 10-30 mM histidine buffer, 60-135 mg / mL of sucrose, and 0.1-0.4 mg / mL of polysorbate 80, with a pH of about 5.7-6.3, wherein the mass ratio of the anti-IL-5 antibody to sucrose is about 0.75-1.

[0032] In some embodiments, the aqueous solution comprises: about 50 mg / mL of an anti-IL-5 antibody, such as mepolizumab, 10-20 mM histidine buffer, 50-70 mg / mL of sucrose, and 0.1-0.4 mg / mL of polysorbate 80, with a pH of about 5.7-6.3.

[0033] In some embodiments, the aqueous solution comprises: about 100 mg / mL of an anti-IL-5 antibody, such as mepolizumab, 10-20 mM histidine buffer, 100-134 mg / mL sucrose, and 0.1-0.4 mg / mL polysorbate 80, with a pH of about 5.7-6.3.

[0034] In some embodiments, the aqueous solution comprises: about 75 mg / mL of an anti-IL-5 antibody, such as mepolizumab, 10-20 mM histidine buffer, 75-100 mg / mL sucrose, and 0.1-0.4 mg / mL polysorbate 80, with a pH of about 5.7-6.3.

[0035] In some embodiments, the aqueous solution comprises: about 75 mg / mL of an anti-IL-5 antibody, such as mepolizumab, about 10-20 mM of histidine buffer, about 80-100 mg / mL of sucrose, and about 0.1-0.4 mg / mL of polysorbate 80, with a pH of about 5.7-6.3.

[0036] In some embodiments, the aqueous solution comprises: about 75 mg / mL of an anti-IL-5 antibody, such as mepolizumab, about 10-20 mM of histidine buffer, about 80-90 mg / mL of sucrose, and about 0.1-0.4 mg / mL of polysorbate 80, with a pH of about 5.7-6.3.

[0037] In some embodiments, the aqueous solution comprises: about 75 mg / mL mepolizumab, about 20 mM histidine buffer, about 80 mg / mL sucrose, and about 0.20 mg / mL polysorbate 80, with a pH of about 5.8-6.1.

[0038] In some embodiments, the aqueous solution comprises: about 75 mg / mL mepolizumab, about 15 mM histidine buffer, about 90 mg / mL sucrose, and about 0.15 mg / mL polysorbate 80, with a pH of about 5.8-6.1.

[0039] In some embodiments, the aqueous solution comprises: about 100 mg / mL mepolizumab, about 20 mM histidine buffer, about 100 mg / mL sucrose, and about 0.20 mg / mL polysorbate 80, with a pH of about 5.8-6.1.

[0040] In some embodiments, the aqueous solution comprises: about 50 mg / mL mepolizumab, about 10 mM histidine buffer, about 60 mg / mL sucrose, and about 0.10 mg / mL polysorbate 80, with a pH of about 5.8-6.1.

[0041] In some embodiments, the aqueous solution comprises: about 75 mg / mL mepolizumab, about 15 mM histidine buffer, about 120 mg / mL sucrose, and about 0.15 mg / mL polysorbate 80, with a pH of about 5.8-6.1.

[0042] In some embodiments, the aqueous solution comprises: about 75 mg / mL mepolizumab, about 15 mM histidine buffer, about 105 mg / mL sucrose, and about 0.15 mg / mL polysorbate 80, with a pH of about 5.8-6.1.

[0043] In some embodiments, the aqueous solution comprises: about 50 mg / mL mepolizumab, about 10 mM histidine buffer, about 100 mg / mL sucrose, and about 0.10 mg / mL polysorbate 80, with a pH of about 5.8-6.1.

[0044] In some embodiments, the aqueous solution comprises: about 75 mg / mL mepolizumab, about 15 mM histidine buffer, about 75 mg / mL sucrose, and about 0.15 mg / mL polysorbate 80, with a pH of about 6.0.

[0045] In some embodiments, the aqueous solution comprises: about 75 mg / mL mepolizumab, about 15 mM histidine buffer, about 60 mg / mL sucrose, and about 0.15 mg / mL polysorbate 80, with a pH of about 6.0.

[0046] In one aspect, the present invention provides the above-mentioned aqueous solution.

[0047] In one aspect, the present invention provides a reconstituted formulation of the above-mentioned lyophilized preparation, which can be directly used for injection. In some embodiments, the lyophilized preparation can be reconstituted with different volumes (e.g., 0.25 mL, 0.5 mL, 1.0 mL, 1.2 mL, 1.5 mL, etc.) of water for injection.

[0048] In some embodiments, the reconstitution formulation of the lyophilized preparation comprises about 100 mg / mL of an anti-IL-5 antibody, such as mepolizumab, about 20-30 mM histidine buffer, about 100-133 mg / mL sucrose, and about 0.1-0.4 mg / mL polysorbate, with a pH of about 5.7-6.3. In some embodiments, the polysorbate is polysorbate 80.

[0049] In some embodiments, the reconstitution formulation of the lyophilized preparation comprises about 100 mg / mL of an anti-IL-5 antibody, such as mepolizumab, about 20 mM histidine buffer, about 106-133 mg / mL of sucrose, and about 0.1-0.4 mg / mL of polysorbate 80, with a pH of about 5.7-6.3.

[0050] In some embodiments, the reconstitution formulation of the lyophilized preparation comprises about 100 mg / mL mepolizumab, about 20 mM histidine buffer, about 100-120 mg / mL sucrose and about 0.1-0.4 mg / mL polysorbate 80, with a pH of about 5.7-6.3.

[0051] In some embodiments, the reconstitution formulation of the lyophilized preparation comprises about 100 mg / mL mepolizumab, about 27 mM histidine buffer, about 100-120 mg / mL sucrose, and about 0.1-0.4 mg / mL polysorbate 80, with a pH of about 5.7-6.3.

[0052] In some embodiments, the reconstitution formulation of the lyophilized preparation comprises about 100 mg / mL mepolizumab, about 27 mM histidine buffer, about 108 mg / mL sucrose, and about 0.27 mg / mL polysorbate 80, with a pH of about 5.8-6.1.

[0053] In some embodiments, the reconstitution formulation of the lyophilized preparation comprises about 100 mg / mL mepolizumab, about 26.7 mM histidine buffer, about 107 mg / mL sucrose, and about 0.27 mg / mL polysorbate 80, with a pH of about 5.8-6.1.

[0054] In some embodiments, the reconstitution formulation of the lyophilized preparation comprises about 100 mg / mL mepolizumab, about 20 mM histidine buffer, about 100 mg / mL sucrose, and about 0.20 mg / mL polysorbate 80, with a pH of about 5.8-6.1.

[0055] In some embodiments, the reconstitution formulation of the lyophilized preparation comprises about 100 mg / mL mepolizumab, about 20 mM histidine buffer, about 120 mg / mL sucrose, and about 0.20 mg / mL polysorbate 80, with a pH of about 5.8-6.1.

[0056] In some embodiments, the osmotic pressure of the reconstituted formulation of the present invention is 300-600 mOsmol / kg. In some embodiments, the osmotic pressure of the reconstituted formulation of the present invention is 300-500 mOsmol / kg. In some embodiments, the osmotic pressure of the reconstituted formulation of the present invention is 400-600 mOsmol / kg.

[0057] On one hand, the lyophilized anti-IL-5 antibody formulation of the present invention exhibits superior stability under light conditions, and after reconstitution, it has a lower osmotic pressure that is closer to the normal physiological osmotic pressure of the human body, reducing injection pain for patients and making it more suitable for clinical use. In some embodiments, the lyophilized formulation of the present invention can be stably stored for at least 12 months under accelerated storage conditions of 25°C and / or 4°C.

[0058] In one aspect, the present invention provides an antibody pharmaceutical product for treating IL-5-related diseases, comprising a lyophilized anti-IL-5 antibody formulation as described above and a container for storing the formulation. The pharmaceutical product may also include an instruction manual. The container may be any container conventionally used in the art for storing pharmaceuticals, such as vials (e.g., vials), ampoules, pouches, etc.

[0059] In some embodiments, the antibody drug product is reconstituted by adding 1.2 mL of water for injection to obtain a drug formulation with an antibody concentration of 100 mg / mL for use.

[0060] In one aspect, the present invention provides the use of the lyophilized formulation, reconstituted formulation, and pharmaceutical product of the anti-IL-5 antibody for the treatment of diseases. In another aspect, the present invention provides the use of the lyophilized formulation, reconstituted formulation, and pharmaceutical product of the anti-IL-5 antibody in the preparation of a medicament for the treatment of diseases, including but not limited to asthma, severe eosinophilic asthma, severe asthma, uncontrolled eosinophilic asthma, eosinophilic asthma, sub-eosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granulomatosis with polyangiitis (EGPA), eosinophilic syndrome, nasal polyposis, bullous pemphigoid, and eosinophilic esophagitis.

[0061] In some embodiments, the lyophilized formulation, reconstituted formulation, and / or pharmaceutical product of anti-IL-5 antibody may be formulated for administration via routes including non-enteral (e.g., intravenous), intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration, or other suitable routes of administration known in the art. In some embodiments, the lyophilized formulation, reconstituted formulation, or pharmaceutical product of anti-IL-5 antibody may be formulated for subcutaneous injection.

[0062] the term

[0063] Unless otherwise stated, each of the following terms shall have the meaning described below.

[0064] definition

[0065] It should be noted that the term “a” entity refers to one or more of the same entity. For example, “an antibody” should be understood as one or more antibodies. Therefore, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably in this document.

[0066] As used herein, the terms “comprising” or “including” mean that compositions and methods include the listed elements, such as components or steps, but do not exclude others. “Substantially composed of” means that compositions and methods exclude other elements that have a fundamental effect on the characterization of the composition, but do not exclude elements that do not substantially affect the composition or method. “Composed of” means excluding elements not specifically listed.

[0067] "Approximately" refers to a typical error range for a given value that is readily known to those skilled in the art. In some embodiments, "approximately" as used herein refers to the described value and its range of ±10%, ±5%, or ±1%.

[0068] In this invention, the amount of buffer refers to the total amount of buffer pairs in the buffer system that makes up the buffer. In some embodiments, molar concentration is used as the unit of amount of buffer, and its value refers to the molar concentration of the buffer pairs in the buffer system. For example, when a histidine buffer is composed of histidine and histidine hydrochloride, a given concentration of histidine buffer (e.g., 20 mM) is the combined concentration of histidine and histidine hydrochloride. In some embodiments, the histidine buffer comprises histidine and histidine hydrochloride. In some embodiments, a 20 mM histidine buffer comprises about 1.24 g / L histidine and about 2.56 g / L histidine hydrochloride monohydrate. In some embodiments, a 26.7 mmol histidine buffer comprises about 1.65 g histidine and about 3.36 g histidine hydrochloride monohydrate.

[0069] The lyophilized or reconstituted formulations of the present invention can be prepared using methods known in the art, such as ultrafiltration, dialysis, freeze-drying, reconstitution / reconstitution, and combinations thereof. A "reconstituted formulation" can be obtained from the lyophilized powder by rapid dissolution after the addition of a reconstitution medium. Suitable reconstitution media include, but are not limited to: water for injection (WFI), antibacterial water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% (w / v) glucose), solutions containing surfactants (e.g., 0.01% (w / v) polysorbate 20), and pH buffer solutions (e.g., acetate buffer solutions).

[0070] In some embodiments, the volume of the lyophilized anti-IL-5 antibody formulation before lyophilization is the same as the volume of the reconstituted formulation before administration. For example, a formulation that was approximately 1.15 mL before lyophilization can be reconstituted to approximately 1.15 mL by adding an appropriate amount of liquid (e.g., water or physiological saline). In some embodiments, the volume of the lyophilized anti-IL-5 antibody formulation before lyophilization is different from the volume of the reconstituted formulation before administration. In some embodiments, an appropriate amount of liquid (e.g., water or physiological saline) is added to the lyophilized formulation to obtain a reconstituted formulation containing approximately 100 mg / mL of anti-IL-5 antibody. For example, an anti-IL-5 antibody formulation in the form of, for example, a 0.25×, 0.5×, or 0.75× dilute solution can be lyophilized and reconstituted to 1× by adding a smaller amount of liquid, for example, 75%, 50%, or a smaller volume than before lyophilization.

[0071] In this document, "stability" or "stability" refers to the fact that in a formulation containing an antibody, the antibody (including its antibody fragments) does not aggregate, or only minimally aggregates, degrades, or fragments under given manufacturing, preparation, transport, and / or storage conditions. A "stable" formulation retains its biological activity under given manufacturing, preparation, transport, and / or storage conditions. The stability of the antibody can be assessed by measuring the degree of aggregation, degradation, or fragmentation of the formulation using techniques such as size exclusion chromatography (SEC or SEC-HPLC), ion exchange chromatography (IEC or IEC-HPLC), reducing (R) or non-reducing (NR) capillary electrophoresis (CE), light inspection and turbidity, insoluble particles, and DLS particle size detection.

[0072] Instability can manifest as: aggregation (e.g., non-covalent soluble aggregation (caused by hydrophobic or charge interactions), covalent soluble aggregation (e.g., disulfide rearrangement / mixing), insoluble aggregation (caused by protein denaturation at liquid / air and liquid / solid interfaces), deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), denaturation, truncation / hydrolysis / fragmentation (e.g., hinge region fragmentation), succinimide formation, N-terminal elongation, C-terminal processing, differential glycosylation, etc.

[0073] "Aggregate", "SEC aggregate" or "soluble aggregate" refers to an antibody protein and / or fragment that are more than one and less than or equal to ten associated together by covalent, ionic or hydrophobic interactions to form a larger protein body.

[0074] The term "antibody" encompasses a wide range of polypeptides that can be distinguished biochemically. Those skilled in the art will understand that heavy chains are categorized as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with further subclasses (e.g., γ1-γ4). The properties of this chain determine the "type" of the antibody, such as IgG, IgM, IgA, IgD, or IgE. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, etc., have been well characterized and their assigned functional specificities are known. All immunoglobulin types are within the scope of this invention. In some embodiments, the immunoglobulin molecule is of the IgG type. The four chains are linked by disulfide bonds in a "Y" configuration, wherein the light chain begins at the "Y" junction and continues through a variable region surrounding the heavy chain.

[0075] Light chains can be divided into kappa (κ) or lambda (λ). Each heavy chain can bind to either a κ or λ light chain. Generally, when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, their light and heavy chains are covalently linked, with the "tail" portions of the two heavy chains linked by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus of the Y-configuration forked end to the C-terminus at the bottom of each chain. The variable region of the immunoglobulin κ light chain is Vκ; the variable region of the immunoglobulin λ light chain is V... λ .

[0076] The variable regions of the light chain (VL) and heavy chain (VH) determine antigen recognition and specificity. The constant regions (CL) of the light chain and (CH) of the heavy chain confer important biological properties, such as secretion, transplacental migration, Fc receptor binding, and complement binding. Conventionally, the constant regions are numbered as they move further away from the antibody's antigen-binding site or N-terminus. The N-terminal portion is the variable region, and the C-terminal portion is the constant region; the CH3 and CL domains contain the carboxyl terms of the heavy and light chains, respectively.

[0077] "Treatment" refers to therapeutic treatments and preventative or preventative measures aimed at preventing, mitigating, improving, and stopping adverse physiological changes or disorders, such as disease progression, including but not limited to the following, whether detectable or undetectable: symptom relief, reduction of disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of the disease state, reduction or disappearance of the disease state (whether partial or complete), and prolongation of the expected lifespan without treatment. Patients requiring treatment include those already suffering from the condition or disorder, those susceptible to the condition or disorder, or those needing prevention of the condition or disorder, as well as those who can or are expected to benefit from the application of the antibodies or compositions disclosed in this invention for detection, diagnostic procedures, and / or treatment.

[0078] DNA encoding the antibody can be designed and synthesized according to the antibody amino acid sequence described herein using conventional methods. This DNA can then be placed into an expression vector, transfected into host cells, and cultured in a culture medium to produce monoclonal antibodies. In some embodiments, the antibody expression vector includes at least one promoter element, an antibody-coding sequence, a transcription termination signal, and a polyA tail. Other elements include an enhancer, a Kozak sequence, and donor and acceptor sites for RNA splicing flanking the insert sequence. Efficient transcription can be achieved using early and late promoters of SV40, early promoters from long terminal repeat sequences of retroviruses such as RSV, HTLV1, HIV, and cytomegalovirus, or other cellular promoters such as the actin promoter. Suitable expression vectors may include pIRES1neo, pRetro-Off, pRetro-On, pLXSN, or pLNCX, pcDNA3.1(+ / -), pcDNA / Zeo(+ / -), pcDNA3.1 / Hygro(+ / -), pSVL, pMSG, pRSVcat, pSV2dhfr, pBC12MI, and pCS2, etc. Commonly used mammalian cell lines include 293 cells, Cos1 cells, Cos7 cells, CV1 cells, mouse L cells, and CHO cells, etc.

[0079] In the following examples, the detection methods, including SEC-HPLC, IEC-HPLC, non-reducing capillary electrophoresis (CE(NR)), and reducing capillary electrophoresis (CE(R)), were all performed according to conventional methods.

[0080] This document is further illustrated by the following examples for illustrative purposes only and should not be construed as limiting the scope or content of this disclosure in any way. The antibody used in the following examples is mepolizumab. Unless otherwise specified, the preparation and detection methods in the following examples are conventional methods in the art.

[0081] Example 1: Preparation of lyophilized formulation

[0082] The pre-lyophilized formulations shown in Table 1 were prepared, filled into glass vials, and lyophilized using manufacturing processes known in the art to obtain lyophilized formulation samples.

[0083] The concentrations and osmotic pressures of the reconstituted formulations are shown in Table 1. The antibody concentrations of all reconstituted formulations are 100 mg / mL. The osmotic pressures of reconstituted formulations 1, 2, 6, 7, and 9 at 0H (hours) are all above 500 mOsmol / kg, which is much higher than the normal physiological osmotic pressure range of the human body. The osmotic pressures of reconstituted formulations 3, 4, 5, 8, 10, and 11 are close to the normal physiological osmotic pressure range of the human body.

[0084] Table 1 Composition of the formulation

[0085] Example 2: Stability assessment at 40℃

[0086] Glass vials containing lyophilized formulation samples were subjected to a forced degradation test at 40°C, with samples taken and tested at 0 days, 4 weeks, and 8 weeks.

[0087] The SEC-HPLC test results are shown in Table 2. The IEC-HPLC test results are shown in Table 3. The CE(NR) test results are shown in Table 4.

[0088] The results showed that all formulations exhibited good stability after being placed at 40°C for 8 weeks.

[0089] Table 2 SEC-HPLC data at 40℃

[0090] Table 3. IEC-HPLC data at 40℃

[0091] Table 4 CE(NR) data at 40℃

[0092] Example 3: Stability assessment under illumination conditions

[0093] The glass vials containing the lyophilized formulation were subjected to a forced degradation test under light conditions (4500±500 lx, 25℃), and samples were taken for testing at 0 days, 2 weeks, and 4 weeks.

[0094] The SEC-HPLC results are shown in Table 5, and the IEC-HPLC and CE(NR) results are shown in Table 6. The results show that, after 4 hours of exposure to light, the monomers in formulations containing phosphate (PB) buffer solution showed a greater decrease compared to formulations containing histidine (His) buffer solution.

[0095] Table 5. SEC-HPLC data under illumination conditions

[0096] Table 6. IEC-HPLC data and CE(NR) data under illumination conditions.

[0097] Example 4: Stability assessment under oscillation conditions

[0098] The glass vials containing the lyophilized formulation were subjected to a forced degradation test under shaking conditions (200 rpm, room temperature), and samples were taken for testing at 0 days and 48 hours.

[0099] The results of SEC-HPLC, IEC-HPLC, and CE(NR) assays are shown in Table 7. After 48 hours of shaking, all formulation samples showed good stability.

[0100] Table 7. SEC-HPLC data, IEC-HPLC data, and CE(NR) data under oscillation conditions.

[0101] Example 5: Stability test at 25℃

[0102] Glass vials containing lyophilized formulation samples were placed at 25°C for long-term stability testing, with samples taken at 0 days, 6 months, and 12 months.

[0103] The SEC-HPLC results are shown in Table 8. After 12 months of observation at 25℃, the His buffer system showed better stability than the PB buffer system.

[0104] The IEC-HPLC results are shown in Table 9. After 12 months of observation at 25℃, the IEC main peak showed a certain degradation trend. Furthermore, the higher the sucrose concentration, the smaller the degradation amplitude of the main peak. Formulations 4 and 5, with relatively low sucrose content, showed the largest degradation amplitude of the main peak. Comparing formulations 2, 6, and 10 with different protein concentrations before lyophilization, it can be seen that there is no significant difference in the degradation trend of the main peak when the protein concentration before lyophilization is 50 mg / mL and 75 mg / mL. The degradation rate of the main peak in formulation 10 (protein concentration before lyophilization is 100 mg / mL) is relatively faster, indicating that the antibody stability is slightly worse when the protein concentration before lyophilization is too high.

[0105] The CE(NR) data are shown in Table 10. After 12 months of observation at 25°C, the main peak content of each formulation showed varying degrees of degradation, with little overall difference.

[0106] The CE(R) data are shown in Table 11. After 12 months of observation at 25°C, the stability of formulation 5 with 6% sucrose content and formulations 7, 8, and 9 with PB buffer was slightly worse than that of other formulations.

[0107] The relative binding activity of each formulation was generally good after 12 months of observation at 25°C.

[0108] Table 8. SEC-HPLC data at 25℃

[0109] Table 9. IEC-HPLC data at 25℃

[0110] Table 10 CE(NR) data at 25℃

[0111] Table 11 CE(R) data at 25℃

[0112] Example 6: Stability of lyophilized formulations and preparation of reconstituted formulations

[0113] After thawing the stock solution, the required formulation buffer solutions for diluting to 100 mg / mL and 75 mg / mL of the finished product were calculated based on the actual stock solution dosage and concentration. The pre-lyophilized formulation aqueous solutions were prepared as shown in Table 12. After thorough mixing, the solutions were sterilized by filtration through a 0.22 μm filter membrane. Samples were taken to test the protein concentration and pH. Samples with a pre-lyophilized protein concentration of 100 mg / mL (formulation 12) were filled at 1.4 mL / bottle, and samples with a pre-lyophilized protein concentration of 75 mg / mL (formulations 1, 3, 5 and 13) were filled at 1.9 mL / bottle. After half-stopping, the solutions were lyophilized. Stability studies were conducted on samples of lyophilized formulations 1, 3, 5, 12, and 13 under light irradiation (4500±500 lx, 25℃), high temperature conditions of 40℃, and accelerated conditions of 25℃. Samples were taken after 14 days of light irradiation (GZ-14d), 4 weeks of high temperature (GW-4W), 8 weeks of high temperature (GW-8W), and 3 months of accelerated conditions (JS-3M), and the lyophilized formulation samples were reconstituted with 1.2 mL of sterile water for injection for testing.

[0114] On day 0 (0d), after 10 days of light exposure and 4 weeks of high temperature, reconstituted formulations 13, 3, and 1 were found to be clear, reconstituted formulation 12 exhibited severe opalescence and had a longer reconstitution time, and reconstituted formulation 5 exhibited obvious opalescence. The freeze-dried formulation 1 was prone to cracking after freeze-drying.

[0115] The results of the forced degradation test are shown in Table 13.

[0116] Table 12 Composition information of the formulation

[0117] Table 13. SEC-HPLC, IEC-HPLC, and CE(NR) data from the forced degradation test.

Claims

1. A lyophilized formulation of an anti-IL-5 antibody comprising: an anti-IL-5 antibody, a histidine buffer, sucrose, and a polysorbate; the heavy chain variable region of the anti-IL-5 antibody comprising the sequence set forth in SEQ ID NO: 1, and the light chain variable region comprising the sequence set forth in SEQ ID NO:

2.

2. The lyophilized formulation of claim 1, the heavy chain of the anti-IL-5 antibody comprising the sequence set forth in SEQ ID NO: 3, and the light chain comprising the sequence set forth in SEQ ID NO:

4.

3. The lyophilized formulation of claim 1, the anti-IL-5 antibody is Mepolizumab.

4. The lyophilized formulation of any one of claims 1-3, wherein the mass ratio of the anti-IL-5 antibody to sucrose is about 0.5-1.5, or about 0.75-1.2, or about 0.75-0.95, or about 0.83-0.

94.

5. The lyophilized formulation of any one of claims 1-4, the ratio of the anti-IL-5 antibody to the histidine buffer is about 3.3 g: 1 mmol to 5 g: 1 mmol, or about 3.5 g: 1 mmol to 5 g: 1 mmol, or about 3.75 g: 1 mmol to 5 g: 1 mmol.

6. The lyophilized formulation of any one of claims 1-5, the mass ratio of the anti-IL-5 antibody to the polysorbate is about 250: 1 to 1000: 1, or about 350: 1 to 500: 1, or about 370: 1 to 500: 1; or, the polysorbate is polysorbate 80.

7. The lyophilized formulation of any one of claims 1-6, comprising at least about 100 mg of the anti-IL-5 antibody, the histidine buffer, sucrose, and the polysorbate, the ratio of the anti-IL-5 antibody to the histidine buffer is about 3.3 g: 1 mmol to 5 g: 1 mmol, the mass ratio of the anti-IL-5 antibody to sucrose is about 0.75-1.2 or 0.8-0.95, and the mass ratio of the anti-IL-5 antibody to the polysorbate is about 250: 1 to 1000: 1; or, the ratio of the anti-IL-5 antibody to the histidine buffer is about 3.75 g: 1 mmol, the mass ratio of the anti-IL-5 antibody to sucrose is about 0.94, and the mass ratio of the anti-IL-5 antibody to the polysorbate is about 375: 1; or, the ratio of the anti-IL-5 antibody to the histidine buffer is about 5 g: 1 mmol, the mass ratio of the anti-IL-5 antibody to sucrose is about 1, and the mass ratio of the anti-IL-5 antibody to the polysorbate is about 500: 1; or, the ratio of the anti-IL-5 antibody to the histidine buffer is about 5 g: 1 mmol, the mass ratio of the anti-IL-5 antibody to sucrose is about 0.83, and the mass ratio of the anti-IL-5 antibody to the polysorbate is about 500:

1.

8. The lyophilized formulation according to any one of claims 1-7, wherein each lyophilized formulation containing about 100g of anti-IL-5 antibody contains about 20-30 mmol of histidine buffer, about 100-133g of sucrose, and about 0.1-0.4g of polysorbate 80; or Approximately 20-30 mmol histidine buffer, approximately 106-120 g sucrose, and approximately 0.1-0.4 g polysorbate 80; or Approximately 27 mmol histidine buffer, approximately 108 g sucrose, and approximately 0.27 g polysorbate 80; or Approximately 26.7 mmol histidine buffer, approximately 107 g sucrose, and approximately 0.27 g polysorbate 80; or Approximately 20 mmol histidine buffer, approximately 100 g sucrose, and approximately 0.2 g polysorbate 80; or Approximately 20 mmol histidine buffer, approximately 120 g sucrose, and approximately 0.2 g polysorbate 80.

9. The lyophilized formulation according to any one of claims 1-8, wherein it is prepared by lyophilizing an aqueous solution, said aqueous solution comprising an anti-IL-5 antibody, a histidine buffer, sucrose, and polysorbate.

10. The lyophilized formulation according to any one of claims 1-9, prepared by lyophilizing an aqueous solution, said aqueous solution comprising: 1) 45-105 mg / mL anti-IL-5 antibody, 10-30 mM histidine buffer, 60-200 mg / mL sucrose, 0.1-0.4 mg / mL polysorbate; or 2) 75-100 mg / mL anti-IL-5 antibody, 10-30 mM histidine buffer, 60-200 mg / mL sucrose, 0.1-0.4 mg / mL polysorbate; or 3) 50-100 mg / mL anti-IL-5 antibody, 10-30 mM histidine buffer, 60-200 mg / mL sucrose, and 0.1-0.4 mg / mL polysorbate 80, wherein the mass ratio of the anti-IL-5 antibody to sucrose is approximately 0.5-1; or 4) 50-100 mg / mL anti-IL-5 antibody, 10-30 mM histidine buffer, 60-135 mg / mL sucrose, and 0.1-0.4 mg / mL polysorbate 80, wherein the mass ratio of the anti-IL-5 antibody to sucrose is approximately 0.75-1; or 5) Approximately 50 mg / mL anti-IL-5 antibody, 10-20 mM histidine buffer, 50-70 mg / mL sucrose, and 0.1-0.4 mg / mL polysorbate 80; or 6) Approximately 100 mg / mL anti-IL-5 antibody, 10-20 mM histidine buffer, 100-134 mg / mL sucrose, and 0.1-0.4 mg / mL polysorbate 80; or 7) Approximately 75 mg / mL anti-IL-5 antibody, 10-20 mM histidine buffer, 75-100 mg / mL sucrose, and 0.1-0.4 mg / mL polysorbate 80; or 8) Approximately 75 mg / mL anti-IL-5 antibody, 10-20 mM histidine buffer, 80-90 mg / mL sucrose, and 0.1-0.4 mg / mL polysorbate 80.

11. The lyophilized formulation of any one of claims 1-10, wherein the anti-IL-5 antibody is mepolizumab.

12. The lyophilized formulation of any one of claims 1-11, prepared by lyophilizing an aqueous solution comprising: 1) about 75 mg / mL mepolizumab, about 20 mM histidine buffer, about 80 mg / mL sucrose, and about 0.20 mg / mL polysorbate 80; or 2) about 75 mg / mL mepolizumab, about 15 mM histidine buffer, about 90 mg / mL sucrose, and about 0.15 mg / mL polysorbate 80; or 3) about 100 mg / mL mepolizumab, about 20 mM histidine buffer, about 100 mg / mL sucrose, and about 0.20 mg / mL polysorbate 80; or 4) about 50 mg / mL mepolizumab, about 10 mM histidine buffer, about 60 mg / mL sucrose, and about 0.10 mg / mL polysorbate 80.

13. The lyophilized formulation of any one of claims 10-12, wherein the aqueous solution has a pH of about 5.3-6.8, or about 5.7-6.3, or about 5.8-6.

1.

14. The aqueous solution of any one of claims 10-13.

15. A reconstituted formulation obtained by reconstituting the lyophilized formulation of any one of claims 1-13.

16. The reconstituted formulation of claim 15, comprising 1) about 100 mg / mL anti-IL-5 antibody, about 20-30 mM histidine buffer, about 100-133 mg / mL sucrose, and about 0.1-0.4 mg / mL polysorbate; or 2) about 100 mg / mL anti-IL-5 antibody, about 20 mM histidine buffer, about 106-133 mg / mL sucrose, and about 0.1-0.4 mg / mL polysorbate 80; or 3) about 100 mg / mL anti-IL-5 antibody, about 27 mM histidine buffer, about 100-120 mg / mL sucrose, and about 0.1-0.4 mg / mL polysorbate 80; or 4) about 100 mg / mL anti-IL-5 antibody, about 20 mM histidine buffer, about 100-120 mg / mL sucrose, and about 0.1-0.4 mg / mL polysorbate 80.

17. The reconstituted formulation of claim 15 or 16, comprising: 1) about 100 mg / mL mepolizumab, about 27 mM histidine buffer, about 108 mg / mL sucrose, and about 0.27 mg / mL polysorbate 80; or 2) about 100 mg / mL mepolizumab, about 26.7 mM histidine buffer, about 107 mg / mL sucrose, and about 0.27 mg / mL polysorbate 80; or 3) about 100 mg / mL mepolizumab, about 20 mM histidine buffer, about 100 mg / mL sucrose, and about 0.20 mg / mL polysorbate 80; or 4) about 100 mg / mL mepolizumab, about 20 mM histidine buffer, about 100 mg / mL sucrose, and about 0.20 mg / mL polysorbate 80. 4) about 100 mg / mL mepolizumab, about 20 mM histidine buffer, about 120 mg / mL sucrose, and about 0.20 mg / mL polysorbate 80.

18. The reconstituted formulation according to any one of claims 15-17 having a pH of about 5.3-6.8, or about 5.7-6.3 or about 5.8-6.

1.

19. Use of the lyophilized formulation according to any one of claims 1-13, the reconstituted formulation according to any one of claims 15-17 for the treatment of a disease.

20. Use of the lyophilized formulation according to any one of claims 1-13, the reconstituted formulation according to any one of claims 15-17 for the manufacture of a medicament for the treatment of a disease.

21. The use according to claim 19 or 20, wherein the disease comprises asthma, severe eosinophilic asthma, severe asthma, uncontrolled eosinophilic asthma, eosinophilic asthma, sub-eosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granulomatosis with polyangiitis, hypereosinophilic syndrome, nasal polyposis, bullous pemphigoid and eosinophilic esophagitis.

Images