WO2021058005A1 - Pharmaceutical composition containing humanized anti-human il-17a monoclonal antibody - Google Patents

Abstract

Provided is a pharmaceutical composition containing a humanized anti-human IL-17A monoclonal antibody, comprising: (a) an anti-human IL-17A monoclonal antibody; and (b) a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier comprises a buffer solution, a stabilizer, and a surfactant.

Description

Pharmaceutical composition of humanized anti-human IL-17A monoclonal antibody Technical field

The present invention relates to the field of pharmaceutical preparations, in particular to a pharmaceutical composition of humanized anti-human IL-17A monoclonal antibody.

Background technique

IL-17A ((Interleukin 17A) is a homodimer composed of two chains of 155 amino acids connected by disulfide bonds, with a molecular weight of 35kDa, which was originally discovered to be secreted by activated CD4+ T cells. This type of feature The subset of T cells that secrete IL-17A sexually is called Th17 cells. In addition to Th17 cells, cytotoxic CD8+ T cells (Tc17), γδ T cells, natural killer T cells (NKT-17) and B cells can also be specified IL-17A is expressed under conditions. Innate immune cells, including monocytes, neutrophils, natural killer cells and lymphoid tissue-inducible (Lti-like) cells can also produce IL-17A.

IL-17 binds to type I cell surface receptors called IL-17R, of which there are at least three: IL-17RA, IL-17RB and IL-17RC. IL-17A and IL-17F combine with IL-17RA and IL-17RC receptor complexes in the form of homodimers or heterodimers to transduce signals, and participate in the body's autoimmune diseases, various inflammatory reactions, and Host immune response against infection.

IL-17A mainly induces signal activation of cells of non-hematopoietic origin, including epithelial cells and stromal cells. A variety of inflammatory factors and chemokines induced by IL-17A can promote the recruitment of a variety of immune cells, thereby promoting autoimmune diseases. Studies have found that IL-17A and IL-17F play their role in promoting inflammation mainly by inducing target cells to express a variety of inflammatory factors and chemokines. IL-17A binds to the cell surface receptor IL-17RA, recruits IL-17RC to form heterodimers, mediates downstream signaling pathways, and also plays an important role in a variety of autoimmune diseases, including autoimmune diseases. Such as Rheumatoid Arthritis (RA) and Multiple Sclerosis (MS), and Inflammatory Boweldisease (IBD), Psoriasis, Systemic lupus Erythematosus , SLE) and type I diabetes (Type1Diabetes, T1D).

Anti-human IL-17A monoclonal antibodies (such as humanized anti-human IL-17A monoclonal antibodies) can prevent and treat IL-17A-mediated diseases, among which humanized anti-human IL-17A monoclonal antibodies are macromolecules Drugs have complex structures, and are affected by various physical and chemical factors in the production, storage, and transportation of drugs. Reactions such as aggregation, hydrolysis, and oxidation occur. The by-products produced will adversely affect the safety and effectiveness of the drug. The development of formulations with excellent stability is very important for clinical medication.

Therefore, there is a need in the art to develop a humanized anti-human IL-17A monoclonal antibody pharmaceutical preparation with excellent stability to improve the stability of the humanized anti-human IL-17A monoclonal antibody.

Summary of the invention

The purpose of the present invention is to provide a pharmaceutical composition containing anti-human IL-17A monoclonal antibody, which can maintain excellent stability of the anti-human IL-17A monoclonal antibody.

In the first aspect of the present invention, there is provided a pharmaceutical composition, the pharmaceutical composition comprising:

(a) Anti-human IL-17A monoclonal antibody; and

(b) A pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier includes a buffer, a stabilizer and a surfactant.

In another preferred embodiment, the anti-human IL-17A monoclonal antibody is a humanized anti-human IL-17A monoclonal antibody.

In another preferred embodiment, the three complementarity determining regions CDRs of the heavy chain variable region of the humanized anti-human IL-17A monoclonal antibody: HCDR1 shown in SEQ ID NO.: 1 and SEQ ID NO.: 2 The HCDR2 shown in the HCDR2 and the HCDR3 shown in SEQ ID NO.: 3, and the three complementary determining regions of the light chain variable region CDR: LCDR1 shown in SEQ ID NO.: 4 and shown in SEQ ID NO.: 5 LCDR2 and SEQ ID NO.: LCDR3 shown in 6.

In another preferred embodiment, the humanized anti-human IL-17A monoclonal antibody preferably has the heavy chain variable region amino acid sequence shown in SEQ ID NO: 7 and the light chain variable region shown in SEQ ID NO: 8 Region amino acid sequence.

In another preferred embodiment, the anti-human IL-17A monoclonal antibody preferably has the heavy chain variable region amino acid sequence shown in SEQ ID NO: 7 and the light chain variable region amino acid sequence shown in SEQ ID NO: 8 .

In another preferred embodiment, the dosage form of the pharmaceutical composition is a liquid preparation.

In another preferred embodiment, the dosage form of the pharmaceutical composition is an injection preparation or an infusion preparation.

In another preferred embodiment, the concentration of the anti-human IL-17A monoclonal antibody is 10-300 mg/mL, preferably 10-200 mg/mL, more preferably 30-180 mg/mL, more preferably 50- 180 mg/mL, more preferably 50-160 mg/mL, more preferably 80-160 mg/mL, most preferably 80-120 mg/mL.

In another preferred embodiment, the buffer is selected from the following group: acetate-acetate buffer, citrate-citrate buffer, phosphate buffer, histidine-acetate buffer Solution, Tris-hydrochloride buffer, phosphate-citrate, or a combination thereof.

In another preferred embodiment, the buffer is selected from the following group: acetate-acetate buffer, phosphate buffer, histidine-acetate buffer, tris (Tris) -Hydrochloride buffer, or a combination thereof.

In another preferred embodiment, the phosphate buffer is sodium dihydrogen phosphate-disodium hydrogen phosphate buffer.

In another preferred embodiment, the buffer is selected from the group consisting of acetate-acetate buffer, citrate-citrate buffer.

In another preferred embodiment, the buffer is selected as acetate-acetate buffer.

In another preferred embodiment, the buffer is selected as sodium acetate-acetate buffer.

In another preferred embodiment, the concentration of the buffer is 5-100 mM, preferably 5-80 mM, more preferably 5-60 mM, more preferably 5-40 mM, more preferably 5-30 mM, more preferably 10-30mM, optimally 15-25mM.

In another preferred embodiment, the solvent of the buffer is water.

In another preferred embodiment, the pH range of the buffer is 5.0-7.2, preferably 5.0-6.5, more preferably 5.3-6.0.

In another preferred embodiment, the stabilizer is selected from the group consisting of sodium chloride, amino acids, sugar alcohols, or combinations thereof.

In another preferred embodiment, the amino acid is selected from the following group: Proline, Arginine, Glycine, Histidine, Methionine, or a combination thereof ;and / or

In another preferred embodiment, the sugar alcohol is selected from the following group: Sucrose, Mannitol, Trehalose, Maltose, Sorbitol, or a combination thereof.

In another preferred embodiment, the sugar alcohol is selected from the group consisting of sucrose, mannitol, trehalose, sorbitol, or a combination thereof.

In another preferred embodiment, the stabilizer is sucrose.

In another preferred embodiment, the sugar alcohol is sucrose.

In another preferred embodiment, the sodium chloride concentration is 50-400 mM, preferably 100-300 mM, more preferably 150-300 mM.

In another preferred embodiment, the amino acid concentration is 10-300mM, preferably 20-250mM, more preferably 20-200mM, more preferably 30-180mM, more preferably 50-150mM, more preferably 80- 120mM.

In another preferred example, the sugar alcohol concentration is 0.1-20wt.%, 2-20wt.%, preferably 2-16wt.%, more preferably 2-14wt.%, more preferably 4-14wt. %, more preferably 5-13 wt.%, most preferably 6-10 wt.%, based on the total weight of the pharmaceutical composition.

In another preferred example, the content of the stabilizer is 0.1-20wt.%, 2-20wt.%, preferably 2-16wt.%, more preferably 2-14wt.%, more preferably 4- 14wt.%, more preferably 5-13wt.%, most preferably 6-10wt.%, based on the total weight of the pharmaceutical composition.

In another preferred example, the concentration of the stabilizer is 10-200g/L, preferably 20-160g/L, more preferably 40-120g/L, more preferably 60-100g/L, most preferably 70-90g/L.

In another preferred embodiment, the surfactant is selected from the following group: polyoxyethylene sorbitan fatty acid ester, polyoxyethylene hydrogenated castor oil, glycerin fatty acid ester, polysorbate, poloxamer, Or a combination.

In another preferred embodiment, the polysorbate is selected from the group consisting of polysorbate 20 (PS-20, Tween-20), polysorbate 40 (PS-40), polysorbate 60 (PS-60), polysorbate 80 (PS-80).

In another preferred embodiment, the poloxamer is selected from the group consisting of poloxamer 188, poloxamer 108, poloxamer 124, or a combination thereof.

In another preferred embodiment, the surfactant is polysorbate.

In another preferred embodiment, the surfactant is polysorbate 80 (PS-80).

In another preferred example, the surfactant content is 0.001-0.2wt.%, preferably 0.001-0.1wt.%, more preferably 0.005-0.1wt.%, more preferably 0.005-0.08wt. %, optimally 0.008-0.05wt.%, based on the total weight of the pharmaceutical composition.

In another preferred embodiment, the pharmaceutically acceptable carrier further includes a chelating agent.

In another preferred embodiment, the chelating agent is selected from the group consisting of disodium ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), or a combination thereof.

In another preferred embodiment, the content of the chelating agent is 0.001-0.01 wt.%, based on the total weight of the pharmaceutical composition.

In another preferred embodiment, the pH range of the pharmaceutical composition is 5.0-7.2, preferably 5.0-6.5, more preferably 5.3-6.0.

In another preferred embodiment, the pharmaceutical composition includes:

Anti-human IL-17A monoclonal antibody 10-200mg/mL Acetate-Acetate Buffer 5-80mM sucrose 20-160g/L PS-80 0.001-0.2wt.% The pH of the pharmaceutical composition is 5.0-7.2 .

In another preferred embodiment, the pharmaceutical composition includes:

Humanized anti-human IL-17A monoclonal antibody 80-160mg/mL Acetate-Acetate Buffer 5-40mM sucrose 40-120g/L PS-80 0.001-0.1wt.% The pH of the pharmaceutical composition is 5.0-6.5 .

In another preferred embodiment, the pharmaceutical composition includes:

Humanized anti-human IL-17A monoclonal antibody 80-120mg/mL Acetate-Acetate Buffer 10-30mM sucrose 60-100g/L PS-80 0.005-0.08wt.% The pH of the pharmaceutical composition is 5.3-6.0 .

In another preferred example, in the pharmaceutical composition, the total weight content (wt.%) of each component is 100%.

The second aspect of the present invention provides a kit containing the pharmaceutical composition according to the first aspect of the present invention, and a container for containing the pharmaceutical composition.

The third aspect of the present invention provides a use of the pharmaceutical composition according to the first aspect of the present invention and/or the kit according to the second aspect of the present invention for preparing (i) preventing and/or treating IL -17A-mediated diseases; and/or (ii) prevention and/or treatment of autoimmune diseases.

In another preferred embodiment, the IL-17A-mediated disease is an autoimmune disease.

In another preferred embodiment, the IL-17A is human IL-17A.

In another preferred example, the autoimmune disease is selected from the group consisting of psoriasis, arthritis (preferably rheumatoid arthritis), multiple sclerosis, compulsive spondylitis, inflammatory bowel disease, Systemic lupus erythematosus, type I diabetes, or a combination thereof.

The fourth aspect of the present invention provides a freeze-dried preparation, which is prepared by the following method:

The pharmaceutical composition according to the first aspect of the present invention is freeze-dried to obtain a freeze-dried preparation.

In another preferred embodiment, the freeze-dried preparation further includes a freeze-dried protective agent.

In another preferred embodiment, the freeze-drying protective agent is selected from the following group: glucose, mannitol, fructose, galactose, or a combination thereof.

The fifth aspect of the present invention provides a method for preventing and/or treating IL-17A-mediated diseases, the method comprising: administering the pharmaceutical composition according to the first aspect of the present invention to a desired subject.

In another preferred embodiment, the IL-17A is human IL-17A.

In another preferred embodiment, the subject is a human or non-human mammal.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as the embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, I will not repeat them here.

detailed description

Through extensive and in-depth research, the inventors unexpectedly developed a pharmaceutical composition that can effectively improve the humanized anti-human IL-17A monoclonal antibody under pressure (high temperature, freeze-thaw and shock). Etc.), the stability under accelerated and long-term refrigeration conditions can improve the safety of clinical use. On this basis, the present invention has been completed.

the term

Unless otherwise defined, the meanings of all technical and scientific terms used herein are the same as those commonly understood by those of ordinary skill in the art to which the present invention belongs.

As used herein, the terms "including", "including", and "containing" are used interchangeably, and include not only closed definitions, but also semi-closed and open definitions. In other words, the term includes "consisting of" and "consisting essentially of".

As used herein, "mM" is a unit of mmol/L, for example, 1 mM=1 mmol/L.

In the pharmaceutical composition of the present invention, the weight content (wt.%) or concentration (such as mM, mg/mL) of each component is based on the weight or volume of the pharmaceutical composition.

As used herein, tris is abbreviated as Tris, that is, "tris" and "Tris" can be used interchangeably.

Pharmaceutical composition

The present invention provides a pharmaceutical composition, characterized in that the pharmaceutical composition comprises:

(a) Anti-human IL-17A monoclonal antibody; and

(b) A pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier includes a buffer, a stabilizer and a surfactant.

The term "pharmaceutically acceptable carrier" refers to: one or more compatible solid, semi-solid, liquid or gel fillers, which are suitable for human or animal use, and must have sufficient purity and low enough toxicity. "Compatibility" means that the components in the pharmaceutical composition and the active ingredients of the drug and their blending with each other without significantly reducing the efficacy of the drug.

In a preferred embodiment of the present invention, the pH range of the pharmaceutical composition is 5.0-7.2, preferably 5.0-6.5, more preferably 5.3-6.0.

Preferably, the dosage form of the pharmaceutical composition is a liquid preparation. More preferably, the dosage form of the pharmaceutical composition is an injection preparation or an infusion preparation.

Anti-human IL-17A monoclonal antibody

The preferred anti-human IL-17A monoclonal antibody of the present invention is a humanized anti-human IL-17A monoclonal antibody. Preferably, the humanized anti-human IL-17A monoclonal antibody (see CN108359011A); patent name: antibody targeted to interleukin 17A, its preparation method and application, the amino acid sequence has SEQ ID NO.: 1. The amino acid sequences of HCDR1, HCDR2, and HCDR3 of SEQ ID NO.: 2 and SEQ ID NO.: 3, and LCDR1 of SEQ ID NO.: 4, SEQ ID NO.: 5 and SEQ ID NO.: 6 respectively LCDR2 and LCDR3 amino acid sequence, the amino acid sequence is shown in the following table:

A further preferred humanized anti-human IL-17A monoclonal antibody has a heavy chain SEQ ID NO.: 7 and a light chain SEQ ID NO.: 8, two amino acid sequences

SEQ ID NO.: 7

Among them, the underline is HCDR1, HCDR2, HCDR3 (SEQ ID NO.: 1, 2 and 3).

SEQ ID NO.: 8

Among them, the underlines are LCDR1, LCDR2, LCDR3 (SEQ ID NO.: 4, 5, and 6).

In a preferred embodiment, the concentration of the anti-human IL-17A monoclonal antibody is 10-300 mg/mL, preferably 10-200 mg/mL, more preferably 30-180 mg/mL, more preferably 50-180 mg /mL, more preferably 50-160mg/mL, more preferably 80-160mg/mL, more preferably 80-120mg/mL.

Buffer

In the pharmaceutical composition of the present invention, preferably, the buffer includes (but is not limited to): acetate-acetate buffer, citrate-citrate buffer, phosphate buffer, Histidine-acetate buffer buffer, Tris-hydrochloride buffer, phosphate-citrate, or a combination thereof.

Preferably, the buffer includes (but is not limited to): acetate-acetate buffer, phosphate buffer, histidine-acetate buffer, tris-salt Acid salt buffer, or a combination thereof.

Typically, the phosphate buffer is sodium dihydrogen phosphate-disodium hydrogen phosphate buffer.

Typically, the buffer is selected from the following group: acetate-acetate buffer, citrate-citrate buffer.

Typically, the buffer is selected as acetate-acetate buffer.

Typically, the buffer is selected as sodium acetate-acetic acid buffer.

In a preferred example, the concentration of the buffer solution is 5-100 mM, preferably 5-80 mM, more preferably 5-60 mM, more preferably 5-40 mM, more preferably 5-30 mM, more preferably 10 -30mM, optimally 15-25mM.

In another preferred embodiment, the solvent of the buffer solution is water commonly used,

stabilizer

In the pharmaceutical composition of the present invention, preferably, the stabilizer includes (but is not limited to): sodium chloride, amino acid, sugar alcohol, or a combination thereof.

Preferably, the amino acids include (but are not limited to): proline, arginine (Arginine), glycine (Glycine), histidine (Histidine), methionine (Methionine), or a combination thereof; and /or

Preferably, the sugar alcohol includes (but is not limited to): sucrose (Sucrose), mannitol (Mannitol), trehalose (Trehalose), maltose (Maltose), sorbitol (Sorbitol), or a combination thereof.

Preferably, the sugar alcohol includes (but is not limited to): sucrose, mannitol, trehalose, sorbitol, or a combination thereof.

Typically, the sugar alcohol is sucrose.

In a preferred example, the stabilizer is sucrose.

In a preferred example, the sodium chloride concentration is 50-400 mM, preferably 100-300 mM, more preferably 150-300 mM.

In a preferred example, the amino acid concentration is 10-300mM, preferably 20-250mM, more preferably 20-200mM, more preferably 30-180mM, more preferably 50-150mM, more preferably 80-120mM .

In a preferred example, the sugar alcohol concentration is 0.1-20wt.%, 2-20wt.%, preferably 2-16wt.%, more preferably 2-14wt.%, more preferably 4-14wt. %, more preferably 5-13 wt.%, most preferably 6-10 wt.%, based on the total weight of the pharmaceutical composition.

In another preferred example, the content of the stabilizer is 0.1-20wt.%, 2-20wt.%, preferably 2-16wt.%, more preferably 2-14wt.%, more preferably 4- 14wt.%, more preferably 5-13wt.%, most preferably 6-10wt.%, based on the total weight of the pharmaceutical composition.

In another preferred example, the concentration of the stabilizer is 10-200g/L, preferably 20-160g/L, more preferably 40-120g/L, more preferably 60-100g/L, most preferably 70-90g/L.

Surfactant

In the pharmaceutical composition of the present invention, preferably, the surfactant includes (but not limited to): polyoxyethylene sorbitan fatty acid ester, polyoxyethylene hydrogenated castor oil, glycerin fatty acid ester, polyoxyethylene Sorbitol ester, poloxamer, or a combination thereof.

Preferably, the polysorbate includes (but is not limited to): polysorbate 20 (PS-20, Tween-20), polysorbate 40 (PS-40), polysorbate 60 ( PS-60), polysorbate 80 (PS-80).

Preferably, the poloxamer includes (but is not limited to): poloxamer 188, poloxamer 108, poloxamer 124, or a combination thereof.

Preferably, the surfactant is polysorbate.

Typically, the surfactant is polysorbate 80 (PS-80).

In a preferred example, the surfactant content is 0.001-0.2wt.%, preferably 0.001-0.1wt.%, more preferably 0.005-0.1wt.%, more preferably 0.005-0.08wt.% , Optimally 0.008-0.05wt.%, based on the total weight of the pharmaceutical composition.

Other pharmaceutically acceptable carriers

In another preferred embodiment of the present invention, the pharmaceutically acceptable carrier further includes a chelating agent.

In another preferred embodiment, the chelating agent includes (but is not limited to): disodium ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), or a combination thereof.

In another preferred embodiment, the content of the chelating agent is 0.001-0.01 wt.%, based on the total weight of the pharmaceutical composition.

A preferred pharmaceutical composition of the present invention includes:

Anti-human IL-17A monoclonal antibody 10-200mg/mL Acetate-Acetate Buffer 5-80mM sucrose 20-160g/L PS-80 0.001-0.2wt.% The pH of the pharmaceutical composition is 5.0-7.2 .

Preferably, the pharmaceutical composition includes:

Humanized anti-human IL-17A monoclonal antibody 80-160mg/mL Acetate-Acetate Buffer 5-40mM sucrose 40-120g/L PS-80 0.001-0.1wt.% The pH of the pharmaceutical composition is 5.0-6.5 .

Preferably, the pharmaceutical composition includes:

Humanized anti-human IL-17A monoclonal antibody 80-120mg/mL Acetate-Acetate Buffer 10-30mM sucrose 60-100g/L PS-80 0.005-0.08wt.%. The pH of the pharmaceutical composition is 5.3-6.0 .

In another preferred embodiment of the present invention, in the pharmaceutical composition, the total weight content (wt.%) of each component is 100%.

Reagent test kit

The present invention also provides a kit containing the pharmaceutical composition of the present invention and a container for containing the pharmaceutical composition.

use

The present invention also provides a use of the pharmaceutical composition and/or kit of the present invention to prepare (i) prevent and/or treat IL-17A-mediated diseases; and/or (ii) prevent And/or drugs to treat autoimmune diseases.

In another preferred embodiment, the IL-17A-mediated disease is an autoimmune disease.

In another preferred example, the autoimmune disease is selected from the group consisting of psoriasis, arthritis (preferably rheumatoid arthritis), multiple sclerosis, compulsive spondylitis, inflammatory bowel disease, Systemic lupus erythematosus, type I diabetes, or a combination thereof.

The present invention also provides a method for preventing and/or treating IL-17A-mediated diseases, the method comprising: administering the pharmaceutical composition according to the present invention to a desired subject.

In another preferred embodiment, the subject is a human or non-human mammal.

In the present invention, the term "prevention" means a method of preventing the onset of a disease and/or its accompanying symptoms or protecting a subject from acquiring the disease. As used herein, "prevention" also includes delaying the onset of the disease and/or its accompanying symptoms and reducing the risk of a subject's disease.

In the present invention, the term "treatment" refers to any treatment of a disease in a mammal, including (but not limited to): (a) inhibiting the disease, that is, slowing down or preventing the development of clinical symptoms; and/or ( b) Alleviate the disease, that is, cause the regression of clinical symptoms, and/or (c) reduce or eliminate the disease and/or its accompanying symptoms.

Lyophilized preparation

The present invention also provides a freeze-dried preparation, which is prepared by the following method: freeze-drying the pharmaceutical composition of the present invention to obtain a freeze-dried preparation.

In another preferred embodiment, the freeze-dried preparation further includes a freeze-dried protective agent.

In another preferred embodiment, the freeze-drying protective agent is selected from the following group: glucose, mannitol, fructose, galactose, or a combination thereof.

The main advantages of the present invention include:

1. The present invention provides a pharmaceutical composition containing an anti-human IL-17A monoclonal antibody (for example, a humanized anti-human IL-17A monoclonal antibody). The pharmaceutical composition can significantly enhance the anti-human IL-17A monoclonal antibody. The stability of the cloned antibody can maintain its stability under pressure (high temperature, freezing and thawing, shaking, etc.), acceleration, and long-term refrigeration.

2. The pharmaceutical composition of the present invention containing anti-human IL-17A monoclonal antibodies (for example, humanized anti-human IL-17A monoclonal antibodies) can enhance high concentrations (greater than 100 mg/ml) of anti-human IL-17A monoclonal antibodies The stability.

3. The pharmaceutical composition of the present invention containing an anti-human IL-17A monoclonal antibody (for example, a humanized anti-human IL-17A monoclonal antibody) can enhance the thermodynamic and chemical stability of the antibody preparation, so that the antibody can be used in a novel preparation Stable storage, while improving the quality of the product, extend the shelf life of the product, and improve the safety of clinical practice.

The present invention will be further explained below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples usually follow conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to manufacturing The conditions suggested by the manufacturer. Unless otherwise specified, percentages and parts are percentages by weight and parts by weight.

Unless otherwise defined, the professional and scientific terms used in the text have the same meaning as those familiar to those skilled in the art. In addition, any method or material similar or equal to the recorded content can be applied to the invention literature. The preferred implementation methods and materials described in this article are for demonstration purposes only.

Example

General measurement method

Protein BEH SEC分析柱(4.6mm×150mm)，通过Waters超高效液相色谱仪器(Acquity H Class)，参照《中华人民共和国药典》(2015版，三部)0514通则高效液相色谱法进行测定，以面积归一法计算纯度。 SEC-UPLC: Use ACQUITY

Protein BEH SEC analytical column (4.6mm×150mm), measured by Waters Ultra-High Performance Liquid Chromatography (Acquity H Class), with reference to "Pharmaceuticals of the People's Republic of China" (2015 Edition, Part Three) 0514 General Principles High Performance Liquid Chromatography. Calculate the purity by the area normalization method.

CEX-HPLC: Use ProPac ^TM WCX-10 analytical column (4mm×250mm), pass Water high performance liquid chromatography instrument (E2695), refer to "People's Republic of China Pharmacopoeia" (2015 edition, three parts) general rules high performance liquid chromatography Measure and calculate the purity by area normalization method.

Tm value determination (IF, full spectrum fluorescence): Use Uncle (Unchained Labs), heating at 0.3°C/min in the range of 25°C to 95°C, and use the full spectrum fluorescence module to calculate the dissolution temperature Tm of different prescriptions.

Average particle size distribution measurement (DLS, dynamic light scattering): Use the dynamic light scattering module of Uncle instrument to measure the average particle size (Z-average) at 25°C for different prescriptions.

Tagg value determination (SLS, static light scattering): Use the Uncle instrument at 25°C to 95°C, heating at 0.3°C/min, and use the static light scattering module to calculate the initial aggregation temperature Tagg of antibody molecules of different prescriptions.

Binding specific activity: the test substance (monoclonal antibody) of different concentrations is combined with the recombinant human interleukin 17A (rh IL-17A) coated on the surface of the well of the ELISA plate, and the goat is labeled with horseradish peroxidase (HRP) Anti-human IgG-Fc antibody is used to determine the amount of bound monoclonal antibody, a sigmoid curve is obtained by four-parameter fitting, and the half maximum effect concentration (EC50) is calculated. Finally, by comparing the EC50 values of the test substance and the reference substance, the relative specific activity (%) is obtained.

Biological specific activity: After mixing different concentrations of the test substance (monoclonal antibody) with a certain amount of recombinant human interleukin 17A (rh IL-17A) and recombinant human tumor necrosis factor alpha (rh TNF-α), add human HT -1080 cells (ATCC) were incubated for 1 day, the IL-6 expression level was measured by (Enzyme-linked immunosorbent assay) ELISA method (Biolegend), the S-shaped curve was obtained by four-parameter fitting, and the half inhibitory concentration (IC50) was calculated. Finally, by comparing the IC50 values of the test substance and the reference substance, the relative specific activity (%) is obtained.

Example 1 Comparison of the stability of different buffer systems

Use WaterSep hollow fiber (Discover 12) to replace the humanized anti-human IL-17A monoclonal antibody (SEQ ID NO.: 7 and SEQ ID NO.: 8) to each screening buffer containing 15 mM (as shown in Table 1 below) , And adjust the antibody concentration to about 5mg/mL with respective buffers, sterilize and filter for later use.

The newly prepared samples were evaluated for the stability of each buffer by Tm, Tagg and DLS. The results are shown in Table 1. At the same time, put the newly prepared sample under various pressure conditions, including: high temperature test (40℃±2℃, placed for three weeks), repeated freezing and thawing (5 cycles, ≤-70℃/5℃±3℃). The stability was evaluated by testing items such as appearance and purity. The results are shown in Table 2.

Table 1 Summary of Tm, Tagg and DLS results of different buffers

Table 2 Summary of appearance and purity results of different buffer systems under various pressure conditions

As can be seen from Table 1, there is little difference in Tm, Tagg and average hydraulic radius of each buffer system.

It can be seen from Table 2 that under freezing and thawing pressure conditions, the citrate-citric acid and phosphoric acid-citrate buffer systems are obviously turbid in appearance and even precipitate, and there is no significant difference in purity between the groups; Under high temperature and pressure conditions, there is no significant difference in appearance and purity of each buffer system group.

In addition, it can be seen from Table 1 and Table 2 that the acetate-acetic acid buffer (pH 5.5) has excellent performance in various stability parameters and pressurization conditions. The subsequent screening of other excipients is based on this Developed on the basis of a buffer system.

Example 2 Comparison of the stability of different stabilizers

Use WaterSep hollow fiber (Discover 12) to replace the humanized anti-human IL-17A monoclonal antibody (SEQ ID NO.: 7 and SEQ ID NO.: 8) into a 20mM acetate-acetic acid (pH5.5) buffer system Add the high-concentration mother liquor of each stabilizer to prepare each stabilizer screening group (as shown in Table 3), adjust the antibody concentration to about 2 mg/mL, and sterilize and filter for use.

Put the newly prepared sample under various pressure conditions, including: high temperature test (40℃±2℃, placed for three weeks), repeated freezing and thawing (5 cycles, ≤-70℃/5℃±3℃). The stability was evaluated through the appearance and purity test items. Some test results are shown in Table 3 below.

Table 3 Summary of appearance and purity results under different stabilizer systems under various pressure conditions

It can be seen from Table 3 that under freezing and thawing pressure, except for the sodium chloride group whose SEC purity is slightly lower than the other groups, there is no significant difference in the other groups; under high temperature pressure, the sodium chloride group appears turbid, which may be Protein aggregation is related, the proportion of the main peak of CEX in each group is reduced by about 10%, but there is no significant difference between the groups, only the purity of the sorbitol group is slightly lower.

In summary, sucrose will be selected as a stabilizer in the follow-up to carry out surfactant screening experiments.

Example 3 Surfactant Screening

Use WaterSep hollow fiber (Discover 12) to replace the humanized anti-human IL-17A monoclonal antibody SEQ ID NO.: 7 and SEQ ID 8) with 20mM acetate-acetic acid (pH5.7) 8wt.% sucrose buffer. In the system, add the high-concentration mother liquor of each surfactant to prepare each surfactant screening group (as shown in Table 4), adjust the antibody concentration to about 100 mg/mL, and sterilize and filter for later use.

Place the freshly prepared samples under each shaking and pressurization conditions, and turn over and shake at room temperature for 3 days (60rpm). The surfactants Tween-20 (PS-20) and Tween-80 (PS-80) are evaluated by the number of insoluble particles. For the protection effect, some test results are shown in the table below.

Table 4 Summary of the number of particles of different surfactants under shock pressure conditions

It can be seen from Table 4 that under the shock pressure, compared with the non-surfactant group, the addition of PS-20 and PS-80 can significantly inhibit the increase in the number of insoluble particles caused by the shock. Further analysis found that the number of insoluble particles decreased with the increase of PS-80 addition, and there was a certain concentration dependence, especially in the number of particles larger than 25μm.

Example 4 Humanized anti-human IL-17A monoclonal antibody pharmaceutical preparation

The humanized anti-human IL-17A monoclonal antibody pharmaceutical preparation was prepared according to the following prescription (as shown in Table 5), and the humanized anti-human IL-17A monoclonal antibody is shown in SEQ ID NO.: 7 and SEQ ID 8:

Table 5 Liquid formulation prescription

Humanized anti-human IL-17A monoclonal antibody 100mg/mL Sodium acetate + acetate buffer 20mM sucrose 80g/L PS-80 0.01wt.% pH 5.7 quantity 1mL

Remarks: The solvent of sodium acetate + acetate buffer is sterile water for injection.

Preparation:

First, replace the humanized anti-human IL-17A monoclonal antibody (SEQ ID NO.: 7 and SEQ ID NO.: 8) with sucrose-containing sodium acetate-acetic acid buffer with Merk’s 30KD ultrafiltration membrane package (PLCTK) Add Tween 80 (PS-80), adjust the antibody concentration to about 100 mg/mL with sodium acetate-acetic acid buffer, and aseptically dispense into prefilled syringes (1.0 mL/piece) to obtain the final liquid preparation. The pH of the obtained liquid preparation was measured to be 5.7.

Example 5

Example 5 investigates the stability of the humanized anti-human IL-17A monoclonal antibody pharmaceutical preparation prepared in Example 4. The stability investigation includes pressurization, acceleration, and long-term stability investigation, and the details are as follows:

5.1 Stability test under pressure

Pressure conditions include: high temperature for 1 month (40℃±2℃), room temperature shaking for 3 days (60rpm), repeated freezing and thawing (5 cycles, ≤-70℃/5℃±3℃ freezing and thawing), etc.

Prepare samples of humanized anti-human IL-17A monoclonal antibody pharmaceutical preparations according to Example 4, store them under various pressure conditions (as shown in Table 6), take samples and submit them for inspection after set time points or treatment times, and pass the appearance , The number and purity of insoluble particles are evaluated for their stability. Some test results are shown in Table 6 below.

Table 6 Summary of stability data of humanized anti-human IL-17A monoclonal antibody pharmaceutical preparations under various pressurized conditions

Remarks: T0 control is the control when the preparation is completed.

It can be seen from Table 6 that the impact items and degrees of various pressure conditions on product quality are different. Freeze-thaw and shock pressure mainly cause an increase in the number of large particles (≥25μm) in insoluble particles, while high-temperature pressure will reduce the purity of the product. The purity of SEC decreases by about 5%, and the proportion of the main peak of CEX significantly decreases by about 15%. In a word, the pharmaceutical composition of the present invention has high stability, can effectively protect the main drug component monoclonal antibody, and thus effectively resist the destructive effects of various pressure conditions.

5.2 Accelerated stability test

Prepare humanized anti-human IL-17A monoclonal antibody pharmaceutical preparation samples according to Example 4, store them under accelerated conditions (25°C±2°C), take samples at the set time point and submit them for testing, and pass insoluble particles, purity and activity Evaluate its stability, and some test results are shown in Table 7 below.

Table 7 Summary of stability data of humanized anti-human IL-17A monoclonal antibody pharmaceutical preparations under accelerated conditions

Remarks: T0 control is the control when the preparation is completed.

It can be seen from Table 7 that accelerated conditions will cause a certain degree of decline in the quality of pharmaceutical preparations. During the entire accelerated test cycle, there were no significant changes in insoluble particles, binding activity, and biological activity. Compared with the T0 sample, the SEC molecular sieve purity decreased by about 2% for the sample accelerated for 3 months, and the main peak content of CEX decreased by about 10%, but still met the quality standard; the sample accelerated for 6 months, the purity of the SEC molecular sieve decreased by about 2%, and the main peak of CEX The content drops by about 15%. In short, the pharmaceutical composition has high stability and can be stored stably for at least 3 months under accelerated conditions at 25°C.

5.3 Long-term stability test

Prepare humanized anti-human IL-17A monoclonal antibody pharmaceutical preparation samples according to Example 4, store them under long-term refrigeration conditions (5°C±3°C), take samples at a set time point and submit them for inspection, and pass insoluble particles, purity, and The stability is evaluated by activity, and some of the test results are shown in Table 8 below.

Table 8 Summary of stability data of humanized anti-human IL-17A monoclonal antibody pharmaceutical preparations under long-term refrigeration conditions

Remarks: T0 control is the control when the preparation is completed.

It can be seen from Table 8 that under long-term refrigeration conditions (5°C±3°C), the stability of the drug formulation is very good, and the half-year samples have no significant changes in insoluble particles, binding activity and biological activity, only the main peak content of CEX A drop of about 2% is far above the quality standard. In short, the pharmaceutical composition has high stability and can be stored stably under long-term refrigeration conditions.

In summary, it can be seen that the humanized anti-human IL-17A monoclonal antibody pharmaceutical preparation prepared in Example 4 has good stability, and the humanized anti-human IL-17A monoclonal antibody under various pressure conditions (high temperature, repeated It maintains good stability under freezing and thawing and shaking). It can be stored stably for at least three months under accelerated conditions (25℃±2℃). At the same time, it exhibits high stability under long-term refrigeration conditions and can be stable. store.

All documents mentioned in the present invention are cited as references in this application, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (15)

A pharmaceutical composition, characterized in that the pharmaceutical composition comprises: (a) Anti-human IL-17A monoclonal antibody; and (b) A pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier includes a buffer, a stabilizer and a surfactant. The pharmaceutical composition of claim 1, wherein the concentration of the anti-human IL-17A monoclonal antibody is 10-300 mg/mL, preferably 10-200 mg/mL, more preferably 30-180 mg /mL, more preferably 50-180mg/mL, more preferably 50-160mg/mL, more preferably 80-160mg/mL, most preferably 80-120mg/mL. The pharmaceutical composition of claim 1, wherein the buffer is selected from the group consisting of acetate-acetate buffer, phosphate buffer, histidine-acetate buffer, three Hydroxymethylaminomethane (Tris)-hydrochloride buffer, or a combination thereof. The pharmaceutical composition of claim 1, wherein the concentration of the buffer is 5-100 mM, preferably 5-80 mM, more preferably 5-60 mM, more preferably 5-40 mM, more preferably 5-30mM, more preferably 10-30mM, most preferably 15-25mM. The pharmaceutical composition of claim 1, wherein the stabilizer is selected from the group consisting of sodium chloride, amino acids, sugar alcohols, or combinations thereof. The pharmaceutical composition of claim 1, wherein the content of the stabilizer is 0.1-20wt.%, 2-20wt.%, preferably 2-16wt.%, more preferably 2-14wt.% %, more preferably 4-14 wt.%, more preferably 5-13 wt.%, most preferably 6-10 wt.%, based on the total weight of the pharmaceutical composition. The pharmaceutical composition of claim 1, wherein the surfactant is selected from the group consisting of polyoxyethylene sorbitan fatty acid ester, polyoxyethylene hydrogenated castor oil, glycerin fatty acid ester, polysorbate Alcohol ester, poloxamer, or a combination thereof. The pharmaceutical composition of claim 1, wherein the content of the surfactant is 0.001-0.2wt.%, preferably 0.001-0.1wt.%, more preferably 0.005-0.1wt.%, More preferably 0.005-0.08 wt.%, most preferably 0.008-0.05 wt.%, based on the total weight of the pharmaceutical composition. The pharmaceutical composition according to claim 1, wherein the pH range of the pharmaceutical composition is 5.0-7.2, preferably 5.0-6.5, more preferably 5.3-6.0. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition comprises: Anti-human IL-17A monoclonal antibody 10-200mg/mL Acetate-Acetate Buffer 5-80mM sucrose 20-160g/L PS-80 0.001-0.2wt.% The pH of the pharmaceutical composition is 5.0-7.2 . The pharmaceutical composition of claim 1, wherein the pharmaceutical composition comprises: Humanized anti-human IL-17A monoclonal antibody 80-120mg/mL Acetate-Acetate Buffer 10-30mM sucrose 60-100g/L PS-80 0.005-0.08wt.% The pH of the pharmaceutical composition is 5.3-6.0 . The pharmaceutical composition of claim 1, wherein the anti-human IL-17A monoclonal antibody preferably has the heavy chain variable region amino acid sequence shown in SEQ ID NO: 7 and SEQ ID NO: 8 The amino acid sequence of the variable region of the light chain. The pharmaceutical composition of claim 5, wherein the sugar alcohol is selected from the group consisting of sucrose, mannitol, trehalose, maltose, sorbitol, or a combination thereof. The pharmaceutical composition of claim 7, wherein the polysorbate is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80 . The antibody pharmaceutical composition according to claim 1 and/or the use of the kit according to claim, characterized in that it is used to prepare (i) drugs for preventing and/or treating IL-17A-mediated diseases; and /Or (ii) drugs for the prevention and/or treatment of autoimmune diseases.