CN117838854A

CN117838854A - Stabilizer composition of antibody medicine and pharmaceutical composition

Info

Publication number: CN117838854A
Application number: CN202311817823.9A
Authority: CN
Inventors: 钱慈; 张贝贝; 郑旖楠; 李璐遥; 汪梦雯; 高栋; 王海彬
Original assignee: Zhejiang Borui Biopharmaceutical Co ltd; Haizheng Biopharmaceutical Co ltd
Current assignee: Zhejiang Borui Biopharmaceutical Co ltd; Haizheng Biopharmaceutical Co ltd
Priority date: 2022-12-30
Filing date: 2023-12-27
Publication date: 2024-04-09

Abstract

The invention discloses a stabilizer composition of an antibody drug and a pharmaceutical composition. The stabilizer composition of the antibody medicine contains a negatively charged buffer agent and a positively charged protein protecting agent. The pharmaceutical composition comprises: antibody class medicine and the stabilizer composition. The stabilizer composition is favorable for better stability of the antibody medicine in the liquid preparation, and ensures that the liquid preparation has lower viscosity under the condition of higher concentration of the antibody medicine.

Description

Stabilizer composition of antibody medicine and pharmaceutical composition

Technical Field

The invention belongs to the technical field of antibodies, and particularly relates to a stabilizer composition of an antibody drug and a pharmaceutical composition.

Background

For antibody-based drugs, the stability of their ready-to-use pharmaceutical products or liquid pharmaceutical compositions can be challenging. Antibodies, which are immunoglobulins in nature, are a protein. The marginal stability of proteins in liquid compositions often prevents long-term storage at room temperature or under refrigerated conditions. Proteins may undergo various physical and chemical reactions in solution (aggregation [ covalent and non-covalent ], deamidation, oxidation, shearing, isomerization, denaturation), leading to increased levels of degradation products and/or loss of biological activity. The safety and effectiveness of the drug are adversely affected by the by-products produced, and the development of a formulation recipe excellent in stability is very important for clinical medication.

Commercial ready-to-use liquid antibody compositions should provide sufficient physical and chemical stability of the antibody during transport and handling to ensure compliance with dosage and product safety requirements when the molecule is administered to a patient. Acceptable liquid antibody compositions must be stable and minimize protein degradation, particularly protein aggregation, to avoid serious immunogenic reactions. The composition must also have an acceptable osmotic pressure and pH for subcutaneous application and have low viscosity and injectability as a prerequisite for preparation (compounding, filtration, filling). Balancing this number of requirements is difficult, making the production of commercially viable aqueous biopharmaceutical compositions a technical challenge.

The Studies monoclonal antibody is a fully humanized monoclonal anti-IL-17A inflammatory cytokine IgG1 antibody, and through combination with IL-17A, the interaction between IL-17A and a receptor thereof is directly blocked, so that immune and inflammatory reactions are changed to achieve the treatment purpose. (active ingredient: secukinumab, studimumab) is the first global heavy-duty biological drug targeting IL-17A developed by North Corp. The stabilizer is preferably trehalose (CN 201580076632.9). Patent CN201910412754.0 prefers xylitol and sorbitol as stabilizers. Patent CN201910935046.5 prefers sucrose as a stabilizer. Patent CN201911239277.9 uses citrate buffer, sucrose, arginine, polysorbate 80 as a stabilizer composition.

Disclosure of Invention

In order to solve the above problems in the prior art, the present invention provides a stabilizer composition for antibody drugs, which comprises a negatively charged buffer and a positively charged protein protectant.

In some embodiments, the negatively charged buffer is selected from one or more of citric acid, acetic acid, phosphoric acid, succinic acid, aspartic acid, glutamic acid, lactic acid; the positively charged protein protectant is selected from one or more of arginine, lysine and histidine.

In some embodiments, the concentration of the negatively charged buffer is 5 to 100mM, preferably 5 to 50mM, more preferably 5 to 35mM, and most preferably 5 to 25mM (such as 10mM or 20 mM); the concentration of the positively charged protein protecting agent is 50-270mM, preferably 80-240mM, more preferably 100-220mM, most preferably 130-190mM (such as 150mM or 175 mM); the antibody medicine is selected from one or more of anti-human IL-17A monoclonal antibody, anti-human HER2 monoclonal antibody and anti-human TNF-alpha monoclonal antibody.

In another aspect, the invention discloses an antibody pharmaceutical composition comprising a negatively charged buffer, a positively charged protein protectant, and an antibody, wherein:

the negatively charged buffer is an acid substantially free of its salt form, the acid being selected from one or more of citric acid, acetic acid, phosphoric acid, succinic acid, aspartic acid, glutamic acid and lactic acid;

the positively charged protein protectant is selected from one or more of arginine, lysine and histidine.

In some embodiments, the acid is citric acid.

In some embodiments, the positively charged protein protectant is arginine.

In some embodiments, the concentration of the acid is 5mM-50mM, preferably 5mM-35mM, more preferably 10mM-20mM; for example, 5mM-15mM, 5mM-10mM, 10mM-15mM, 8mM-12mM; in some embodiments, the concentration of the acid is about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 11mM, about 12mM, about 13mM, about 14mM, about 15mM, about 18mM, about 20mM, or any value in between; more preferably 5mM, 10mM or 20mM.

In some embodiments, the concentration of the positively charged protein protectant is 100mM-220mM, preferably 150mM-200mM, more preferably 150mM-170mM; for example, 100mM-200mM, 120mM-200mM, 140mM-180mM, 170mM-180mM; in some embodiments, the concentration of the positively charged protein protecting agent is about 150mM, about 160mM, about 170mM, about 180mM, about 190mM, about 140mM, about 145mM, about 165mM, about 175mM, or any value in between any two values; more preferably 150mM or 170mM.

In some embodiments, the antibody class drug is selected from one or more of an anti-human IL-17A monoclonal antibody, an anti-human HER2 monoclonal antibody, an anti-human TNF-a monoclonal antibody. In some specific embodiments, the antibody is an anti-human IL-17A monoclonal antibody comprising an amino acid sequence as set forth in SEQ ID NO:1 and a heavy chain as set forth in SEQ ID NO:2, and a light chain as shown in FIG. 2. In some specific embodiments, the antibody is secukinumab. In some specific embodiments, the antibody is an anti-human HER2 monoclonal antibody comprising the amino acid sequence as set forth in SEQ ID NO:3 and a heavy chain as set forth in SEQ ID NO: 4.

In some embodiments, the concentration of the antibody drug is 10-300mg/mL, preferably 10-250mg/mL, more preferably 30-250mg/mL, and most preferably 135-165mg/mL.

In some embodiments, the concentration of the antibody is 50mg/mL-200mg/mL, preferably 50mg/mL-60mg/mL or 90mg/mL-160mg/mL, more preferably 100mg/mL-150mg/mL. Such as 90mg/mL to 180mg/mL, 90mg/mL to 170mg/mL, 90mg/mL to 155mg/mL, 95mg/mL to 155mg/mL; in some embodiments, the concentration of the antibody is about 90mg/mL, about 95mg/mL, about 100mg/mL, about 140mg/mL, about 1450mg/mL, about 150mg/mL, about 160mg/mL, about 155mg/mL, or any value therebetween. Preferably 100mg/mL or 150mg/mL.

In some embodiments, the aforementioned antibody pharmaceutical compositions further comprise a surfactant.

In some embodiments, the surfactant is selected from polysorbate 20, polysorbate 80, and poloxamer 188. In some embodiments, the surfactant is at a concentration of 0.1mg/mL to 0.6mg/mL, e.g., 0.1mg/mL to 0.6mg/mL, 0.15mg/mL to 0.55mg/mL, 0.2mg/mL to 0.5mg/mL. Preferably 0.2mg/mL.

In some embodiments, the aforementioned antibody pharmaceutical composition further comprises an antioxidant.

In some embodiments, the antioxidant is selected from methionine, glutathione, cysteine, cystine, or ascorbic acid. In some embodiments, the antioxidant is at a concentration of 20mM-30mM, e.g., 20mM-28mM, 22mM-26mM; preferably 25mM.

In some embodiments, the pH of the aforementioned antibody pharmaceutical composition is from 5.6 to 6.0, e.g., from 5.7 to 6.0, from 5.8 to 6.0, from 5.6 to 5.9; in some specific embodiments, the pH of the antibody pharmaceutical composition is about 5.6, about 5.7, about 5.8, about 5.9, about 6.0; preferably 5.8.

In some embodiments, the aforementioned antibody pharmaceutical composition comprises:

a) 50mg/mL-200mg/mL of antibody;

b) 5mM-50mM acetic acid;

c) Arginine at 100mM-220 mM; and

d) 0.1mg/mL-0.6mg/mL polysorbate 80; wherein the pH of the antibody pharmaceutical composition is 5.6-6.0.

a) 50mg/mL-200mg/mL of antibody;

b) 5mM-50mM citric acid;

c) Arginine at 100mM-220 mM;

d) 0.1mg/mL-0.6mg/mL polysorbate 20; and

e) Methionine at 20mM-30 mM; wherein the pH of the antibody pharmaceutical composition is 5.6-6.0.

a) 90mg/mL-160mg/mL of antibody;

b) 5mM-35mM citric acid;

c) 150mM-200mM arginine;

d) 0.1mg/mL-0.6mg/mL polysorbate 20; and

a) 150mg/mL of antibody;

b) 10mM citric acid;

c) 150mM-170mM arginine;

d) 0.2mg/mL polysorbate 20; and

e) 25mM methionine; and the pH of the antibody pharmaceutical composition is 5.6-6.0.

a) 90mg/mL-160mg/mL of antibody;

b) 5mM-35mM citric acid;

c) 150mM-200mM arginine;

d) 0.1mg/mL-0.6mg/mL polysorbate 20; and

e) Methionine at 20mM-30 mM; wherein the pH of the antibody pharmaceutical composition is 5.6-6.0, and the antibody is an anti-human IL-17A monoclonal antibody, which comprises the amino acid sequence shown in SEQ ID NO:1 and a heavy chain as set forth in SEQ ID NO:2, and a light chain as shown in FIG. 2.

a) 150mg/mL of the antibody, and,

b) 10mM citric acid;

c) 150mM-170mM arginine;

d) 0.2mg/mL polysorbate 20; and

e) 25mM methionine; and the pH of the antibody pharmaceutical composition is 5.6-6.0, wherein the antibody is an anti-human IL-17A monoclonal antibody comprising the amino acid sequence as shown in SEQ ID NO:1 and a heavy chain as set forth in SEQ ID NO:2, and a light chain as shown in FIG. 2.

a) 150mg/mL of the antibody, and,

b) 10mM citric acid;

c) 150mM arginine;

d) 0.2mg/mL polysorbate 20; and

a) 150mg/mL of the antibody, and,

b) 10mM citric acid;

c) 170mM arginine;

d) 0.2mg/mL polysorbate 20; and

a) 50mg/mL-60mg/mL of antibody; wherein the antibody is an anti-human Her2 monoclonal antibody comprising the amino acid sequence as set forth in SEQ ID NO:3 and a heavy chain as set forth in SEQ ID NO:4, and a light chain represented by the formula 4

b) 10mM-20mM acetic acid;

c) 170mM-180mM arginine; and

d) 0.15mg/mL-0.55mg/mL polysorbate 80; wherein the pH of the antibody pharmaceutical composition is 5.6-6.0.

a) 50mg/mL of antibody; wherein the antibody is an anti-human Her2 monoclonal antibody comprising the amino acid sequence as set forth in SEQ ID NO:3 and a heavy chain as set forth in SEQ ID NO:4, and a light chain represented by the formula 4

b) 20mM acetic acid;

c) 175M arginine; and

d) 0.2mg/mL polysorbate 80; wherein the pH of the antibody pharmaceutical composition is 5.6-6.0.

In some embodiments, the aforementioned antibody drug combination has an osmolality ranging from 285 to 310mOsmol/kg and a viscosity of less than 7.2cp. Preferably, the viscosity is 2.5 to 6.9cP, 2.9 to 6.7cP, or 1.65 to 6.1cP. In some embodiments, the viscosity is 2.9cp, 6.1cp, or 6.7cp.

The osmotic pressure molar concentration range of normal human blood is 285-310 mOsmol/kg, the liquid preparation provides a medicine composition which is isotonic with human body, has better stability and lower viscosity, and is favorable for the administration of the liquid preparation by subcutaneous injection.

In some embodiments, the antibody pharmaceutical compositions of the invention are liquid formulations.

In another aspect, the invention provides a lyophilized formulation obtained by freeze-drying an antibody pharmaceutical composition according to any one of the preceding claims.

In a further aspect, the invention provides the use of an antibody pharmaceutical composition as defined in any preceding claim or a lyophilized formulation as defined above in the manufacture of a medicament for the treatment of a disease associated with IL-17A and/or IL-17F.

In some embodiments, the disease is selected from psoriasis, psoriatic arthritis, ankylosing spondylitis, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, osteoarthritis, and inflammatory bowel disease.

In the antibody pharmaceutical composition of the invention, negatively charged citric acid is combined with positively charged arginine (which is a basic amino acid, also known as an amino acid base) to maintain the pH of the liquid formulation.

In another aspect, the invention provides a method of preparing an antibody pharmaceutical composition of any of the preceding claims, the method comprising the step of replacing an antibody solution with a solution comprising a negatively charged buffer and a positively charged protein protectant, wherein:

the negatively charged buffer is an acid substantially free of its salt form, the acid being selected from one or more of citric acid, acetic acid, phosphoric acid, succinic acid, aspartic acid, glutamic acid and lactic acid; the positively charged protein protectant is selected from one or more of arginine, lysine and histidine.

In some embodiments, the invention provides a method of preparing an antibody pharmaceutical composition of any of the preceding claims, the method comprising the step of replacing the antibody solution with a solution comprising citric acid and arginine, or a solution of acetic acid and arginine.

In some embodiments, the method replaces the antibody solution with a solution comprising 5mM-50mM citric acid and 5mM-40mM arginine.

In some embodiments, the method replaces the antibody solution with a solution comprising 5mM-15mM citric acid and 9mM-30mM arginine.

In some embodiments, the method replaces the antibody solution with a solution comprising 5mM-15mM citric acid and 12mM-30mM arginine.

In some embodiments, the method replaces the antibody solution with a solution comprising 5mM-10mM citric acid and 12mM-24mM arginine.

In some embodiments, the method replaces the antibody solution with a solution comprising 10mM citric acid and 24mM arginine.

In some embodiments, the method replaces the antibody solution with a solution comprising 10mM acetic acid and 9.2mM arginine.

The present invention provides a method for preparing the antibody pharmaceutical composition of any one of the preceding claims, wherein the ultrafiltration step does not require additional adjustment of the pH of the antibody solution. In the liquid ultrafiltration liquid exchange process, the combination of citric acid and arginine is used as a replacement liquid. The liquid preparation has more excellent stability under the same osmotic pressure and lower viscosity.

The invention can provide more excellent antibody drug stability under the condition of the same osmotic pressure, and the viscosity of the liquid preparation under the condition of higher antibody concentration is lower.

In the invention, in the preparation with the same osmotic pressure in the preparation prescription development process, the combination of the acid without salt and the stabilizer with single positive charge is used, the usage amount of the stabilizer is more than that of other buffers (such as citric acid-sodium citrate buffer) containing salt, and the stabilizer with more usage amount is favorable for stabilizing the antibody and reducing the viscosity. Mechanically, the method comprises the following steps: the charged stabilizer has larger ionic strength, and is combined to the surface of the protein, so that electrostatic force repulsive force between the proteins is reduced or hydrophobic acting force between the proteins is reduced, and the purposes of stabilizing the antibody and reducing the viscosity are achieved.

The conception, specific structure, and technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, features, and effects of the present invention.

Drawings

Fig. 1 is a superposition of polymer peaks in SEC spectra of formulation 1 and formulation 2 at an elevated temperature of 14 days.

Fig. 2 is a superposition of IEC maps for formulation 1 and formulation 2 at high temperature for 7 days.

Fig. 3 is a superposition of RP spectra of formulation 1 and formulation 2 at high temperature for 14 days.

Detailed Description

The term "comprising" encompasses both the meaning of "comprising" and "consisting of … …", e.g. a composition "comprising" X may consist of X alone, as well as some other ingredients, e.g. x+y.

"composition" means a mixture comprising one or more of the compounds described herein or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to organisms, facilitate the absorption of active ingredients and thus exert biological activity. As used herein, "pharmaceutical composition," "formulation," and "prescription" are used interchangeably. The pharmaceutical compositions described herein are in the form of solutions, wherein the solvent is water unless otherwise specified.

"buffering agent" generally refers to a substance that is resistant to pH changes by the action of its acid-base conjugate components, and in the present invention, the buffering agent is an acid that is substantially free of its salt form, which interacts with a positively charged protein protectant to act as a buffer. In the present invention, a positively charged protein protectant (an amino acid base such as arginine) is combined with an acid that is substantially free of its salt form to maintain pH.

By "substantially free of an acid in its salt form" is meant that the acid, which acts as a buffer in the liquid pharmaceutical composition, is present in the absence of any salt form thereof. Acid-containing buffers commonly used in liquid pharmaceutical compositions are prepared with salts of the acids or a combination of the acids and salt forms thereof. Thus, for example, the buffer is prepared with an acid and its counter ion, such as sodium, potassium, ammonium, calcium or magnesium. Such a citric acid buffer typically contains a salt of citric acid, such as citric acid-sodium citrate. Acids suitable for use in formulating the stabilized polypeptide-containing liquid pharmaceutical compositions of the present invention include, but are not limited to, citric acid, acetic acid, phosphoric acid, succinic acid, aspartic acid, glutamic acid, and lactic acid, with citric acid being more preferred.

"positively charged protein protectant" refers to an "amino acid base" that is an amino acid or combination of amino acids, wherein any given amino acid is present in its free base form or in its salt form. Preferred amino acids for use in preparing the compositions of the present invention are amino acids with charged side chains, such as arginine.

Surfactants suitable for use in the compositions disclosed herein include, but are not limited to, nonionic surfactants, ionic surfactants, zwitterionic surfactants, and combinations thereof. Typical surfactants for use in the present invention include, but are not limited to, sorbitan fatty acid esters (e.g., sorbitan monocaprylate, sorbitan monolaurate, sorbitan monopalmitate), sorbitan trioleate, glycerol fatty acid esters (e.g., glycerol monocaprylate, glycerol monomyristate, glycerol monostearate), polyglycerol fatty acid esters (e.g., decaglyceryl monostearate, decaglyceryl distearate, decaglyceryl monolinoleate), polyoxyethylene sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate, polyoxyethylene sorbitan tristearate), polyoxyethylene sorbitol fatty acid esters (e.g., polyoxyethylene sorbitol tetrastearate, polyoxyethylene sorbitol tetraoleate), polyoxyethylene glycerol fatty acid esters (e.g., polyoxyethylene glyceryl monostearate), polyethylene glycol fatty acid esters (e.g., polyethylene glycol distearate), polyoxyethylene alkyl ethers (e.g., polyoxyethylene lauryl ether), polyoxyethylene polyoxypropylene alkyl ethers (e.g., polyoxyethylene polyoxypropylene glycol, polyoxyethylene polyoxypropylene propyl ether, polyoxyethylene polyoxypropylene cetyl ether), polyoxyethylene alkylphenyl ethers (e.g., polyoxyethylene nonylphenyl ether), polyoxyethylene castor oil (e.g., polyoxyethylene castor oil, hydrogenated castor oil), polyoxyethylene beeswax derivatives (e.g., polyoxyethylene sorbitol beeswax), polyoxyethylene lanolin derivatives (e.g., polyoxyethylene lanolin), and polyoxyethylene fatty acid amides (e.g., polyoxyethylene stearic acid amide); C10-C18 alkyl sulfates (e.g., sodium cetyl sulfate, sodium lauryl sulfate, sodium oleyl sulfate), polyoxyethylene C10-C18 alkyl ether sulfates having an average addition of 2 to 4 moles of ethylene oxide units (e.g., sodium polyoxyethylene lauryl sulfate), and C1-C18 alkyl sulfosuccinates (e.g., sodium lauryl sulfosuccinate salt); and natural surfactants such as lecithin, glycerophospholipids, sphingomyelins (e.g., sphingomyelins), and sucrose esters of C12-C18 fatty acids. The composition may comprise one or more of these surfactants. Preferred surfactants are polyoxyethylene sorbitan fatty acid esters, such as polysorbate 20, 40, 60 or 80. Polysorbate 20 (e.g., at a concentration of about 0.2 mg/mL) is particularly suitable.

By "lyophilized formulation" is meant a pharmaceutical composition or a formulation of a solution or a pharmaceutical composition obtained after a vacuum freeze-drying step of the pharmaceutical composition or the formulation of a solution in liquid or solution form.

The term "about" or "approximately" as used herein means that the value is within an acceptable error range for the particular value being determined by one of ordinary skill in the art, the value depending in part on how the measurement or determination is made (i.e., the limits of the measurement system). For example, "about" in each implementation in the art may mean within 1 or exceeding a standard deviation of 1. Alternatively, "about" or "substantially comprising" may mean a range of up to ±20%, e.g., a pH of about 5.5 means a pH of 5.5±1.1. Furthermore, the term may mean at most one order of magnitude or at most 5 times the value, especially for biological systems or processes. Unless otherwise indicated, when a particular value is found in this application and in the claims, the meaning of "about" or "consisting essentially of" should be assumed to be within the acceptable error range for that particular value.

The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of the antibody heavy chain. In some embodiments, the Fc region of a human IgG heavy chain is defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxy terminus. Suitable native sequence Fc regions for antibodies described herein include human IgG1, igG2 (IgG 2A, igG 2B), igG3, and IgG4. The numbering convention for the Fc region is EU index, unless otherwise indicated. The C-terminus of the Fc region may be the complete C-terminus ending with the amino acid residue PGK, or may be a truncated C-terminus, e.g., with one or two C-terminal amino acid residues removed in the truncated C-terminus. Thus, in some embodiments, the composition of an intact antibody may include a population of antibodies that have all K447 residues and/or g446+k447 residues removed. In some embodiments, the composition of intact antibodies may include a population of antibodies that do not remove the K447 residue and/or the g446+k447 residue. In some embodiments, the composition of intact antibodies has an antibody population with and without a mixture of antibodies with the K447 residue and/or the g446+k447 residue. The pharmaceutical composition described herein is capable of achieving a stable effect: wherein the antibody substantially retains its physical and/or chemical stability and/or biological activity after storage, e.g., the pharmaceutical composition substantially retains its physical and chemical stability and its biological activity after storage. The shelf life is generally selected based on the predetermined shelf life of the pharmaceutical composition. There are a number of analytical techniques for measuring protein stability that measure stability after storage at a selected temperature for a selected period of time.

A stable pharmaceutical antibody formulation is one in which no significant change is observed in the following cases: preserving at refrigeration temperature (2-8deg.C) for at least 3 months, at least 6 months, at least 1 year, and up to 2 years. In addition, stable liquid formulations include those that: which exhibits the desired characteristics after a period of time including 1 month, 3 months, 6 months, or 1 month at 40 ℃ of storage at 25 ℃. Typical acceptable criteria for stability are as follows: typically no more than about 10%, e.g., no more than about 5%, of the antibody monomers degrade as measured by SEC-HPLC. The pharmaceutical antibody formulation was colorless, or clear to slightly milky, by visual analysis. The concentration, pH and osmolality of the formulation have a variation of no more than + -10%. It is generally observed that no more than about 10%, such as no more than about 5%, truncations, typically form no more than about 10%, such as no more than about 5%, of aggregates.

An antibody "retains its physical stability" in a pharmaceutical formulation if it does not exhibit a significant increase in aggregation, precipitation and/or denaturation after visual inspection of color and/or clarity, or as measured by UV light scattering, size Exclusion Chromatography (SEC) and Dynamic Light Scattering (DLS). The change in protein conformation can be assessed by fluorescence spectroscopy (which determines the tertiary structure of the protein) and by FTIR spectroscopy (which determines the secondary structure of the protein).

An antibody "retains its chemical stability" in a pharmaceutical formulation if it does not exhibit a significant chemical change. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that often alter the chemical structure of proteins include hydrolysis or truncation (assessed by methods such as size exclusion chromatography and SDS-PAGE), oxidation (assessed by methods such as peptide spectroscopy in combination with mass spectrometry or MALDI/TOF/MS), deamidation (assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide spectroscopy, isoaspartic acid measurement, etc.), and isomerization (assessed by measuring isoaspartic acid content, peptide spectroscopy, etc.).

An antibody "retains its biological activity" in a pharmaceutical formulation if the biological activity of the antibody at a given time is within a predetermined range of biological activities exhibited when the pharmaceutical formulation is prepared. The biological activity of an antibody may be determined, for example, by an antigen binding assay.

In this context, unless otherwise indicated or clearly unsuitable, proportions, including percentages, are by weight.

Herein, "/" means either not contained or not detected.

"administration" and "treatment" when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid refers to the contact of an exogenous drug, therapeutic, diagnostic, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "administration" and "treatment" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell includes contacting a reagent with the cell, and contacting the reagent with a fluid, wherein the fluid is in contact with the cell. "administration" and "treatment" also mean in vitro and ex vivo treatment of, for example, a cell by an agent, diagnosis, binding composition, or by another cell. "treatment" when applied to a human, veterinary or research subject refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

By "treating" is meant administering to a subject an internally or externally used therapeutic agent, such as a composition comprising any of the binding compounds disclosed herein, that has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the subject or population being treated to induce regression of such symptoms or to inhibit the development of such symptoms to any clinically measurable extent. The amount of therapeutic agent (also referred to as a "therapeutically effective amount") effective to alleviate any particular disease symptom can vary depending on a variety of factors, such as the disease state, age, and weight of the subject, and the ability of the drug to produce a desired therapeutic effect in the subject. Whether a disease symptom has been reduced can be assessed by any clinical test method that a physician or other healthcare professional typically uses to assess the severity or progression of the symptom. Although embodiments (e.g., methods of treatment or articles of manufacture) disclosed herein may be ineffective in alleviating each symptom of the target disease, they should alleviate the symptom of the target disease in a statistically significant number of subjects, as determined by any statistical test methods known in the art, such as Student t-test, chi-square test, U-test according to Mann and Whitney, kruskal-Wallis test (H test), jonckheere-Terpstra test, and Wilcoxon test.

An "effective amount" comprises an amount sufficient to ameliorate or prevent a symptom or condition of a medical disorder. An effective amount is also meant to be an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular subject or veterinary subject may vary depending on the following factors: for example, the condition to be treated, the general health of the subject, the route and dosage of administration, and the severity of the side effects. An effective amount may be the maximum dose or regimen that avoids significant side effects or toxic effects.

The invention is further described with reference to the following detailed description in order to make the technical means, the inventive features, the achieved objects and the effects of the invention easy to understand. The present invention is not limited to the following examples.

The detection method used in the embodiment of the application comprises the following steps:

SEC detection:

the instrument is an Agilent 1260 mixed module liquid chromatography system; analytical column TSKgel G3000SW _XL 7.8X100 mm; mobile phase is 0.15mol/L phosphate, ph=6.5; the flow rate is 0.4ml/min; column temperature is 30 ℃; the detection wavelength is 220nm; the running time is equal gradient running for 40min.

IEC detection:

the instrument is an Agilent 1260 mixed module liquid chromatography system; the analytical column is Thermo ProPac ^TM WCX-104X 250mm; mobile phase a was 0.025mol/L sodium dihydrogen phosphate-sodium hydroxide, ph=6.0, mobile phase B was 0.025mol/L sodium dihydrogen phosphate-sodium hydroxide, 0.25mol/L sodium chloride, ph=6.0; the flow rate is 1.0ml/min; the detection wavelength is 220nm; column temperature 25 ℃; the run time was 40min and the run gradient is shown in table 1.

TABLE 1 gradient of mobile phase elution

Time (min)	Mobile phase a (%)	Mobile phase B (%)
			0	90	10
30.0	63	37
			30.1	0	100
32.0	0	100
			32.1	90	10
40.0	90	10

RP detection:

the instrument is an Agilent 1260 mixed module liquid chromatography system; the analytical column was Agilent 300SB-C8, 4.6X105 mm,5 μm; mobile phase a is 0.1% aqueous trifluoroacetic acid and mobile phase B is 0.1% acetonitrile trifluoroacetic acid; the flow rate is 1.0ml/min; the detection wavelength is 280nm; column temperature 80 ℃; the run time was 50min and the run gradient is shown in table 2.

TABLE 2 gradient of mobile phase elution

Time (min)	Mobile phase a (%)	Mobile phase B (%)
			0	75	25
4.0	75	25
			35.0	58	42
37.0	75	25
			40.0	5	95
42.0	75	25
			50.0	75	25

CE detection:

the instrument is an Agilent 7100 capillary electrophoresis apparatus; the effective length of the capillary tube is 220mm, and the total length is 305mm; -10.0kv sample introduction for 40s; operating voltage-15.0 kv; running for 40min; detection wavelength 214nm; column temperature 20 ℃.

The anti-human IL-17A monoclonal antibody used in the invention is Stuzumab, and the sequence of the anti-human IL-17A monoclonal antibody is as follows:

heavy chain of secukinumab (SEQ ID NO: 1):

light chain of Stuzumab (SEQ ID NO: 2):

example 1 formulation recipe and preparation

1.1. Preparation of formulation 1

Formulation 1 was a stock solution of Studies consisting of 150mg/ml anti-human IL-17A monoclonal antibody, 20mM histidine-HCl, 200mM trehalose, 5mM methionine, 0.2mg/ml polysorbate 80, pH5.8. The solvent was water and the pH was adjusted with hydrochloric acid.

The preparation process comprises the following steps: the purified anti-human IL-17A monoclonal antibody is ultrafiltered and concentrated to 50-80 mg/ml, and a 20mM histidine-histidine hydrochloride buffer system is used for ultrafiltering and replacing 6-15 times of volume, and then ultrafiltering and concentrating is carried out to 175-215 mg/ml to obtain concentrated solution. Adding the prepared first buffer solution (which needs to be confirmed by calculation) for dilution into the concentrated solution, uniformly mixing and filtering to obtain the anti-human IL-17A monoclonal antibody protein stock solution with the concentration of 150mg/ml, and then filling to obtain the preparation 1.

The 20mM histidine-histidine hydrochloride buffer system was 20mM histidine and the pH was adjusted to 5.6 with hydrochloric acid.

The first buffer for dilution contains histidine, trehalose, methionine and polysorbate 80. Because of the difference in concentration of the concentrates, it is necessary to calculate to formulate and add the first buffer for dilution, thereby obtaining formulation 1 as described above.

1.2. Preparation of formulation 2

Formulation 2 consisted of 150mg/ml anti-human IL-17A monoclonal antibody, 10mM citric acid, 150mM arginine, 25mM methionine, 0.2mg/ml polysorbate 20, pH5.8. The solvent is water, and the pH is adjusted by citric acid.

The preparation process comprises the following steps: the purified anti-human IL-17A monoclonal antibody is ultrafiltered and concentrated to 50-80 mg/ml, 10mM citric acid and 24mM arginine buffer system is used for ultrafiltering and replacing 6-15 times of volume, and then ultrafiltering and concentrating is carried out to 175-215 mg/ml to obtain concentrated solution. Adding a prepared second buffer solution (comprising citric acid, arginine, methionine and polysorbate 20, wherein the concentration of each component is required to be confirmed through calculation) for dilution into the concentrated solution, uniformly mixing and filtering to obtain an anti-human IL-17A monoclonal antibody protein stock solution with the concentration of 150mg/ml, and then filling to obtain the preparation 2.

The 10mM citric acid+24 mM arginine buffer system was an aqueous solution containing 10mM citric acid and 24mM arginine at a pH of 5.6 (without additional pH adjustment).

The second buffer for dilution contains citric acid, arginine, methionine and polysorbate 20. Because of the difference in concentration of the concentrates, it was calculated to formulate and add the second buffer for dilution to obtain formulation 2 as described above.

Example 2: comparison of Properties of formulation 1 and formulation 2

The measurement was carried out using a freezing point depression osmometer (2020 edition "Chinese pharmacopoeia general rule" 0632) and a pressure-driven microaerometer (U.S. pharmacopoeia <914> viscosity-pressure-driven method).

The results show that: formulation 1 had an osmotic pressure of 306mOsmol/kg and a viscosity of 7.2cP; formulation 2 had an osmotic pressure of 302mOsmol/kg and a viscosity of 6.1cP.

As can be seen from comparison of the osmolalities of preparation 1 and preparation 2, both were within the osmolality range of normal human blood (285-310 mOsmol/kg). The low viscosity preparation is easier to carry out the production process (lower shearing force when using peristaltic pump, lower back pressure when using membrane filtration and difficult to block) in the production process, and is more beneficial to self injection of patients (tissue back pressure and injection pain can be generated due to the excessive viscosity). Thus, formulation 2 is easier to manufacture due to its lower viscosity and is more beneficial for administration via the subcutaneous route (reduced injection pain) than formulation 1.

The main peak of formulation 2 (i.e., anti-human IL-17A monoclonal antibody) was found to be of higher purity and the polymer and fragment peaks were lower under the various treatment conditions shown in table 3, as measured by Size Exclusion Chromatography (SEC), ion Exchange Chromatography (IEC), reverse phase chromatography (RP) and capillary electrophoresis Chromatography (CE), indicating that formulation 2 had more excellent stability (see table 4, fig. 1-3). As can be seen from fig. 1, the peak of the polymer of formulation 2 is lower than that of formulation 1, indicating that formulation 2 has fewer polymers than formulation 1, and formulation 2 is safer and more stable. As can be seen in FIGS. 2 and 3, the main peak of preparation 2 is significantly higher than that of preparation 1, indicating that the purity of the anti-human IL-17A monoclonal antibody in preparation 2 is higher.

TABLE 3 multiple processing scenarios

Table 4 comparison of stability of formulations 1 and 2 under various treatment conditions

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Note that: N/A indicates no detection and no data.

Example 3 Effect of different buffers on the stability of Stuzumab

As shown in table 5, the following formulations were prepared:

TABLE 5 preparations 5-7

Note that: citric acid-sodium citrate: after weighing the citric acid formulation, the pH was adjusted using sodium hydroxide, and the buffer was defined as a citric acid-sodium citrate buffer.

The preparation process of the preparation 5 comprises the following steps: ultrafiltering and concentrating the purified anti-human IL-17A monoclonal antibody to obtain the final product

50-80 mg/ml, ultrafiltration and displacement by using a 10mM citric acid+24 mM arginine buffer system for 6-15 times of volume, and ultrafiltration concentration to reduce the concentration of the antibody to 175-215 mg/ml to obtain concentrated solution. Adding a prepared second buffer solution (containing citric acid, arginine, methionine and polysorbate 20, wherein the concentration of each component is required to be confirmed through calculation) for dilution into the concentrated solution, uniformly mixing and filtering to obtain an anti-human IL-17A monoclonal antibody protein stock solution with the concentration of 150mg/ml, and then filling to obtain the preparation 5. The viscosity of the preparation 5 is 6.7cp, and the osmotic pressure meets the requirement. Preparation 6 and preparation 7 are prepared by a similar process to preparation 5, and will not be described again here, except that the final components and proportions are shown in table 5.

The formulations were subjected to shaking, high temperature and freeze thawing (see table 3 for processing conditions) and the effect of different formulation formulations on the stability of secukinumab under different conditions, including Size Exclusion Chromatography (SEC), ion Exchange Chromatography (IEC) and capillary electrophoresis Chromatography (CE), results are given in table 6 below.

TABLE 6 stability analysis results for formulations 5-7

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The results show that:

preparation 5 had a higher purity of the main peak at each test item, especially after 10 days of light treatment. The peak of the polymer in SEC test for formulation 5 was significantly lower compared to formulations 6 and 7, while the main peak was higher; in IEC tests, the main peak height is higher, while the acidic and basic peaks are lower; in CE testing, the fragment peaks and the polymers were lower in height, while the main peaks were more prominent. These results indicate that citric acid buffer can better maintain the stability of the secukinumab than citrate buffer, and formulation 5 has significant advantages in terms of safety and stability.

Example 4 Effect of buffer concentration on formulation stability

To further examine whether the concentration of citric acid buffer would affect the stability of secukinumab, formulation 8 was prepared, which contained the following components: 100mg/mL of Stuzumab, 20mM citric acid, 200mM arginine, 0.5mg/mL polysorbate 80, pH6.0.

The preparation process comprises the following steps: the purified anti-human IL-17A monoclonal antibody is ultrafiltered and concentrated to 50-80 mg/ml, 10mM citric acid and 24mM arginine buffer system is used for ultrafiltering and replacing 6-15 times of volume, and then ultrafiltering and concentrating is carried out to 175-215 mg/ml to obtain concentrated solution. Adding the prepared second buffer solution (which needs to be confirmed by calculation) for dilution into the concentrated solution, uniformly mixing and filtering to obtain the anti-human IL-17A monoclonal antibody protein stock solution with the concentration of 100mg/ml, and then filling to obtain the preparation 8.

The osmotic pressure of formulation 8 was measured to be 298mOsm/kg and the viscosity was measured to be 2.9cP.

Formulation 8 was subjected to shaking, high temperature and freeze thawing (see table 3 for treatment conditions) and the effect of different formulation formulations on the stability of secukinumab under different conditions, including Size Exclusion Chromatography (SEC), ion Exchange Chromatography (IEC) and capillary electrophoresis Chromatography (CE), results are shown in table 7.

TABLE 7 comparison of stability of formulation 8 under various treatment conditions

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The results show that formulation 8 shows excellent stability under various conditions, demonstrating that 20mM citric acid can well maintain stability of the secukinumab.

Example 5: preparation of anti-human HER2 monoclonal antibody preparation 3 and preparation 4

Formulation 3 consisted of 50mg/ml anti-human HER2 monoclonal antibody (amino acid sequence see U.S. Pat. No. 3, 5821337A, SEQ ID NO:4 of the present invention), 20mM acetic acid, 235mM sucrose, 0.2mg/ml polysorbate 80, pH6.0. The solvent was water and the pH was adjusted with sodium hydroxide.

The anti-human HER2 monoclonal antibody is trastuzumab, which has the sequence:

trastuzumab heavy chain (SEQ ID NO: 3):

trastuzumab light chain (SEQ ID NO: 4):

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the preparation process comprises the following steps: the purified anti-human HER2 monoclonal antibody is ultrafiltered and concentrated to 20-30 mg/ml, the volume of the purified anti-human HER2 monoclonal antibody is ultrafiltered and replaced by 8-12 times by using a 10mM acetic acid-sodium hydroxide buffer system, and then the purified anti-human HER2 monoclonal antibody is ultrafiltered and concentrated to 80-120 mg/ml to obtain concentrated solution. And adding the prepared third buffer solution (which needs to be confirmed by calculation) for dilution into the concentrated solution, uniformly mixing and filtering to obtain 50mg/ml of anti-human HER2 monoclonal antibody protein stock solution, and then filling to obtain the preparation 3.

The 10mM acetic acid-sodium hydroxide buffer system was an aqueous solution containing 10mM acetic acid and adjusted to pH5.8 with sodium hydroxide.

The third buffer for dilution contains acetic acid, sucrose and polysorbate 80. Because of the difference in concentration of the concentrates, it was calculated to formulate and add the third buffer for dilution to obtain formulation 3 as described above.

Formulation 4 consisted of 50mg/ml anti-human HER2 monoclonal antibody (amino acid sequence see U.S. Pat. No. 3, 5821337A, SEQ ID NO. 4 of the present invention), 20mM acetic acid, 175mM arginine, 0.2mg/ml polysorbate 80, pH6.0. The solvent is water and acetic acid is used to adjust the pH.

The preparation process comprises the following steps: the purified anti-human HER2 monoclonal antibody is ultrafiltered and concentrated to 20-30 mg/ml, and the volume of the purified anti-human HER2 monoclonal antibody is ultrafiltered and replaced by 8-12 times by using a 10mM acetic acid+9.2 mM arginine buffer system, and then the purified anti-human HER2 monoclonal antibody is ultrafiltered and concentrated to 80-120 mg/ml to obtain concentrated solution. And adding the prepared fourth buffer solution (which needs to be confirmed by calculation) for dilution into the concentrated solution, uniformly mixing and filtering to obtain 50mg/ml of anti-human HER2 monoclonal antibody protein stock solution, and then filling to obtain the preparation 4.

The 10mM acetic acid-arginine buffer system was an aqueous solution containing 10mM acetic acid and 9.2mM arginine, at which point the pH was 5.8 (no additional pH adjustment was required).

The fourth buffer for dilution contains acetic acid, arginine and polysorbate 80. Because of the difference in concentration of the concentrates, it was calculated to formulate and add the fourth buffer for dilution to obtain formulation 4 as described above.

Example 6: comparison of Properties of formulation 3 and formulation 4

Formulation 3 is a prior art formulation and formulation 4 is a formulation of the present invention. Detection is carried out by using an osmometer and a micro-viscosimeter, and the detection is carried out by: formulation 3 had an osmotic pressure of 326mOsmol/kg and a viscosity of 1.91cP; the osmotic pressure of formulation 4 was 312mOsmol/kg and the viscosity was 1.65cP.

As can be seen from comparison of the osmolalities of formulations 3 and 4, both were within the ideal osmolality range (300.+ -.30 mOsmol/kg). The low viscosity preparation is easier to carry out the production process (lower shearing force when using peristaltic pump, lower back pressure when using membrane filtration and difficult to block) in the production process, and is more beneficial to self injection of patients (tissue back pressure and injection pain can be generated due to the excessive viscosity). Thus, formulation 4 is easier to manufacture due to its lower viscosity and is more beneficial for administration via the subcutaneous route (reduced injection pain) than formulation 3.

The main peak of formulation 4 (i.e., anti-human HER2 monoclonal antibody) was found to be higher in purity and lower in the polymer peak (see table 8) under the various treatment conditions shown in table 3, indicating that formulation 4 had more excellent stability.

TABLE 8 comparison of stability of formulations 3 and 4 under various treatment conditions

Note that: N/A indicates no detection and no data.

The foregoing describes in detail preferred embodiments of the present invention. It should be understood that numerous modifications and variations can be made in accordance with the concepts of the invention without requiring creative effort by one of ordinary skill in the art. Therefore, all technical solutions which can be obtained by logic analysis, reasoning or limited experiments based on the prior art by the person skilled in the art according to the inventive concept shall be within the scope of protection defined by the claims.

Claims

1. An antibody pharmaceutical composition comprising a negatively charged buffer, a positively charged protein protectant, and an antibody, wherein:

2. The antibody pharmaceutical composition of claim 1, wherein:

the concentration of the acid is 5mM-50mM, preferably 5mM-35mM, more preferably 10mM-20mM;

the concentration of the positively charged protein protecting agent is 100mM-220mM, preferably 150mM-200mM, more preferably 150mM-170mM; and/or

The antibody is selected from one or more of an anti-human IL-17A monoclonal antibody, an anti-human HER2 monoclonal antibody and an anti-human TNF-alpha monoclonal antibody; preferably, the antibody is an anti-human IL-17A monoclonal antibody comprising the amino acid sequence as set forth in SEQ ID NO:1 and a heavy chain as set forth in SEQ ID NO:2, and a light chain as shown in FIG. 2.

3. The antibody pharmaceutical composition of claim 1 or 2, wherein the concentration of the antibody is 50mg/mL to 200mg/mL, preferably 90mg/mL to 160mg/mL, more preferably 100mg/mL to 150mg/mL.

4. The antibody pharmaceutical composition of any one of claims 1-3, further comprising a surfactant selected from polysorbate 20, polysorbate 80, and poloxamer 188; preferably, the concentration of the surfactant is from 0.1mg/mL to 0.6mg/mL, preferably 0.2mg/mL.

5. The antibody pharmaceutical composition of any one of claims 1-4, further comprising an antioxidant selected from methionine, glutathione, cysteine, cystine, and ascorbic acid; preferably, the concentration of the antioxidant is 20mM-30mM, preferably 25mM.

6. The antibody pharmaceutical composition of any one of claims 1 to 5, wherein the pH of the antibody pharmaceutical composition is 5.6-6.0, preferably 5.8.

7. The antibody pharmaceutical composition of any one of claims 1-6 comprising:

a) 90mg/mL-160mg/mL of antibody;

b) 5mM-35mM citric acid;

c) 150mM-200mM arginine;

d) 0.1mg/mL-0.6mg/mL polysorbate 20; and

e) Methionine at 20mM-30 mM; wherein the pH of the antibody pharmaceutical composition is 5.6-6.0; preferably, the antibody pharmaceutical composition comprises:

a) 150mg/mL of antibody;

b) 10mM citric acid;

c) 150mM-170mM arginine;

d) 0.2mg/mL polysorbate 20; and

8. The antibody pharmaceutical composition of any one of claims 1-7, wherein the pharmaceutical combination has an osmolality in the range of 285-310 mOsmol/kg and a viscosity of less than 7.2cp; preferably, the viscosity is 1.65cP-6.1cP.

9. A lyophilized formulation obtained by freeze-drying the antibody pharmaceutical composition of any one of claims 1-8.

10. Use of an antibody pharmaceutical composition according to any one of claims 1-8 or a lyophilized formulation according to claim 9 in the manufacture of a medicament for the treatment of a disease associated with IL-17A and/or IL-17F; preferably, the disease is selected from psoriasis, psoriatic arthritis, ankylosing spondylitis, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, osteoarthritis and inflammatory bowel disease.