CN118045173A - Stable formulations containing anti-sclerostin antibodies and methods of making and using same - Google Patents

Abstract

The present invention relates to stable formulations containing high concentrations of an anti-sclerostin antibody, comprising a therapeutically effective dose of an anti-sclerostin antibody or antigen-binding fragment thereof, histidine-hcl buffer and calcium chloride, said formulations having low viscosity, long-term stability and good solubility. The invention also provides a preparation method of the preparation and application of the preparation in preparing medicines for treating, preventing and/or improving any disease or symptom related to the sclerostin.

Description

Stable formulations containing anti-sclerostin antibodies and methods of making and using same

Technical Field

The invention relates to the field of medicines, in particular to a stable preparation containing an anti-sclerostin antibody, a preparation method thereof and application of the stable preparation in medicines for treating, preventing or improving diseases related to sclerostin activity.

Background

Osteoporosis (Osteoporosis, OP), including post-menopausal women's osteoporosis (Postmenopausal Osteoporosis, PMO) and senile osteoporosis, is a systemic bone metabolic disorder characterized by low bone mass and deterioration of bone microstructure, resulting in decreased bone strength, increased bone fragility, and susceptibility to fracture. It is counted that about 2 hundred million people worldwide have osteoporosis, and the incidence rate thereof has jumped to the seventh place of common diseases and frequently occurring diseases. The prevalence rate of osteoporosis of aged women over 60 years old in China is up to 60%, and the prevalence rate of men is also 40-50%.

Sclerostin (Sclerostin) is used as a new biological target for drug development, the principle is that osteoporosis can be treated by regulating osteoblast anabolism, and the target fills the blank in the field of treating osteoporosis by regulating bone metabolism.

At present, clinical reports of anti-sclerostin antibodies Romosozumab jointly developed by Amgen and UCB show that the safety and tolerance are good, and the bone density of a tested body is obviously improved compared with that of a blank group. Two monoclonal antibodies were separately introduced by Lilly and Novartis. The antibodies are applicable to osteoporosis/osteoporosis, bone injury/related bone disease treatment and the like.

However, antibody drugs have large molecular weight, complex structure, easy degradation, polymerization, or occurrence of undesired chemical modification, etc., and monoclonal antibodies are widely used as biotherapeutic agents, and because of the presence of extracellular matrix, the mobility of the drug is limited, and the subcutaneous injection volume is generally limited to 1-2mL under normal conditions, and the concentration of antibody preparation needs to reach more than 90mg/mL to meet the requirement of clinical drug dosage, and the requirement of solubility is high.

WO2019020069 discloses SOST antibody pharmaceutical compositions and uses thereof, the compositions comprising acetate buffer, disaccharide, polysorbate 80, calcium salt, etc., which can improve the stability of the antibody, but do not relate to the solubility of the antibody.

Therefore, there is a need to develop an anti-sclerostin antibody formulation having low viscosity, long-term stability, and good solubility.

Disclosure of Invention

The invention starts from solving the defects of the prior art, and provides a stable preparation containing an anti-sclerostin antibody, which has the characteristics of low viscosity, high concentration of the antibody, long-term stability, good solubility, high patient compliance and the like, and is suitable for subcutaneous injection.

The above object of the present invention is achieved by the following technical scheme:

in one aspect, the invention provides a stable formulation comprising an anti-sclerostin antibody, comprising:

An anti-sclerostin antibody or antigen-binding fragment thereof at a concentration of 50-200 mg/mL;

a pharmaceutically acceptable buffer at a concentration of 10-50 mmol/L; and

Calcium chloride with the concentration of 10-30 mmol/L; wherein the anti-sclerostin antibody is selected from Romosozumab antibodies, the buffer is selected from histidine-histidine hydrochloride, and the pH value of the preparation is 4.5-6.5.

In some embodiments, the Romosozumab antibody, or antigen-binding fragment thereof, is at a concentration of 80-100mg/mL. In a specific embodiment, the concentration of the anti-sclerostin antibody or antigen-binding fragment thereof is from 90 to 95mg/mL. In a specific embodiment, the concentration of the anti-sclerostin antibody or antigen-binding fragment thereof is 90mg/mL. In a specific embodiment, the concentration of the anti-sclerostin antibody or antigen-binding fragment thereof is 95mg/mL.

As a preferable technical scheme of the invention, the concentration (mol) ratio of the histidine-histidine hydrochloride to the calcium chloride is 0.8:1-8:1, in one specific embodiment the histidine-hcl to calcium chloride concentration (molar) ratio is 1.76:1. the concentration of the pharmaceutically acceptable buffer is 10-50mmol/L. In one embodiment, the buffer concentration is 20-40mmol/L. In a specific embodiment, the buffer concentration is 40mmol/L.

The concentration of calcium chloride is preferably 15-25mmol/L, in one embodiment 15-23mmol/L, and in one particular embodiment 15mmol/L or 22.7mmol/L.

The pH value of the stable liquid preparation is 4.5-6.5. In one embodiment, the stable liquid formulation has a pH of 4.5 to 6.0. In a specific embodiment, the pH of the stable liquid formulation is 4.5,5.5 or 6.0. In one embodiment, the stable liquid formulation has a pH of 4.7 to 5.5. In one embodiment, the stable liquid formulation has a pH of 5.0 to 5.5. In one embodiment, the stable liquid formulation has a pH of 5.0 to 5.2. In a specific embodiment, the pH of the stable liquid formulation is 5.0,5.2 or 5.5.

Histidine has an imidazole group in the molecular structure, has pH sensitivity at about 6.0, can generate protonic action when the pH of the environment is less than 6.0, and is hydrophilic; deprotonation occurs when the ambient pH is > 6.0, and is rendered hydrophobic.

Although histidine is pH sensitive and prone to oxidation, the inventors have surprisingly found that when the formulation of the invention uses histidine-histidine hydrochloride as a buffer system and the pH is maintained between 4.7 and 5.5, it has good compliance and quality stability, e.g. the pain sensation in patients can be significantly reduced upon subcutaneous administration compared to the use of acetate buffer system with lower pH (pKa 4.76), whereas for chronic disease patients requiring chronic administration, the use of liquid formulation of histidine/histidine hydrochloride and calcium chloride can improve patient compliance; in addition, when a histidine/histidine hydrochloride buffer system is selected and the pH value is 4.7-5.5, the antibody viscosity of the liquid preparation can be reduced to below 15cp, even below 10cp, even below 5cp, the degradation rate of the antibody in the storage process can be reduced, and the liquid preparation has better stability.

In addition, when histidine-histidine hydrochloride and calcium chloride are selected, the histidine-histidine hydrochloride and the calcium chloride have good promotion and synergistic effects in increasing the solubility and stability of the system protein.

As a preferred technical scheme of the invention, the preparation also comprises a surfactant with the concentration of 0-0.1 mg/mL; wherein the surfactant is selected from polysorbate 80 or polysorbate 20, preferably polysorbate 20, and preferably at a concentration of 0.01-0.09mg/mL, and in one embodiment, preferably at a concentration of 0.03-0.09mg/mL, more preferably at 0.03mg/mL, 0.06mg/mL or 0.09mg/mL.

The invention is found through a large number of experiments: when polysorbate 80 or polysorbate 20 is selected as the surfactant of the liquid formulation of the present invention, aggregation of the anti-sclerostin antibody or antigen-binding fragment thereof of the present invention during agitation and shaking can be reduced, the antibody is prevented from being adsorbed to the surface of the container, and the requirement of subcutaneous injection administration of the liquid formulation of the present invention can be satisfied. In addition, the polysorbate 80 has the characteristics of low freezing point (the solid state at other normal temperature) and convenient use because of double bonds in the structure. In addition, the ability of polysorbate 80 or polysorbate 20 to stabilize antibodies is also related to their concentration. The concentration of polysorbate 80 or polysorbate 20 in the liquid formulation of the present invention above 0.01 (mg/ml) can inhibit the aggregation of antibodies caused by shaking, and below that concentration polysorbate 80 or polysorbate 20 cannot reduce the protein instability caused by shaking, while above 0.1 (mg/ml) cannot significantly improve the stability of antibodies.

As a preferred embodiment of the present invention, the formulation further comprises an excipient having a concentration of 10-80mg/mL, the excipient being selected from any one of proline, sucrose, trehalose, sodium chloride, sorbitol, mannitol, or arginine hydrochloride, or a combination thereof. In a specific embodiment, the excipient is sucrose at a concentration of 10-80mg/ml, preferably 45-75mg/ml, more preferably 45mg/ml, 60mg/ml or 75mg/ml.

As a preferred technical scheme of the invention, the preparation is a liquid preparation or a freeze-dried preparation, the freeze-dried preparation is a reconstituted liquid preparation, the viscosity of the liquid preparation at 25 ℃ is less than 15cp, preferably the viscosity at 25 ℃ is less than 10cp, and the osmotic pressure of the stable liquid preparation is 220-400mOsmol/kg. In one embodiment, the stable liquid formulation has an osmotic pressure of 250 to 350mOsm/Kg. In one embodiment, the stabilized liquid formulation has an osmotic pressure of 290 to 320mOsm/Kg at 25℃and in one embodiment, the stabilized liquid formulation has an osmotic pressure of 296mOsm/Kg at 25 ℃.

The invention is found through a large number of experiments: when sucrose is selected as one of the excipients of the present invention, not only can the osmotic pressure of the liquid formulation of the present invention be maintained at 250-300mOsm/Kg due to the biocompatibility of sucrose, but also a hydration layer can be formed on the surface of the anti-sclerostin antibody or antigen-binding fragment thereof to stabilize the antibody protein without damaging its structure, and interactions of the antibody protein itself or other substances can be prevented from acting thereon, so that the viscosity and denaturation of the high concentration antibody in the liquid formulation can be reduced, thereby being advantageous for improving the stability and fluidity of the liquid formulation and compliance of patients when subcutaneously administered. Sucrose is used as one of the most suitable excipients of the preparation, so that hemolysis or irritation can be avoided during administration, and the use safety and stability of the injection preparation are effectively ensured.

In one embodiment, the stable liquid formulation has a purity of the anti-sclerostin antibody or antigen-binding fragment thereof reduced by no more than 10%, e.g., no more than 5%, 4%, 3%, 2%, 1.0%, 0.5%, 0.2% or 0.1% as measured by a reduced CE-SDS method (rCE-SDS) and a non-reduced CE-SDS (nrCE-SDS) after storage at 2-8 ℃ for at least 1 month, at least 3 months, at least 6 months, or longer, or after storage at about 25 ℃ for 1 month, at least 6 months, or longer.

The stable formulation is a liquid pharmaceutical formulation, preferably an injection, more preferably a subcutaneous injection or an intramuscular injection, most preferably a subcutaneous injection.

In a preferred embodiment, the stable liquid formulation of the present invention comprises:

(1) Antibody or antigen binding fragment thereof at a concentration of 90mg/mL-95 mg/mLRomosozumab;

(2) The concentration is 40mmol/L histidine, histidine hydrochloride and 22.7mmol/L calcium chloride;

(3) Polysorbate 20 at a concentration of 0.03mg/mL to 0.09 mg/mL;

(4) Sucrose with concentration of 45mg/mL-75 mg/mL; wherein the pH value of the liquid preparation is 4.7-5.5.

In a more preferred embodiment, the stable liquid formulation of the present invention comprises:

(1) Romosozumab antibodies or antigen-binding fragments thereof at a concentration of 90mg/mL or 95 mg/mL;

(2) The concentration is 40mmol/mL histidine, histidine hydrochloride and 22.7mmol/mL calcium chloride;

(3) Polysorbate 20 at a concentration of 0.06 mg/mL; and

(4) Sucrose at a concentration of 45 mg/mL; wherein the pH value of the preparation is 4.7-5.5.

In a second aspect, the present invention provides a prefilled syringe loaded with a stable formulation as described above, which is a liquid formulation, which can be administered parenterally, preferably subcutaneously or intramuscularly.

In a third aspect, the present invention provides the use of a stable formulation as described above in the manufacture of a medicament for the treatment, prevention or amelioration of any sclerostin-related disease or condition, selected from osteoporosis or osteopenia.

Compared with the prior art, the invention has the following advantages and beneficial effects:

(1) The liquid preparation containing high-concentration anti-sclerostin antibody has the characteristics of low viscosity and high solubility, meets the requirement of clinical medicine dosage, meets the viscosity limit of the antibody required for subcutaneous delivery by using a syringe at ambient temperature, and is particularly suitable for subcutaneous injection or intramuscular injection administration;

(2) The invention provides a liquid preparation containing high-concentration anti-sclerostin antibody, which has high physical and chemical stability, is not easy to generate aggregates, particle precipitates and charge heterosomes when being stored for a long time (such as more than 1 month, especially more than 3 months or 6 months), therefore, the liquid preparation provided by the invention is not only suitable for long-term storage, but also can ensure the biological activity of the antibody, and the quality safety, effectiveness and high consistency of the liquid preparation prepared into clinical medicines;

(3) The liquid preparation containing the high-concentration anti-sclerostin antibody has the advantages of simple components, safety, no toxicity, less aggregate and particle sediment, high biological activity, simple and easy production method and easy quality control;

(4) The liquid preparation containing the high-concentration anti-sclerostin antibody has the characteristics of high concentration of antibody and low viscosity, and the pH value is maintained between 4.5 and 6.5 (especially 5.0 and 5.5), so that the liquid preparation is suitable for subcutaneous injection administration, not only meets the requirement of chronic disease patients on domestic medication, but also can reduce the administration pain to a lower value in the administration process, and improves the medication compliance of the patients.

Detailed Description

The following description of the application is merely illustrative of various embodiments of the application. Therefore, the specific modifications discussed herein should not be construed as limiting the scope of the claims. Numerous equivalents, variations and modifications will readily occur to those skilled in the art without departing from the scope of the present application, and it is to be understood that such equivalent embodiments are included within the scope of the present application. All documents cited in this application, including publications, patents and patent applications, are incorporated by reference in their entirety.

Definition of the definition

The terms used in the present invention have the definitions listed below. If no definition is given herein, the terms used in the present invention have meanings commonly understood by those of ordinary skill in the art.

The term "about" as used herein, when used in reference to a particular value or range of values listed, means that the value may vary from the particular value listed by + -20% or + -10%, including + -5%, + -1%, and + -0.1%, as appropriate for performing the disclosed method.

For purposes of explaining the present specification, the following definitions will be used, and terms used in the singular form may also include the plural, and vice versa, as appropriate. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

"Treating" or "treatment" of a disease or condition refers to alleviating a disease or condition, reducing the rate at which a disease or condition arises or progresses, reducing the risk of developing a disease or condition, or delaying the development of a condition associated with a disease or condition, reducing or terminating a condition associated with a disease or condition, producing complete or partial reversal of a disease or condition, curing a disease or condition, or a combination thereof.

"Preventing" includes inhibition of the occurrence or progression of a disease or disorder or a symptom of a particular disease or disorder. In some embodiments, the subject with a family history is a candidate for a prophylactic regimen. In general, the term "prevention" refers to administration of a drug prior to the occurrence of a sign or symptom, particularly in a subject at risk.

"Anti-sclerostin-related disease or condition" refers to a disease or condition caused or characterized by a change in sclerostin, such as achondroplasia, collarbone-skull hypoplasia, endogenous chondrioma, fibrodysplasia, gaucher's disease, hypophosphorous rickets, marfan's syndrome, hereditary multiple exogenous bone verrucosis, neurofibromas, osteogenesis imperfecta, osteosclerosis, fragile bone sclerosis, sclerotic lesions, pseudoarthropathy, suppurative osteomyelitis, periodontal disease, antiepileptic drug-induced bone loss, primary or secondary hyperparathyroidism, familial hyperparathyroidism syndrome, weightless induced bone loss, male osteoporosis, postmenopausal bone loss, osteoarthritis, renal bone disease, osmotic bone disease, oral bone loss, mandibular necrosis, juvenile pecies disease, limb bone striated hypertrophy, metabolic bone disease, mastocytosis, sickle cell disease, sickle cell anemia/disease bone loss associated with organ transplantation, bone loss associated with kidney transplantation, systemic lupus erythematosus, ankylosing spondylitis, epilepsy, juvenile arthritis, thalassemia, mucopolysaccharidosis, fabry's disease, turner's syndrome, down's syndrome, cresoft's syndrome, leprosy, perthe's disease, juvenile idiopathic scoliosis, infant-onset multisystemic inflammatory disease, winchester syndrome, door's disease, wilson's disease, ischemic bone disease (e.g., sev-peck-pecies disease or regional migratory osteoporosis), anemic status, steroid-induced conditions, glucocorticoid-induced bone loss, heparin-induced bone loss, bone marrow disorders, necrosis, malnutrition, calcium deficiency, osteoporosis, reduced bone mass, alcoholism, chronic liver disease, post-menopausal status, chronic inflammatory conditions, rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, inflammatory colitis, crohn's disease, diluted menstruation, no menstruation, pregnancy, diabetes, hyperthyroidism, thyroid disorders, parathyroid disorders, cushing's disease, acromegaly, hypogonadism, disuse or inactivity, reflex sympathetic atrophy syndrome, regional osteoporosis, osteomalacia, joint replacement-related bone loss, HIV-related bone loss, growth hormone deficiency-related bone loss, cystic fibrosis-related bone loss, chemotherapy-related bone loss, tumor-induced bone loss, cancer-related bone loss, hormone-deprived bone loss (hormone ablative bone loss), multiple myeloma, drug-induced bone loss, anorexia, disease-related facial bone loss, disease-related mandible bone loss, disease-related skull loss, disease-related bone loss, age-related bone loss, aging-related bone loss of the head, aging-related bone loss of the jaw aging, aging-related bone loss of the aging, or space-related bone loss of the jaw aging. Methods for identifying/diagnosing the above-mentioned diseases or symptoms are known in the art.

The term "subject" includes any human or non-human animal. "non-human animals" include all vertebrates, such as mammals (including, but not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., monkeys), rabbits, and rodents (e.g., mice and rats)), and non-mammals (e.g., poultry, amphibians, and reptiles). In some embodiments, the subject is a human.

The term "therapeutically effective amount" or "effective dose" refers to a dose or concentration effective to achieve prevention or amelioration of symptoms associated with a disease or disorder and/or lessening the severity of a disease or disorder at a desired dose for a desired period of time. The therapeutically effective amount of the formulations, antibodies, or antigen binding fragments thereof, or compositions of the invention may vary depending on a variety of factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount may also be considered to be any toxic or detrimental effect of the formulation, antibody or antigen-binding fragment thereof or composition that is less than a therapeutically beneficial effect.

The term "formulation" refers to a composition comprising at least one active ingredient and at least one inactive ingredient suitable for administration to animals, preferably mammals (including humans). "liquid formulation" refers to a formulation in liquid form. The composition of the formulation of the present invention may be as shown in the liquid formulation embodiments described hereinbefore. The liquid preparation of the present invention is preferably an injection, more preferably a subcutaneous injection or intramuscular injection, and most preferably a subcutaneous injection.

The term "buffer" refers to a pH buffer. Preferably, the buffer is capable of maintaining the pH of the liquid formulation of the present invention at about 4.5 to 6.5, preferably about 4.7 to 5.5, more preferably 5.0 to 5.2, more preferably 5.0 to 5.5. The buffer is present in the liquid formulation at a concentration of about 10-50mmol/L, preferably at a concentration of 20-40mmol/L, more preferably at a concentration of 40mmol/L. The buffering agent is selected from histidine-histidine hydrochloride.

The term "excipient" refers to a non-therapeutic agent that can be added to a formulation to provide desired characteristics (e.g., consistency, increased stability) and/or to regulate osmotic pressure, which non-therapeutic agent is added to the formulation to stabilize the physicochemical and biological characteristics of the active ingredient. Examples of common excipients include, but are not limited to, sugars, polyols, amino acids, surfactants, and polymers. Preferably the excipient is sucrose.

The term "osmolyte" is a molecule that contributes to the osmolarity of a solution. By "osmolality adjusting agent" in accordance with the present invention is meant an agent that is capable of adjusting, altering or optimizing the osmolality of the anti-sclerostin antibody liquid formulation of the present invention, preferably, the osmolality of the liquid formulation of the present invention is adjusted, not only to maintain the isotonicity of the liquid formulation, but also to maximize the stability of the anti-sclerostin antibody or antigen binding fragment thereof of the present invention, while minimizing patient discomfort upon administration. Examples of osmolality adjusting agents suitable for varying the osmolality include, but are not limited to, amino acids (proline, arginine, cysteine, histidine, etc.), salts (sodium chloride, potassium chloride, calcium chloride, etc.) and/or sugars (sucrose, trehalose, glucose, mannitol, sorbitol, and the like), with the preferred osmolality adjusting agent being calcium chloride.

The term "surfactant" refers to a substance that, when added in small amounts, causes a significant change in the interfacial state of the solution system. For example, proteins (e.g., antibodies) can be protected from air/solution interface induced stress, solution/surface induced stress, thereby reducing aggregation of the protein or the formation of particulates in the formulation. Exemplary surfactants include, but are not limited to, nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (e.g., polysorbate 80, polysorbate 60, polysorbate 40, or polysorbate 20), polyethylene-polypropylene copolymers, polyethylene-polypropylene glycols, polyoxyethylene-stearates, polyoxyethylene alkyl ethers such as polyoxyethylene monolauryl ether, alkylphenyl polyoxyethylene ether (Triton-X), polyoxyethylene-polyoxypropylene copolymers (poloxamer, pluronic), sodium Dodecyl Sulfate (SDS). Preferred surfactants of the invention are nonionic surfactants such as polysorbate 80 or polysorbate 20. Preferably the surfactant is polysorbate 20 (PS 20), the concentration of the surfactant in the liquid formulation is 0-0.1 (mg/mL), preferably 0.01-0.09 (mg/mL), preferably 0.03-0.09 (mg/mL), more preferably 0.03 (mg/mL), 0.06 (mg/mL) and 0.09 (mg/mL).

The term "viscosity" may be "kinematic viscosity" or "absolute viscosity". "kinematic viscosity" is a measure of the resistive flow of a fluid under the influence of gravity. When two fluids of the same volume are placed in two identical capillary viscometers, respectively, and flow by gravity, the viscous fluid takes longer to flow through the capillary than the less viscous fluid. "absolute viscosity", sometimes referred to as dynamic viscosity or simple viscosity, is the product of the kinematic viscosity and the fluid density (absolute viscosity = kinematic viscosity x density). The dimension of the kinematic viscosity is L2/T (where L is length and T is time). Typically, kinematic viscosity is expressed in centistokes (cSt). The International units of kinematic viscosity are mm2/s (i.e., lcSt). Absolute viscosity is expressed in centipoise (cP) units. The units in international units of absolute viscosity are millipascal-seconds (mPa-s), where 1 cp= lmPa-s.

The term "low level viscosity" refers to an absolute viscosity of less than about 15 cp. "medium horizontal viscosity" refers to an absolute viscosity of between about 15cp and about 35 cp. The absolute viscosity of the liquid formulations of the antibodies of the present application is about 3.8cp, 4.2cp, 6.0cp, about 8.0cp, about 9.0cp, about 10.0cp, about 11.0cp, or about 12.0cp, as measured using standard viscosity measurement techniques, indicating that the liquid formulations of the present application have a "low level viscosity". In some embodiments, the inventors of the present application have surprisingly found that proline may not only act as an osmotic regulator, but may also reduce the viscosity of the liquid formulation of the antibodies of the present application when formulated with an anti-sclerostin antibody and a buffer.

The term "isotonic" means that the liquid formulation has substantially the same osmotic pressure as human blood. Isotonic formulations generally have an osmotic pressure of about 250 to 300 mOsm. Isotonicity can be measured using a vapor pressure or freezing point depression osmometer.

The term "vehicle" refers to a substance used to mix, disperse, solubilize a test or control without affecting the test results. Solvents useful in the present invention include, but are not limited to, water for injection, salts (e.g., physiological saline), saccharides (e.g., dextrose injection), organic solvents for injection (including, but not limited to, oil for injection, ethanol, propylene glycol, and the like), or combinations thereof.

By "stable" antibody formulation is meant a formulation in which the antibody substantially retains its physicochemical stability and/or biological activity during manufacturing and/or during storage. Antibody formulations can be considered stable even if the contained antibody fails to retain 100% of its physicochemical properties or biological functions after storage for a certain period of time. For example, the formulation can maintain more than 90% of the antibody structure or function after storage for a period of time, and can be considered a "stable" formulation. Criteria for stability e.g. liquid formulations are visually colorless, or clear to slightly milky; the antibody concentration, pH value and osmotic pressure of the preparation change by less than +/-10%; the biological activity of the antibody is 80-140%, preferably 90-120% of that of the reference antibody; the antibody monomer degradation of the formulation is no more than 10%, preferably 5%, more preferably 3%; the aggregate formed in the formulation is not more than 10%, preferably 5%, more preferably 3%.

No significant increase in aggregation, precipitation and/or denaturation of the antibodies in the formulation is indicated if the color and/or clarity of the formulation is visually observed, or detected by Differential Scanning Calorimetry (DSC), size exclusion chromatography (SEC-HPLC) and Dynamic Light Scattering (DLS), indicating that the antibodies in the formulation "retain their physical stability. An antibody "retains its chemical stability" if no significant chemical change occurs in the antibody in the formulation, its chemical structure remains intact. Most of the disruption of chemical stability can be attributed to the formation of covalent modifications of the protein (e.g., covalent aggregates, degradants, or charge isomers) and non-covalent modifications of the protein (e.g., non-covalent aggregates). The changes in the chemical structure of the antibody protein (e.g., variants of different molecular weights or charges) can be determined using methods known to those skilled in the art. Such methods include, but are not limited to, detection of hydrolysates of antibodies using SEC-HPLC and sodium dodecyl sulfate capillary gel electrophoresis (CE-SDS), detection of degraded fragments of antibodies and other protein molecules having a molecular weight less than that of antibodies using non-reducing calipers, or detection of charge isomers of antibodies using cation exchange chromatography (CE-HPLC). An antibody "retains its biological activity" if the biological activity of the antibody in the formulation during storage is still within the range of biological activity exhibited by the formulation at the time of its preparation. Methods for detecting the biological activity of an antibody include, but are not limited to, antigen binding ELISA assays or cell activity assays of an antibody.

By "pharmaceutically acceptable" is meant that the carrier, vehicle, diluent, adjuvant and/or salt is, in general, chemically and/or physically compatible with the other ingredients in the formulation and physiologically compatible with the subject.

Detailed Description

The present invention will be described in further detail with reference to examples, but embodiments of the invention are not limited thereto.

Meaning of chinese and english abbreviations:

HPLC (High Performance Liquid Chromatography): high performance liquid chromatography

CEX (function-exchange): cation exchange

SEC (Size-Exclusion Chromatography): size exclusion chromatography

CEX-HPLC: cation exchange chromatography

SEC-HPLC: size exclusion chromatography

CE (Capillary Electrophoresis): capillary electrophoresis

SDS (Sodium Dodecyl Sulfate): sodium dodecyl sulfate

CE-SDS: sodium dodecyl sulfate capillary gel electrophoresis

The anti-sclerostin antibody used in the examples of the present invention was obtained by screening the antibody romosozumab in examples 1 to 5 of patent application number CN200680024330.8, and constructing a CHO cell line capable of stably expressing the anti-sclerostin antibody of the present invention, and obtaining a supernatant after cell culture, and purifying the supernatant by steps such as affinity chromatography, ion exchange, ultrafiltration, etc., to obtain a stock solution of the anti-sclerostin antibody (Romosozumab is also referred to as antibody a in the present invention), wherein the purity of antibody a is 99.1 to 100.0%.

Example 1 buffer system screening experiments.

The experiment adopts an antibody A with the concentration of 22.7mM calcium ions and the concentration of 90mg/ml to carry out the stability experiment of the antibody A, and the stability of the protein of the antibody A under the combination condition of different candidate buffer systems pH5.0 is examined, and the stability experiment is shown in Table 1 in detail.

Table 1 shows the stability test of antibody A

As is clear from Table 1, the protein content of antibody A in Fa1 histidine-histidine hydrochloride and calcium chloride was changed to a minimum value and the stability was improved as compared with the buffer systems Fa2-Fa 6.

Example 2pH screening experiments.

Based on the screening experiment results of the buffer system in the example 1, the stability of samples of 40mM histidine-histidine hydrochloride buffer system and 22.7mM calcium chloride in different pH ranges (pH 5.0-5.5) is examined, and the pH value experimental screening scheme is shown in Table 2 in detail. Sample detection was performed after 4 weeks of incubation at 40℃and the stability of the different formulations was assessed by visual inspection, purity (SEC-HPLC), charge-coupled-exosomes (CEX-HPLC), disulfide bond isomerism, purity (nr-CE-SDS).

According to the pH range screening scheme, concentrating the stock solution by using an ultrafiltration membrane bag, changing the stock solution to a corresponding buffer system, regulating the concentration of calcium ions, filtering the protein concentration and the pH value of the system by using a 0.2 mu m PES filter, sequentially subpackaging the obtained liquid medicine into sterile penicillin bottles of 4ml to 10ml, and capping by plugging and rolling.

Table 2 shows the pH experimental screening protocol

The appearance, purity (SEC-HPLC), charge-coupled isomer (CEX-HPLC), purity (nr-CE-SDS) and disulfide isomerization of antibody A were examined according to the protocol of Table 2, and the experimental results are shown in tables 3 and 4.

Table 3 shows the pH screening results

Table 4 shows the pH screening results

As is clear from the experimental results in tables 3 and 4, the antibody A was colorless and micro-emulsion optical liquid, and no visible particles were found, and the differences in the changes in the purity (SEC-HPLC), charge heterosomes (CEX-HPLC) and purity (nr-CE-SDS) were small when the buffer was histidine-histidine hydrochloride and calcium chloride, and the pH was 5.0 to 5.5, in the F1-F3, after the examination at 40℃for 4 weeks.

Wherein, the pH is 5.0, the variation of the purity (SEC-HPLC), the charge heterosomes (CEX-HPLC) and the purity (nr-CE-SDS) is the smallest, and the stability is the best.

As is clear from Table 3, the amount of change in the purity of F3 (SEC-HPLC) after 4 weeks was 2.2, while the amount of change in comparative example 2 was 2.8, which revealed that the purity of comparative example 2 was reduced by 27.3% as compared with F3, and the stability of antibody A in the F3 buffer system was better than that of comparative example 2.

As is further evident from Table 4, the amount of change in disulfide isomerism (B/A) of F2 after 4 weeks was 1.1, while the amount of change in comparative example 1 was 2.7, and the amount of change in disulfide isomerism (B/A) of F2 after 4 weeks was more than twice as small as that of comparative example 1, so that the stability of antibody A in F2 buffer system was better than that of comparative example 1.

EXAMPLE 3 solubility study

90Mg/ml of antibody A,40mM histidine-histidine hydrochloride, pH5.0 was selected, the effect of different concentrations of calcium ions (10 mM,19.6mM,22.7mM,42.6mM,50mM,80.3 mM) on the solubility of the system was studied, the optimum calcium ion concentration was determined, and the sample was left to stand at 5℃for 48 hours, then the clarity of the sample was observed and the protein content of the supernatant was tested, and the results of examination of the calcium ion concentration and the solubility of the system were shown in Table 8.

Table 5 shows the results of examination of the calcium ion concentration and the solubility of the system

As is clear from Table 5, the viscosity of antibody A in the buffer system with the calcium ion concentration of not less than 22.7mM is 3.2cP, the viscosity of the buffer system with the calcium ion concentration increased is not changed obviously, and the solubility of the protein content of the buffer system is not changed obviously.

The experiment adopts the concentration of 22.7mM calcium ions to carry out the solubility experimental study of the antibody A, the solubility of the antibody A protein under the combined condition of a buffer system pH of 5.0 after 2-8-2W is examined, and the solubility experiment is shown in Table 6.

Table 6 shows the solubility test of antibody A

As shown in Table 6, after the antibody A is placed at 2-8 ℃ for 2 weeks, the solubility of the antibody A in the F12 buffer system is higher than 100mg/mL, the preparation concentration of the antibody A can reach more than 100mg/mL, the requirement of clinical medicine dosage is met, and the solubility of the antibody A in histidine-histidine hydrochloride alone and calcium chloride alone is not good in combination of 40mM histidine-histidine hydrochloride and 22.7mM calcium chloride, so that the combination of histidine-histidine hydrochloride and calcium chloride has good promotion and synergy in terms of solubility.

Example 4 screening of the content of excipients (sucrose and polysorbate 20).

The stability of samples of different concentrations of sucrose (45 mg/ml,60mg/ml,75 mg/ml) and polysorbate 20 (0.03 mg/ml,0.06mg/ml,0.09 mg/ml) were investigated with a buffer system of 40mM histidine-hcl+22.7 mM calcium chloride, the formulation combinations are detailed in Table 7, the samples were placed under accelerated conditions for sampling after 4 weeks at 5℃and 40℃respectively, the appearance, protein concentration, purity (SEC-HPLC), charge-heterosomes (CEX-HPLC), insoluble Microparticles (MFI) were examined for evaluation of the stability of the different formulations, and the results are presented in Table 8.

Table 7 shows the combination of the screening prescription for the content of the auxiliary materials

Table 8 shows the results of the screening for the content of the auxiliary materials

As is evident from the above analysis, antibody A has better stability when the buffer is 40mM histidine-histidine hydrochloride and 22.7mM calcium chloride, the sucrose concentration is 45-75mg/ml, and polysorbate 20 is 0.03-0.09 mg/ml.

The detection results of the zero-point osmotic pressure screening of auxiliary materials (sucrose and polysorbate 20) are shown in the following table 9, and the results show that F13 (the sucrose content is 60 mg/ml) and the osmotic pressure of the system can reach 396mOmsom/kg; f14 (sucrose content: 45 mg/ml), the osmotic pressure of the system was 296mOmsom/kg; all approach the isotonic condition (280-310 mOmsom/kg) of human body, and has better stability.

Table 9 shows the osmotic pressure test results

As is clear from Table 9 above, when the buffer is histidine-histidine hydrochloride and calcium chloride, the sucrose concentration is 45mg/ml, and polysorbate 20 is 0.06mg/ml, the osmotic pressure of the system is closest to the isotonic conditions of human body, and the system has better stability.

EXAMPLE 5 Long-term stability Studies of liquid formulations containing anti-sclerostin antibodies

According to the screening results, antibody A with a prescription of 90mg/mL, 40mM histidine-histidine hydrochloride, 22.7mM calcium chloride, 45mg/mL sucrose, 0.06mg/mL polysorbate 20 and pH of 5.0 were selected to prepare semi-finished products, and the semi-finished products were filled into 1mL elongated prefilled syringes (with injection needles) with matched rubber plugs, and the semi-finished products were placed at 2-8deg.C and 25+ -2deg.C, respectively, and subjected to storage stability studies for 1 month, 3 months, and 6 months, respectively, with the stability test results shown in tables 10-11.

Table 10 shows a 6 month stability test of the liquid preparation of the anti-sclerostin antibody at 2-8deg.C

Table 11 shows a 6 month stability test of the liquid preparation of the anti-sclerostin antibody at 25 DEG C

As can be seen from tables 10 and 11, the liquid formulations of antibody A of the present invention showed no significant change in appearance, pH, SEC-HPLC, non-reducing CE-SDS and biological activity after storage at 2-8℃and 25℃for 1 month, 3 months and 6 months; the visible foreign matters, the insoluble particles and the sterility meet the requirements of pharmacopoeia; the CEX-HPLC assay (sample subjected to CPB cleavage) shows slight changes in the charge heterogeneity of the antibody, which are mainly caused by deamidation and heavy chain N-ring denaturation, these changes being typical modifications and conformational changes of the antibody, and these changes have no effect on its biological activity. Therefore, the antibody liquid preparation can be stored for 6 months at 2-8 ℃ and 25 ℃, so that the long-term stability of the medicine stored at low temperature is ensured, the difference between preparation batches is small, the antibody biological activity is high, and the quality safety of long-term administration of patients is met.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (11)

1. A stable formulation comprising:

An anti-sclerostin antibody or antigen-binding fragment thereof at a concentration of 50-200 mg/mL;

a pharmaceutically acceptable buffer at a concentration of 10-50 mmol/L; and

Calcium chloride with the concentration of 10-30 mmol/L; wherein the anti-sclerostin antibody is selected from Romosozumab antibodies, the buffer is selected from histidine-histidine hydrochloride, and the pH value of the preparation is 4.5-6.5.

2. The stable formulation of claim 1, wherein the Romosozumab antibody concentration is preferably 80-100mg/mL, more preferably 90mg/mL or 95mg/mL.

3. The stable formulation of claim 1, wherein the buffer concentration is 20-40mmol/L.

4. The stable formulation of claim 1, wherein the molar ratio of histidine-histidine hydrochloride to calcium chloride is 0.8:1-8:1, preferably in a molar ratio of 1.76:1.

5. The stable formulation of claim 1, further comprising:

Surfactant with concentration of 0-0.1 mg/mL; wherein the surfactant is selected from polysorbate 80 or polysorbate 20, preferably polysorbate 20, and the concentration of the surfactant is preferably 0.01-0.09mg/mL, more preferably 0.03mg/mL, 0.06mg/mL or 0.09mg/mL.

6. The stable formulation of claim 1, further comprising:

an excipient having a concentration of 10-80mg/mL, said excipient being selected from any one or a combination of proline, sucrose, sodium chloride, sorbitol, mannitol, or arginine hydrochloride, preferably said excipient is sucrose, said sucrose concentration being preferably 10-80mg/mL sucrose, more preferably 45mg/mL sucrose.

7. The stable formulation according to claim 1, wherein the formulation has a pH of 4.7-5.5, preferably a pH of 5.0-5.2, more preferably a pH of 5.0, 5.2 or 5.5.

8. The stable formulation according to claim 1, wherein the formulation is a liquid formulation, or a lyophilized formulation, the lyophilized formulation being a reconstituted liquid formulation, the liquid formulation having a viscosity of less than 15cp at 25 ℃, preferably less than 10cp at 25 ℃, the liquid formulation having an osmotic pressure of 220-400 mOsm/Kg, preferably 250-350mOsm/Kg, more preferably 290-320mOsm/Kg at 25 ℃.

9. The stable formulation of claim 1, comprising:

(1) Romosozumab antibodies or antigen-binding fragments thereof at a concentration of 90mg/mL to 95 mg/mL;

(2) Histidine-histidine hydrochloride at a concentration of 40mmol/mL and calcium chloride at a concentration of 22.7 mmol/mL;

(3) Polysorbate 20 at a concentration of 0.03mg/mL to 0.09 mg/mL; and

(4) Sucrose with concentration of 45mg/mL-75 mg/mL; wherein the pH value of the preparation is 4.7-5.5.

10. A prefilled syringe, characterized in that it is filled with a stable formulation according to any one of claims 1-9, which is a liquid formulation, which can be administered parenterally, preferably subcutaneously or intramuscularly.

11. Use of a stable formulation according to any one of claims 1-9 in the manufacture of a medicament for the treatment, prevention or amelioration of any sclerostin-related disease or symptom, selected from osteoporosis or osteopenia.