A romosozumab antibody stable formulation and preparation method and use thereof

Abstract

The present application relates to a kind of Romosozumab antibody stable preparation, the preparation includes the therapeutically effective dose of Romosozumab antibody or its antigen fragment, pharmaceutically acceptable cosolvent hydroxypropyl-β-cyclodextrin and surfactant polysorbate 20 and polysorbate 80, the preparation has long-term stability and good solubility.The present application also provides the preparation method of the preparation, and its use in the preparation of the drug for treating, preventing and / or improving any with anti-sclerostin related disease or symptom.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, more particularly, the present application relates to a stable formulation of antibody Romosozumab and a preparation method thereof, and the use of the stable formulation in a medicament for treating, preventing or improving a disease related to sclerostin activity. BACKGROUND

[0002] Osteoporosis (OP) includes postmenopausal osteoporosis (PMO) and senile osteoporosis, which is a systemic bone metabolic disorder characterized by low bone mass and bone microstructure degradation, resulting in decreased bone strength, increased bone fragility, and increased risk of fractures. According to statistics, there are about 200 million people worldwide with osteoporosis, and its incidence has jumped to the seventh place among common diseases and frequently-occurring diseases.

[0003] Sclerostin as a new biological target for drug development, its principle is to treat osteoporosis by regulating osteoblast synthesis metabolism, which fills the gap in the field of treating osteoporosis by regulating bone metabolism.

[0004] Currently, the clinical report of Romosozumab, an anti-sclerostin antibody jointly launched by Amgen and UCB companies, shows that it has good safety and tolerability, and the bone density of the subjects is significantly improved compared with the blank group. Two mAbs drugs launched by Lilly and Novartis companies respectively. The indications of these antibodies are all osteoporosis / osteoporosis and bone injury / related bone disease treatment, etc.

[0005] However, antibody drugs have large molecular weight and complex structure, are prone to degradation, aggregation or unwanted chemical modification, and are unstable. In addition, monoclonal antibodies are widely used as biological therapeutics, and due to the presence of extracellular matrix, the drug flowability is limited, and in general, the subcutaneous injection volume is generally limited to 1-2 mL, and the antibody formulation concentration needs to reach 90 mg / mL or more to meet the clinical drug dosage requirements, and the solubility requirement is higher.

[0006] WO2019020069 discloses a Romosozumab antibody drug composition and its use, the composition includes acetate buffer, disaccharide, polysorbate 80, calcium salt, etc., which can improve the stability of the antibody, but the solubility of the antibody is not involved. SUMMARY

[0007] The present application provides a stable formulation containing anti-sclerostin antibody, which has good solubility and long-term stability.

[0008] The above object of the present application is achieved by the following technical solutions.

[0009] In one aspect, the present application provides a stable formulation containing anti-sclerostin antibody, which comprises:

[0010] a concentration of 50-200 mg / mL Romosozumab antibody or antigenic fragment thereof;

[0011] a concentration of 10-200 mmol / L of a pharmaceutically acceptable cosolvent; and

[0012] a concentration of 0.01-1 mg / mL of a surfactant; wherein the cosolvent is selected from hydroxypropyl-β-cyclodextrin, the surfactant is polysorbate 20 and polysorbate 80, the volume ratio of polysorbate 20 and polysorbate 80 is 1:1-1:9, and the pH value of the formulation is 4.5-5.5.

[0013] As a preferred technical solution of the present application, the volume ratio of polysorbate 20 and polysorbate 80 is 1:1-1:6, in a specific embodiment, the volume ratio of polysorbate 20 and polysorbate 80 is 1:1-1:4, in a specific embodiment, the volume ratio of polysorbate 20 and polysorbate 80 is 1:4, and in a specific embodiment, the volume ratio of polysorbate 20 and polysorbate 80 is 1:1.75.

[0014] In some embodiments, the concentration of the Romosozumab antibody or antigenic binding fragment thereof is 50-120 mg / mL. In a specific embodiment, the concentration of the anti-sclerostin antibody or antigenic binding fragment thereof is 90-100 mg / mL. In a specific embodiment, the concentration of the anti-sclerostin antibody or antigenic binding fragment thereof is 90 mg / mL. In a specific embodiment, the concentration of the anti-sclerostin antibody or antigenic binding fragment thereof is 95 mg / mL. In a specific embodiment, the concentration of the anti-sclerostin antibody or antigenic binding fragment thereof is 100 mg / mL.

[0015] As a preferred technical solution of the present application, the concentration of hydroxypropyl-β-cyclodextrin is 40-100 mmol / L, preferably 50-60 mmol / L, and more preferably 60 mmol / L; the concentration of polysorbate 20 / polysorbate 80 is 0.1-0.9 mg / mL, preferably 0.5-0.9 mg / mL, and more preferably 0.6 mg / mL.

[0016] As a preferred technical solution of the present application, the preparation further comprises a buffer with a concentration of 1-100 mmol / L; wherein the buffer is selected from acetic acid-sodium acetate, and the concentration of the acetic acid-sodium acetate is preferably 10-50 mmol / L, more preferably 40 mmol / L.

[0017] As a preferred technical solution of the present application, the preparation further comprises calcium chloride with a concentration of 1-20 mmol / L, preferably a concentration of 5-15 mmol / L, and more preferably a concentration of 15 mmol / L.

[0018] As a preferred technical solution of the present application, the preparation further comprises:

[0019] an excipient with a concentration of 10-80 mg / mL, the excipient being selected from any one or a combination of proline, sucrose, sodium chloride, sorbitol, mannitol, or arginine hydrochloride, preferably the excipient is sucrose, and the concentration of the sucrose is preferably 10-80 mg / mL sucrose, and more preferably 45 mg / mL sucrose.

[0020] As a preferred technical solution of the present application, the pH value of the stable liquid preparation is 4.5-5.5. In one embodiment, the pH value of the stable liquid preparation is 5.0-5.2. In a specific embodiment, the pH value of the stable liquid preparation is 5.0, 5.2, or 5.5.

[0021] As a preferred technical solution of the present application, the preparation comprises:

[0022] (1) Romosozumab antibody or an antigen-binding fragment thereof with a concentration of 90 mg / mL-100 mg / mL;

[0023] (2) acetic acid-sodium acetate with a concentration of 40 mmol / mL and calcium chloride with a concentration of 15 mmol / mL;

[0024] (3) polysorbate 20 / polysorbate 80 with a concentration of 0.5-0.9 mmol / mL and hydroxypropyl-β-cyclodextrin with a concentration of 50-90 mmol / mL; and

[0025] (4) sucrose with a concentration of 45 mg / mL-60 mg / mL; wherein the pH value of the preparation is 4.5-5.2.

[0026] In a more preferred embodiment, the stable liquid preparation of the present application comprises:

[0027] (1) Romosozumab antibody or an antigen-binding fragment thereof with a concentration of 95 mg / mL or 100 mg / mL;

[0028] (2) Acetic acid-sodium acetate at a concentration of 40 mmol / mL and calcium chloride at a concentration of 15 mmol / mL;

[0029] (3) Polysorbate 20 / polysorbate 80 at a concentration of 0.6 mg / mL (1 :1.75 by volume) and hydroxypropyl-β-cyclodextrin at a concentration of 60 mmol / mL; and

[0030] (4) Sucrose at a concentration of 45 mg / mL; wherein the pH of the formulation is 4.5-5.2.

[0031] In a second aspect, the present application provides a pre-filled syringe loaded with the above-mentioned stable formulation, which is a liquid formulation, for parenteral administration, preferably subcutaneous or intramuscular injection.

[0032] In a third aspect, the present application provides use of the above-mentioned stable formulation in the manufacture of a medicament for treating, preventing or ameliorating any disease or condition associated with sclerostin, selected from osteoporosis or osteopenia.

[0033] The present application has the following advantages and beneficial effects over the prior art:

[0034] A stable formulation of Romosozumab antibody is provided, which has the characteristics of low viscosity, high solubility and long-term stability, not only meeting the requirements of clinical drug dosage, but also meeting the antibody viscosity limit required for subcutaneous delivery using a syringe at ambient temperature, especially suitable for subcutaneous injection or intramuscular injection administration. DETAILED DESCRIPTION

[0036] The following description of the application is only for illustrating various embodiments of the application. Therefore, the specific modifications discussed herein should not be understood as limiting the scope of the application. Those skilled in the art can easily derive various equivalents, variations and modifications without departing from the scope of the application, and it should be understood that such equivalent embodiments are within the scope of the present application. All documents cited in the present application, including published publications, patents and patent applications, are incorporated by reference in their entirety.

[0037] Definitions

[0038] The terms used in the present application have the following definitions. If no definition is given in the text, the terms used in the present application have the meanings commonly understood by those skilled in the art.

[0039] The term "about" used in the present application, when used in connection with a specific numerical value or numerical range, means that the numerical value can vary by ±20% or ±10%, including ±5%, ±1% and ±0.1%, of the specific numerical value listed, as appropriate to carry out the disclosed methods.

[0040] For the purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural; and vice versa. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0041] “Treatment” of a disease or condition refers to reducing a disease or condition, decreasing the rate of progression of a disease or condition, decreasing the risk of developing a disease or condition, or delaying the development of symptoms associated with a disease or condition, reducing or terminating symptoms associated with a disease or condition, producing a complete or partial reversal of a disease or condition, curing a disease or condition, or a combination thereof.

[0042] “Prevention” includes inhibition of the onset or progression of a disease or condition or symptoms of a particular disease or condition. In some embodiments, a subject with a family history is a candidate for a prophylactic regimen. Generally, the term “prevention” refers to the administration of a drug prior to the onset of signs or symptoms, particularly in a subject at risk.

[0043] "Disorder or condition associated with sclerostin" refers to a disease or condition caused or characterized by changes in sclerostin, such as achondroplasia, cleidocranial dysostosis, enchondromatosis, fibrous dysplasia, Gaucher disease, hypophosphatemic rickets, Marfan syndrome, hereditary multiple exostoses, neurofibromatosis, osteogenesis imperfecta, osteopetrosis, osteopoikilosis, sclerotic lesions, pseudarthrosis, pyogenic osteomyelitis, periodontal disease, antiepileptic drug-induced bone loss, primary or secondary hyperparathyroidism, familial hyperparathyroidism syndrome, weightlessness-induced bone loss, male osteoporosis, postmenopausal bone loss, osteoarthritis, renal osteodystrophy, osteitis fibrosa cystica, oral bone loss, mandibular osteonecrosis, juvenile Paget's disease, melorheostosis, metabolic bone disease, mastocytosis, 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 disease, Turner syndrome, Down syndrome, Cleri- Filtration syndrome, leprosy, Perthe's disease, juvenile idiopathic scoliosis, infantile onset multisystem inflammatory disease, Winchester syndrome, Menkes disease, Wilson's disease, ischemic bone disease (e.g., Caffey-Hyde-Parkinson disease or regional migratory osteoporosis), anemic states, conditions caused by steroids, glucocorticoid-induced bone loss, heparin-induced bone loss, bone marrow disorders, scurvy, malnutrition, calcium deficiency, osteoporosis, osteopenia, alcoholism, chronic liver disease, postmenopausal state, chronic inflammatory conditions, rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, inflammatory colitis, Crohn's disease, oligomenorrhea, amenorrhea, pregnancy, diabetes, hyperthyroidism, thyroid disorders, parathyroid disorders, Cushing's disease, acromegaly, hypogonadism, disuse or immobilization, reflex sympathetic dystrophy syndrome, regional osteoporosis, osteomalacia, bone loss associated with joint replacement, bone loss associated with HIV, bone loss associated with growth hormone deficiency, bone loss associated with cystic fibrosis, bone loss associated with chemotherapy, tumor-induced bone loss, bone loss associated with cancer, hormone ablative bone loss, multiple myeloma, drug-induced bone loss, anorexia nervosa, facial bone loss associated with disease, skull bone loss associated with disease, mandibular bone loss associated with disease, calvarial bone loss associated with disease, bone loss associated with aging, facial bone loss associated with aging, skull bone loss associated with aging, mandibular bone loss associated with aging, calvarial bone loss associated with aging, or bone loss associated with space travel. Methods of identifying / diagnosing the above diseases or conditions are known in the art.

[0044] The term "subject" includes any human or non-human animal. "Non-human animal" includes all vertebrates, e.g., mammals (including, but not limited to, primates, farm animals, such as cows, sheep, cats, dogs, and horses, laboratory animals, such as rabbits, and rodents, e.g., mice and rats), and non-mammals, such as avians, amphibians, and reptiles. In some embodiments, the subject is a human.

[0045] The term "therapeutically effective amount" or "effective dose" refers to an amount or concentration of a formulation, antibody or antigen-binding fragment thereof, or composition effective to achieve the prevention or amelioration of symptoms associated with a disease or condition and / or to reduce the severity of a disease or condition for the desired period of time. The therapeutically effective amount of a formulation, antibody or antigen-binding fragment thereof, or composition of the present application can vary according to 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 is also one in which any toxic or detrimental effects of the formulation, antibody or antigen-binding fragment thereof, or composition are outweighed by the therapeutically beneficial effects.

[0046] The term "formulation" refers to a composition comprising at least one active ingredient and at least one inactive ingredient suitable for administration to an animal, preferably a mammal, including a human. The term "liquid formulation" refers to a formulation in liquid form. The formulation of the present application can be composed as indicated above for the liquid formulation embodiments. The liquid formulation of the present application is preferably an injection, more preferably a subcutaneous or intramuscular injection, most preferably a subcutaneous injection.

[0047] The term "buffer" refers to a pH buffer. Preferably, the buffer is capable of maintaining the pH of the liquid formulation of the present application at about 4.5-5.5, preferably about 4.5-5.2, more preferably 5.0-5.2. The concentration of the buffer in the liquid formulation is about 10-50 mmol / L, preferably 20-40 mmol / L, more preferably 40 mmol / L. The buffer is selected from the group consisting of acetic acid-sodium acetate.

[0048] The term "excipient" refers to a non-therapeutic agent that can be added to a formulation to provide a desired property (e.g., consistency, increased stability) and / or to adjust the osmotic pressure, which, when added to a formulation, is capable of stabilizing the physicochemical and biological properties of the active ingredient. Examples of commonly used excipients include, but are not limited to, sugars, polyols, amino acids, surfactants, and polymers. A preferred excipient is sucrose.

[0049] The term "osmotic pressure regulator" is a molecule that contributes to the osmotic pressure concentration of a solution. The "osmotic pressure regulator" of the present invention refers to a molecule that is capable of adjusting, changing or optimizing the osmotic degree of the anti-sclerostin antibody liquid formulation of the present invention, preferably, the osmotic pressure concentration 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 also minimizing the discomfort of the patient at the time of administration. Examples of osmotic pressure regulators suitable for changing the osmotic pressure concentration 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), and the preferred osmotic pressure regulator is calcium chloride.

[0050] 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, it can protect proteins (e.g., antibodies) from air / solution interface-induced stress, solution / surface-induced stress, thereby reducing protein aggregation or the formation of particulate matter in the formulation. Exemplary surfactants include, but are not limited to, non-ionic surfactants such as polyoxyethylene sorbitan fatty acid esters (e.g., polysorbate 80, polysorbate 60, polysorbate 40, or polysorbate 20), polyethylene-polypropylene copolymers, polyethylene-polypropylene glycol, polyoxyethylene-stearate, polyoxyethylene-alkyl ethers, e.g., polyoxyethylene monolauryl ether, alkylphenyl polyoxyethylene ether (Triton-X), polyoxyethylene-polyoxypropylene copolymers (poloxamer, Pluronic), sodium dodecyl sulfate (SDS). The preferred surfactant of the present invention is a non-ionic surfactant such as polysorbate 80 and polysorbate 20. The preferred surfactants are polysorbate 80 (PS80) and polysorbate 20 (PS20), and the concentration of the surfactant in the liquid formulation is 0.01-1 (mg / mL), preferably 0.01-0.9 (mg / mL), preferably 0.1-0.9 (mg / mL), more preferably 0.1 (mg / mL) or 0.6 (mg / mL).

[0051] The term "viscosity" can be "kinematic viscosity" or "absolute viscosity." "Kinematic viscosity" is a measure of the resistance of a fluid to flow under the influence of gravity. When two identical volumes of fluid are placed in two identical capillary viscometers and allowed to flow by gravity, the viscous fluid takes longer to flow through the capillary than a less viscous fluid. "Absolute viscosity," sometimes called dynamic viscosity or simply viscosity, is the product of kinematic viscosity and fluid density (absolute viscosity = kinematic viscosity x density). The dimension of kinematic viscosity is L2 / T (where L is length and T is time). Typically, kinematic viscosity is expressed in centistokes (cSt). The SI unit of kinematic viscosity is mm2 / s (i.e., lcSt). Absolute viscosity is expressed in units of centipoise (cP). The SI unit of absolute viscosity is millipascal-seconds (mPa-s), where 1 cP = lmPa-s.

[0052] The term "low level viscosity" refers to an absolute viscosity of less than about 15 cp. "Moderate level viscosity" refers to an absolute viscosity of between about 15 cp and about 35 cp. The absolute viscosity of the antibody liquid formulations of the present application are about 3.8 cp, 4.2 cp, 6.0 cp, about 8.0 cp, about 9.0 cp, about 10.0 cp, about 11.0 cp, or about 12.0 cp when measured using standard viscosity measurement techniques, indicating that the liquid formulations of the present application have a "low level viscosity." In some embodiments, the present inventors have surprisingly found that proline not only acts as an osmotic pressure modifier, but also reduces the viscosity of the antibody liquid formulations of the present application when formulated with anti-sclerostin antibodies and a buffer.

[0053] The term "isotonic" refers to a liquid formulation having an osmotic pressure substantially the same as human blood. Isotonic formulations generally have an osmotic pressure of about 250-300 mOsm. Isotonicity can be measured using a vapor pressure or freezing point depression osmometer.

[0054] The term "vehicle" refers to a substance used to mix, disperse, or dissolve a test or control substance without affecting the results of the test. Vehicles that can be used in the present application include, but are not limited to, water for injection, salts (e.g., normal saline), sugars (e.g., dextrose injection), organic solvents for injection (including, but not limited to, oils for injection, ethanol, propylene glycol, etc.), or combinations thereof.

[0055] A "stable" antibody formulation is one in which the antibody remains substantially chemically and / or biologically active throughout the manufacturing process and / or during storage. An antibody formulation can be considered stable even if the antibody contained therein does not retain 100% of its chemical and / or biological properties after a certain period of storage. For example, an antibody formulation can be considered stable if it retains more than 90% of its structure or function after a certain period of storage. The criteria for stability include, for example, the formulation is colorless or clear to slightly opalescent to the naked eye; the antibody concentration, pH, and osmolality of the formulation do not change by more than ±10%; the antibody biological activity is 80-140%, preferably 90-120% of that of a reference antibody; the antibody monomer degradation of the formulation does not exceed 10%, preferably 5%, more preferably 3%; and the aggregate formation in the formulation does not exceed 10%, preferably 5%, more preferably 3%.

[0056] An antibody in a formulation is said to "retain its physical stability" if there is no significant increase in aggregation, precipitation, and / or denaturation of the antibody in the formulation as observed by the naked eye for color and / or clarity, or by differential scanning calorimetry (DSC), size exclusion chromatography (SEC-HPLC), and dynamic light scattering (DLS). An antibody in a formulation is said to "retain its chemical stability" if there is no significant chemical change in the antibody, and its chemical structure remains intact. Most of the chemical instability is attributed to the formation of covalent modifications of the protein (e.g., covalent aggregates, degradation products, or charge variants) and non-covalent modifications of the protein (e.g., non-covalent aggregates). 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 hydrolysis products of the antibody using SEC-HPLC and capillary electrophoresis with sodium dodecyl sulfate (CE-SDS), detection of degradation fragments of the antibody using non-reducing Caliper, detection of other protein molecules with a smaller molecular weight than the antibody using cation exchange chromatography (CE-HPLC), or detection of charge variants of the antibody using cation exchange chromatography (CE-HPLC). An antibody in a formulation is said to "retain its biological activity" if its biological activity remains within the range of biological activity exhibited by the antibody when the formulation was prepared, during the period of storage. Methods for determining the biological activity of an antibody include, but are not limited to, antigen binding ELISA assays or cell-based activity assays for the antibody.

[0057] "Pharmaceutically acceptable" means that the identified carrier, vehicle, diluent, adjuvant, and / or salt is chemically and / or physiologically compatible with the other ingredients of the formulation and physiologically compatible with the subject. DETAILED DESCRIPTION

[0058] The application is further described in connection with the following examples, which are not intended to limit the scope of the application.

[0059] Meanings of English abbreviations:

[0060] HPLC (High Performance Liquid Chromatography): high performance liquid chromatography

[0061] CEX (Cation-exchange): cation exchange

[0062] SEC (Size-Exclusion Chromatography): size exclusion chromatography

[0063] CEX-HPLC: cation exchange chromatography

[0064] SEC-HPLC: size exclusion chromatography

[0065] The anti-sclerostin antibody used in the embodiments of the present application is screened by the method for the antibody romosozumab in Examples 1-5 of patent application No. CN200680024330.8, and a CHO cell strain capable of stably expressing the anti-sclerostin antibody of the present application is constructed, and after cell culture, the supernatant is taken, and the anti-sclerostin antibody (Romosozumab is also referred to as antibody A in the present application) of the present application is purified by steps of affinity chromatography, ion exchange and ultrafiltration, etc. to obtain the anti-sclerostin antibody (Romosozumab is also referred to as antibody A in the present application) of the present application, wherein the purity of the antibody A is 99.1-100.0%.

[0066] Example 1 Screening of cosolvents

[0067] In this experiment, the solubility experiment of the antibody A was carried out under the condition of 15 mM calcium chloride, pH 5.0, and the solubility of the antibody A under the condition of different candidate cosolvents was investigated, and the experimental results are shown in Table 1.

[0068] Table 1 is the solubility of the antibody A in different cosolvents

[0069]

[0070] As can be seen from Table 1, the solubility of the antibody A in Fa1 and Fa2 is better, wherein the solubility of the antibody A in hydroxypropyl-β-cyclodextrin and calcium chloride is 81.62 mg / mL, which is better than the combination of other cosolvents and calcium chloride.

[0071] Example 2 Study of surfactants

[0072] The solubility of antibody A was studied by the experimental scheme in Table 2, and the solubility of antibody A under the conditions of room temperature (25℃), 15mM calcium chloride, pH 5.0, hydroxypropyl-β-cyclodextrin, polysorbate 80, polysorbate 20 and their combinations was investigated. The solubility experiment results are shown in Table 2.

[0073] Table 2 is the solubility experiment of antibody A

[0074]

[0075] As shown in Table 2, a certain proportion of polysorbate 20 / polysorbate 80 surfactant and hydroxypropyl-β-cyclodextrin have a synergistic effect on the solubility of antibody A, and the use of both can increase the solubility of antibody A. Specifically, F9-F12 all have excellent solubility effect.

[0076] Example 3 stability experiment screening

[0077] The stability of antibody A was studied by the experimental scheme in Table 3, and the stability of antibody A under the conditions of 25℃, 15mM calcium chloride, pH 5.0 was investigated. The concentration of antibody A was 100mg / mL, and the stability experiment results are shown in Table 3, in which hydroxypropyl-β-cyclodextrin and polysorbate 20 / polysorbate 80 (volume ratio 1:1.75 and 1:4).

[0078] Table 3 is the stability experiment results

[0079]

[0080] As shown in Table 3, antibody A has better stability in the solutions of hydroxypropyl-β-cyclodextrin, polysorbate 20 / polysorbate 80 (volume ratio 1:1.75), acetic acid-sodium acetate and calcium chloride.

[0081] Example 4 calcium ion concentration screening.

[0082] Select 100mg / ml of antibody A, 60mM hydroxypropyl-β-cyclodextrin+10mM polysorbate 20 / polysorbate 80 (volume ratio 1:1.75)+40mM acetic acid-sodium acetate, pH 5.0, study the effect of different concentrations of calcium ions (5mM, 10mM, 15mM, 20mM, 25mM) on the viscosity of the system, and determine the best calcium ion concentration. The results are shown in Table 4.

[0083] Table 4 is the effect of calcium ion concentration on viscosity

[0084]

[0085]

[0086] As shown in Table 4, when the concentration of calcium ions is 15 mM, the viscosity of the system solution is 3.2, and the viscosity of the system solution does not change significantly with the increase of the concentration of calcium ions.

[0087] Example 5: Screening of the content of adjuvants (sucrose and polysorbate 20 / polysorbate 80 in a volume ratio of 1:1.75).

[0088] When the concentration of antibody A is 100 mg / mL, the stability of samples with different concentrations of sucrose (45 mg / ml, 60 mg / ml) and polysorbate 20 / polysorbate 80 (0.1 mg / ml, 0.6 mg / ml, 0.9 mg / ml) in a 60 mM hydroxypropyl-β-cyclodextrin + 15 mM calcium chloride, pH 5.0 buffer system is investigated, and the prescription combinations are shown in Table 5. The samples are placed under accelerated conditions, and sampling is performed after 4 weeks of storage at 25°C. The appearance, purity (SEC-HPLC), charge heterogeneity (CEX-HPLC), and insoluble particles (MFI) are investigated to evaluate the stability of different prescription formulations, and the results are shown in Table 6.

[0089] Table 5 is the prescription combination for screening of the content of adjuvants

[0090]

[0091] Table 6 is the screening result of the content of adjuvants

[0092]

[0093]

[0094] From the above analysis, it can be seen that when the prescription is 40 mM acetic acid-sodium acetate + 60 mM hydroxypropyl-β-cyclodextrin + 15 mM calcium chloride, the stability of antibody A is good when the concentration of sucrose is 45-60 mg / ml and the concentration of polysorbate 20 / polysorbate 80 is 0.1-0.9 mg / ml.

[0095] The results of the osmotic pressure detection of the screening of adjuvants (sucrose and polysorbate 20 / polysorbate 80) are shown in Table 7. The results show that the osmotic pressure of F4 (sucrose content of 60 mg / ml and polysorbate 20 / polysorbate 80 content of 0.1 mg / ml) will reach 391 mOmsom / kg, and the osmotic pressure of F2 (sucrose content of 45 mg / ml and polysorbate 20 / polysorbate 80 content of 0.6 mg / ml) is 292 mOmsom / kg, which is close to the isotonic condition of the human body (280-310 mOmsom / kg) and has better stability.

[0096] Table 7 is the osmotic pressure test results

[0097]

[0098] From the above table 7, when the prescription is 40mM acetic acid-sodium acetate + 60mM hydroxypropyl-β-cyclodextrin + 15mM calcium chloride, the sucrose concentration is 45mg / ml, and the polysorbate 20 / polysorbate 80 (volume ratio = 1:1.75) is 0.6mg / ml, the osmotic pressure of the system is closest to the isotonic condition of the human body, and has better stability.

[0099] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. A stable formulation characterized in that, Comprising: Romosozumab antibody or antigen binding fragment thereof at a concentration of 90-100 mg / mL; a pharmaceutically acceptable cosolvent at a concentration of 50-60 mmol / L, the cosolvent being selected from hydroxypropyl-β-cyclodextrin; a surfactant at a concentration of 0.1-0.9 mg / mL; wherein the surfactant is polysorbate 20 and polysorbate 80 at a volume ratio of 1:4 or 1:1.75; calcium chloride at a concentration of 5-15 mmol / L; and an excipient at a concentration of 10-80 mg / mL, the excipient being sucrose; the formulation having a pH of 5.0-5.2, wherein the formulation does not comprise methionine.

2. The stable formulation of claim 1, wherein, The concentration of the polysorbate 20 / polysorbate 80 is 0.5-0.9 mg / mL.

3. The stable formulation of claim 2, wherein, The concentration of the polysorbate 20 / polysorbate 80 is 0.6 mg / mL.

4. The stable formulation of claim 1, wherein, The formulation further comprises: a buffer at a concentration of 10-50 mmol / L, the buffer being selected from acetic acid-sodium acetate.

5. The stable formulation of claim 4, wherein, The concentration of the acetic acid-sodium acetate is 40 mmol / L.

6. The stable formulation of claim 1, wherein, The concentration of the sucrose is 45 mg / mL or 60 mg / mL.

7. The stable formulation of claim 1, wherein, The formulation comprises: (1) Romosozumab antibody or antigen binding fragment thereof at a concentration of 100 mg / mL; (2) acetic acid-sodium acetate at a concentration of 40 mmol / mL and calcium chloride at a concentration of 15 mmol / mL; (3) polysorbate 20 / polysorbate 80 at a concentration of 0.6 mg / mL and hydroxypropyl-β-cyclodextrin at a concentration of 60 mmol / mL; and (4) sucrose at a concentration of 45 mg / mL-60 mg / mL; wherein the formulation has a pH of 5.0-5.

2.

8. A pre-filled syringe characterized in that, The pre-filled syringe is loaded with a stable formulation as claimed in any one of claims 1-7, the formulation being a liquid formulation, for parenteral administration.

9. The pre-filled syringe of claim 8, the parenteral administration being selected from subcutaneous or intramuscular injection.

10. Use of a stable formulation as claimed in any one of claims 1-7 in the manufacture of a medicament for the treatment, prevention or amelioration of any disease or condition associated with sclerostin, the disease being selected from osteoporosis or osteopenia.

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