An adalimumab composition

Abstract

The present application belongs to the field of pharmacy, and provides an adalimumab composition, wherein aspartic acid is used as a buffer system. The adalimumab pharmaceutical composition has high physical and chemical stability, and can be stored for a long time.

Description

An adalimumab composition Technical Field

[0001] This invention belongs to the field of pharmaceutical science, specifically relating to adalimumab compositions. Background Technology

[0002] Adalimumab (CAS # 331731-18-1) is a fully human monoclonal antibody that specifically binds to TNF-α in the body, preventing TNF-α from binding to its cell surface receptors, thereby blocking the biological activity of TNF-α. This reduces inflammation and osteoclast activation, ultimately controlling and alleviating symptoms. It can be used to treat rheumatoid arthritis, psoriasis, ankylosing spondylitis, ulcerative colitis, and Crohn's disease.

[0003] However, like other monoclonal antibody drugs, adalimumab is relatively unstable. It is susceptible to various chemical and physical degradations, especially its highly fragile higher-order structures, which are prone to structural changes such as denaturation, aggregation, and precipitation. These degradation or unstable products can significantly impact the safety of biopharmaceuticals. In particular, some protein aggregates can trigger an immune response in the human body, which may reduce the efficacy of the biopharmaceutical in mild cases and even cause death in severe cases. Therefore, polymers are considered a critical quality attribute (CQA) for biopharmaceutical safety, directly affecting the safety of adalimumab. Antibody drugs not only need to achieve high purity during production but also need to maintain structural stability during transportation, storage, and use, which is especially important for products intended for long-term use, such as adalimumab.

[0004] Humira, the original adalimumab product developed and marketed by AbbVie ® This is an aqueous injection containing adalimumab 50 mg / mL, sodium chloride 6.16 mg / mL, sodium citrate 0.35 mg / mL, citrate 1.3 mg / mL, sodium dihydrogen phosphate dihydrate 0.86 mg / mL, disodium hydrogen phosphate dihydrate 1.53 mg / mL, mannitol 12 mg / mL, and polysorbate 80 1 mg / mL, with a solution pH of 5.2. Adalimumab is the active ingredient, sodium chloride is an osmotic pressure regulator, citrate and phosphate are buffers, mannitol provides structural stability, and polysorbate 80 is a surfactant.

[0005] CN201080030083.9 discloses an adalimumab drug formulation that does not contain sodium chloride but contains a high concentration (>20 mg / ml) of polyols such as mannitol. This formulation is different from the already marketed Humira. ®The formulation exhibits higher stability under high-temperature conditions. However, the buffer system in the above formulation is relatively complex, consisting of two buffer solutions, making preparation inconvenient; if it contains citrate, it can cause injection pain at the injection site, reducing patient comfort.

[0006] CN201310611288.1 discloses a stable adalimumab composition using a buffer solution of 0.8-6.2 mg / ml histidine. However, the disclosed formulation contains NaCl as an osmotic pressure regulator, which may lead to problems such as precipitation and gelation (KCl also has this problem).

[0007] Therefore, it remains necessary to develop a novel adalimum antibody formulation to improve the stability of adalimum antibodies, thereby enhancing the uniformity and consistency of product quality and improving its stability in use. Summary of the Invention

[0008] After repeated experimental research and extensive screening of the components and contents of various buffer systems, the inventors made a surprising discovery: in drug compositions containing adalimumab, the use of an aspartic acid buffer system can greatly improve the stability of the adalimumab drug composition, which has a very broad market application prospect.

[0009] Therefore, the present invention provides an adalimumab composition comprising adalimumab and a buffer, wherein the buffer is aspartic acid.

[0010] In a preferred embodiment, the composition further includes a stabilizer and a surfactant.

[0011] In a preferred embodiment, the composition is a liquid formulation.

[0012] In a preferred embodiment, the concentration of adalimumab in the composition is 5 to 100 mg / mL, for example, 20 mg / mL to 100 mg / mL, 50 mg / mL to 100 mg / mL, 60 mg / mL to 100 mg / mL, 70 mg / mL to 100 mg / mL, 80 mg / mL to 100 mg / mL, 10 mg / mL to 90 mg / mL, 20 mg / mL to 80 mg / mL, or 30 mg / mL to 50 mg / mL; in some specific embodiments, the concentration of adalimumab is about 10 mg / mL, about 20 mg / mL, about 30 mg / mL, about 50 mg / mL, about 60 mg / mL, about 65 mg / mL, about 70 mg / mL, about 75 mg / mL, about 80 mg / mL, about 85 mg / mL, about 90 mg / mL, about 95 mg / mL, about 100 mg / mL, or any value between any two values. The preferred concentrations are 5 mg / mL, 50 mg / mL, or 100 mg / mL.

[0013] In a preferred embodiment, the concentration of aspartic acid is 5 to 20 mM, for example, 5 mM to 15 mM, 5 mM to 10 mM, 10 mM to 15 mM, or 8 mM to 12 mM; in some specific embodiments, the concentration of the buffer is about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 18 mM, about 20 mM, or any value between any two values; preferably 5 mM, 10 mM, or 20 mM.

[0014] In a preferred embodiment, the stabilizer is sucrose, trehalose dihydrate, or mannitol; preferably, the concentration of the stabilizer is 43-91 mg / mL; for example, 45 mg / mL to 91 mg / mL, 50 mg / mL to 91 mg / mL, 60 mg / mL to 83 mg / mL, 70 mg / mL to 90 mg / mL, 70 mg / mL to 80 mg / mL, or 43 mg / mL to 60 mg / mL; in some specific embodiments, the concentration of the stabilizer is about 83 mg / mL, about 97.3 mg / mL, about 43.7 mg / mL, or any value between any two values. Preferably, it is 83 mg / mL sucrose, 90.7 mg / mL trehalose dihydrate, or 43.7 mg / mL mannitol.

[0015] In a preferred embodiment, the surfactant is polysorbate 80; preferably, the concentration of polysorbate 80 is 0.8 mg / mL to 2 mg / mL, for example, 0.8 mg / mL to 1.5 mg / mL, 0.8 mg / mL to 1.2 mg / mL, or 0.8 mg / mL to 1 mg / mL. Preferably, it is 1 mg / mL.

[0016] In a preferred embodiment, the pH value of the composition is 4.8 to 5.6, for example 4.8 to 5.5, 4.8 to 5.4, 4.8 to 5.2, or 5.2 to 5.6; in some specific embodiments, the pH value of the buffer is about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, or about 5.6; preferably 4.8, 5.2, or 5.6.

[0017] In a preferred embodiment, the composition comprises: 5 mg / mL to 100 mg / mL adalimumab, 80 mg / mL to 90 mg / mL sucrose, 0.8 mg / mL to 2 mg / mL polysorbate 80, and 5 mM to 20 mM aspartic acid, with a pH of 4.8 to 5.6.

[0018] In a preferred embodiment, the composition comprises: 5 mg / mL to 100 mg / mL adalimumab, 83 mg / mL sucrose, 0.8 mg / mL to 2 mg / mL polysorbate 80 and 5 mM to 20 mM aspartic acid, with a pH of 4.8 to 5.6.

[0019] In a preferred embodiment, the composition comprises: 5 mg / mL to 100 mg / mL adalimumab, 83 mg / mL sucrose, 1 mg / mL polysorbate 80 and 5 mM to 20 mM aspartic acid, with a pH of 4.8 to 5.6.

[0020] In a specific embodiment, the composition is any one of the following:

[0021] (1) 50 mg / mL adalimumab, 83 mg / mL sucrose, 1 mg / mL polysorbate 80 and 20 mM aspartic acid, pH 5.2;

[0022] (2) 5 mg / mL adalimumab, 83 mg / mL sucrose, 1 mg / mL polysorbate 80 and 20 mM aspartic acid, pH 5.2;

[0023] (3) 5 mg / mL adalimumab, 83 mg / mL sucrose, 1 mg / mL polysorbate 80 and 20 mM aspartic acid, pH 4.8;

[0024] (4) 5 mg / mL adalimumab, 83 mg / mL sucrose, 1 mg / mL polysorbate 80 and 20 mM aspartic acid, pH 5.6;

[0025] (5) 5 mg / mL adalimumab, 83 mg / mL sucrose, 1 mg / mL polysorbate 80 and 5 mM aspartic acid, pH 5.2;

[0026] (6) 5 mg / mL adalimumab, 83 mg / mL sucrose, 1 mg / mL polysorbate 80 and 10 mM aspartic acid, pH 5.2;

[0027] (7) 100 mg / mL adalimumab, 83 mg / mL sucrose, 1 mg / mL polysorbate 80 and 20 mM aspartic acid, pH 5.2;

[0028] (8) 5 mg / mL adalimumab, 90.7 mg / mL trehalose dihydrate, 1 mg / mL polysorbate 80 and 20 mM aspartic acid, pH 5.2;

[0029] (9) 5 mg / mL adalimumab, 43.7 mg / mL mannitol, 1 mg / mL polysorbate 80 and 20 mM aspartic acid, pH 5.2.

[0030] In a second aspect, a lyophilized formulation containing adalimumab is provided, said lyophilized formulation being obtained by freeze-drying the composition described above herein.

[0031] In a third aspect, the use of aspartic acid as a buffer in the preparation of adalimumab compositions or lyophilized formulations containing adalimumab as described in this invention is provided.

[0032] In a fourth aspect, a method for preparing the composition of the present invention is provided, comprising accurately weighing various components according to the formulation of the composition, preparing it with water for injection, and adjusting the pH value.

[0033] In a fifth aspect, the invention provides the use of adalimumab compositions or lyophilized formulations containing adalimumab in the preparation of medicaments for treating inflammatory diseases. In some embodiments, the inflammatory disease is selected from rheumatoid arthritis, ankylosing spondylitis, psoriasis, psoriatic arthritis, juvenile idiopathic arthritis, Crohn's disease, ulcerative colitis, hidradenitis suppurativa, and uveitis.

[0034] In a sixth aspect, a method for treating and / or preventing inflammatory diseases is also provided, comprising administering to a subject in need an effective amount of the aforementioned adalimumab composition or lyophilized formulation or a reconstituted solution of the lyophilized formulation for treatment and / or prevention. In some embodiments, the subject is a human subject.

[0035] In a seventh aspect, the adalimumab composition of the present invention can be used as a medicine. In some embodiments, it is used as a medicine for treating inflammatory diseases.

[0036] In this invention, the adalimumab comprises a light chain and a heavy chain, the amino acid sequence of which is shown in SEQ ID NO: 1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO: 2. The preparation and reconstitution of the lyophilized formulation described in this invention are conventional techniques in the art.

[0037] This invention significantly improves the physical and chemical stability of adalimumab drug compositions by using an aspartic acid buffer system, especially the stability under high temperature and light conditions, enabling the prepared adalimumab compositions to be stored stably for a long period of time. Attached Figure Description

[0038] Figure 1 shows the DSC experiment T at 0 for the three formulations. onset Comparison chart;

[0039] Figure 2 shows the DSC experiment T at 0 for the three formulations. m1 Comparison chart. Detailed Implementation

[0040] The present invention will be described below through specific embodiments. Unless otherwise stated, the instruments and reagents used in the present invention are conventional instruments and reagents that can be obtained commercially; the methods used are conventional technical methods, and those skilled in the art can undoubtedly know how to implement the present invention and obtain the corresponding results based on the content described in the embodiments.

[0041] The term “comprising” encompasses both the meaning of “including” and “consisting of”. For example, a composition that “comprising” X may consist of only X or may include other components, such as X+Y.

[0042] “Composition” means a mixture containing one or more of the compounds described herein or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity. In this document, “pharmaceutical composition,” “composition,” “formulation,” and “prescription” are used interchangeably. Unless otherwise specified, the solvent in the solution form of the pharmaceutical compositions described herein is water.

[0043] "Buffer" refers to a substance that is resistant to pH changes through the action of its acid-base conjugate components. Suitable buffering agents for use in the compositions disclosed herein include, but are not limited to, histidine buffer, citrate buffer, phosphate (e.g., sodium or potassium) buffer, succinate (e.g., sodium) buffer, acetate buffer, Tris buffer, glycine, arginine, aspartic acid, and combinations thereof. Preferred buffer is aspartic acid.

[0044] Surfactants suitable for use in the compositions disclosed herein include, but are not limited to, nonionic surfactants, ionic surfactants, amphoteric surfactants, and combinations thereof. Typical surfactants used in this 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), and polyoxyethylene sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monostea ... Polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate, polyoxyethylene sorbitan tristearate, polyoxyethylene sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan tetrastearate, polyoxyethylene sorbitan tetraoleate), polyoxyethylene glycerol fatty acid esters (e.g., polyoxyethylene glycerol monostearate), polyethylene glycol fatty acid esters (e.g., polyethylene glycol distearate), polyoxyethylene alkyl ethers (e.g., polyoxyethylene alkyl ethers). Polyoxyethylene lauryl ether, polyoxyethylene polyoxypropylene alkyl ether (e.g., polyoxyethylene polyoxypropylene glycol, polyoxyethylene polyoxypropylene propyl ether, polyoxyethylene polyoxypropylene cetyl ether), polyoxyethylene alkylphenyl ether (e.g., polyoxyethylene nonylphenyl ether), polyoxyethylene hydrogenated castor oil (e.g., polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil), polyoxyethylene beeswax derivatives (e.g., polyoxyethylene sorbitan beeswax), polyoxyethylene lanolin derivatives (e.g., polyoxyethylene lanolin), and polyoxyethylene fatty acid amides (e.g., polyoxyethylene... The composition may contain: stearamide; C10-C18 alkyl sulfates (e.g., sodium cetyl sulfate, sodium lauryl sulfate, sodium oleyl sulfate), polyoxyethylene C10-C18 alkyl ether sulfates (e.g., polyoxyethylene sodium lauryl sulfate) having an average addition of 2 to 4 moles of ethylene oxide units, and C1-C18 alkyl sulfosuccinates (e.g., sodium lauryl sulfosuccinate); and natural surfactants such as lecithin, glycerophospholipids, sphingomyelin (e.g., sphingomyelin), and sucrose esters of C12-C18 fatty acids. The composition may contain one or more of these surfactants. Preferred surfactants are polyoxyethylene sorbitan fatty acid esters, such as polysorbate 20, 40, 60, or 80. Polysorbate 80 (Tween 80) (e.g., at a concentration of about 1 mg / mL) is particularly suitable.

[0045] "Lyophilized formulation" refers to a formulation or pharmaceutical composition obtained by a vacuum freeze-drying step after a liquid or solution form of a pharmaceutical composition or solution preparation.

[0046] As used herein, the terms “about” or “approximately” mean a numerical value within an acceptable margin of error for a specific value determined by a person skilled in the art, the numerical value depending in part on how it is measured or determined (i.e., the limits of the measurement system). For example, in every practice in the art, “about” may mean within or above a standard deviation of 1. Alternatively, “about” or “substantially comprises” may mean a range of up to ±20%, for example, about 5.5 pH means pH 5.5 ± 1.1. Furthermore, particularly for biological systems or processes, the term may mean up to an order of magnitude or up to five times the numerical value. Unless otherwise stated, when a specific value appears in this application and claims, the meaning of “about” or “substantially comprises” should be assumed to be within an acceptable margin of error for that specific value.

[0047] The pharmaceutical compositions described herein achieve a stable effect: the antibodies contained therein substantially retain their physical and / or chemical stability and / or biological activity after storage; for example, the pharmaceutical compositions substantially retain their physical and chemical stability and their biological activity after storage. The storage period is generally selected based on the intended shelf life of the pharmaceutical composition. Currently, various analytical techniques are available for measuring protein stability, which can measure stability after storage at a selected temperature for a selected period of time.

[0048] Stable drug antibody formulations are those in which no significant changes are observed when stored at refrigerated temperatures (2-8°C) for at least 3 months, at least 6 months, at least 1 year, and up to 2 years. Additionally, stable liquid formulations include those that exhibit the desired characteristics after storage at 25°C for 1 month, 3 months, or 6 months, or at 40°C for 1 month. Typical acceptable criteria for stability are as follows: degradation of antibody monomers typically not exceeding about 10%, e.g., not exceeding about 5%, as determined by SEC-HPLC. By visual analysis, the drug antibody formulation is colorless or clear to slightly milky white. The concentration, pH, and osmotic pressure of the formulation exhibit variations not exceeding ±10%. Truncation typically not exceeding about 10%, e.g., not exceeding about 5%, and aggregation typically not exceeding about 10%, e.g., not exceeding about 5%, are observed.

[0049] If, after visual inspection of color and / or clarity, or by means of UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS), the antibody does not show significant increase in aggregation, precipitation, and / or denaturation, then the antibody “retains its physical stability” in the pharmaceutical formulation. Changes in protein conformation can be evaluated by fluorescence spectroscopy (which determines the tertiary structure of the protein) and by FTIR spectroscopy (which determines the secondary structure of the protein).

[0050] If an antibody does not show significant chemical changes, then the antibody "retains its chemical stability" in the pharmaceutical formulation. Chemical stability can be assessed by detecting and quantifying the chemically altered form of the protein. Degradation processes that frequently alter the chemical structure of a protein include hydrolysis or truncation (evaluated by methods such as size exclusion chromatography and SDS-PAGE), oxidation (evaluated by methods such as peptide mapping combined with mass spectrometry or MALDI / TOF / MS), deamidation (evaluated by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, and isofpartate measurement), and isomerization (evaluated by measuring isofpartate content, peptide mapping, etc.).

[0051] If the antibody's biological activity at a given time is within a predetermined range of the biological activity exhibited when the pharmaceutical formulation is prepared, then the antibody "retains its biological activity" in the pharmaceutical formulation. The biological activity of an antibody can be determined, for example, by an antigen-binding assay.

[0052] In this document, unless otherwise specified or clearly inappropriate, all proportions, including percentages, are by weight.

[0053] In this article, " / " indicates that it is not present or not detected.

[0054] When applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, "giving" and "treatment" refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Giving" and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and cells, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cells. "Giving" and "treatment" also mean the treatment of, for example, cells, by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo. When applied to humans, veterinary, or research subjects, "treatment" refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.

[0055] "Treatment" means administering an oral or topical therapeutic agent, such as a composition comprising any of the compounds disclosed herein, to a subject who has symptoms of one or more diseases, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, a therapeutic agent is administered in a treated subject or population in an amount that effectively relieves symptoms of one or more diseases to induce regression of such symptoms or inhibit their progression to any clinically measurable extent. The amount of a therapeutic agent that effectively relieves any specific disease symptom (also referred to as a "therapeuticly effective amount") can vary depending on a variety of factors, such as the subject's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the subject. Whether the disease symptoms have been relieved can be evaluated using any clinical test that a physician or other healthcare professional typically uses to assess the severity or progression of the symptoms. Although the implementation methods disclosed herein (e.g., treatments or products) may be ineffective in alleviating symptoms of each target disease, they should reduce symptoms of the target disease in a statistically significant number of subjects, as determined by any statistical test known in the art, such as the Student t-test, chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

[0056] An "effective dose" includes an amount sufficient to improve or prevent the symptoms or condition of a medically diagnosed disease. An effective dose also means an amount sufficient to allow or facilitate diagnosis. The effective dose for a specific subject or veterinary subject can vary depending on factors such as the condition to be treated, the subject's overall health, the route and dosage of administration, and the severity of side effects. An effective dose can be the maximum dose or administration regimen that avoids significant side effects or toxicity.

[0057] The "Tm value" refers to the thermal denaturation temperature of a protein, which is the temperature at which half of the protein unfolds, at which point the protein's spatial structure is disrupted. Therefore, the higher the Tm value, the higher the protein's thermal stability. Typically, antibodies have three transition regions: Tm1 relative to the CH2 domain of the Fc region, Tm2 relative to the Fab region, and Tm3 relative to the CH3 domain of the Fc region.

[0058] "Tonset" is T 起始 , is the temperature at which a protein begins to unfold.

[0059] This document also covers kits for treating various inflammatory diseases, such as psoriasis. These kits generally include at least one composition disclosed herein, along with instructions for use. The instructions disclose suitable techniques for providing stable compositions to patients as part of a dosing regimen. These kits may also contain other agents for treating inflammatory diseases such as psoriasis (e.g., immunosuppressants, DMARDs, pain controllers, steroids, nonsteroidal anti-inflammatory drugs (NSAIDs), etc.) for use in combination with the contained composition (i.e., simultaneous or sequential [before or after]).

[0060] Stability testing experiment:

[0061] Adalimumab drug composition solutions were prepared using water for injection, and stability studies were conducted on each formulation.

[0062] In this invention, the stability test can be carried out from the following aspects:

[0063] Vibration test: Vibrate for 3 days under sealed, light-proof conditions at 25℃ and 300rpm.

[0064] Freeze-thaw test: 3 freeze-thaw cycles, with the freeze-thaw conditions being -20℃ for 2 days and thawing at room temperature (approximately 25℃) for 2 days.

[0065] Light exposure test: Place the sample in a light box (5℃±3℃, 4500Lx±500Lx) and take the sample after 10 days.

[0066] High temperature test: The samples were placed in an incubator (40℃±3℃, protected from light) and samples were taken after 7 days and 14 days.

[0067] Accelerated 1-month test: Place the sample in a constant temperature and humidity chamber (25℃±3℃, protected from light) and take the sample after 1 month.

[0068] Three copies of the sample were placed in parallel for each condition.

[0069] Reagents and testing instruments

[0070] Adalimumab, prepared according to CN1300173C, has the following sequence:

[0071] Light chain:

[0072] DIQMTQSPSSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ IDNO: 1)

[0073] Heavy chain:

[0074] EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 2)

[0075] Reagents:

[0076] Sugar, purchased from Merck KGaA;

[0077] Trehalose dihydrate was purchased from Hayashibara Co., Ltd., Japan.

[0078] Polysorbate 80 was purchased from JT Baker (product number 4117-04) or Nanjing Well Pharmaceutical Group Co., Ltd.

[0079] instrument:

[0080] The Agilent 1200 liquid chromatograph was purchased from Agilent Technologies, Inc., USA.

[0081] SEC-HPLC column: TSK-GEL G3000 SWXL, 7.8×300mm, from TOSOH, Japan; mobile phase: 0.08mol / L sodium dihydrogen phosphate, 0.5mol / L sodium chloride buffer, pH 7.0; flow rate: 0.5ml / min; column temperature: room temperature; run time: 30min; wavelength: 280nm.

[0082] IEC-HPLC column: Propac WCX-10, 4.0 mm, Thermo Fisher Scientific (USA) 250 mm; Mobile phase A: 0.01 mol / L disodium hydrogen phosphate, pH=7.5; Mobile phase B: 0.01 mol / L disodium hydrogen phosphate, 0.5 mol / L sodium chloride, pH=5.5; Column temperature: 30℃; Wavelength: 280 nm; Run time: 30 min; Run gradient is shown in Table 1 below.

[0083] Table 1 Operating Conditions

[0084]

[0085] The differential scanning calorimeter (DSC) was purchased from Malvern.

[0086] CE-SDS: The instrument was an Agilent 7100 capillary electrophoresis system; effective capillary length 220 mm, total length 305 mm; injection voltage -10.0 kV for 40 s; operating voltage -15.0 kV; running time 40 min; detection wavelength 214 nm; column temperature 20 ℃.

[0087] method

[0088] Protein content determination method: Protein content was determined using the Unchained Labs Lunatic high-throughput protein detection system. The absorbance of the sample at 280 nm was measured, and an extinction coefficient of 1.369 L was used. g -1 cm -1 Calculations are performed to obtain the protein content.

[0089] Method for calculating relative protein content: Using the sum of the peak areas of all peaks in SEC-HPLC at 0 as the benchmark, calculate the relative ratio of the sum of the peak areas of all peaks in SEC-HPLC under various experimental conditions to this benchmark.

[0090] Example 1

[0091] This example compares the effects of aspartic acid (formulation 1), histidine (formulation 2), and phosphate / citric acid (the buffer system used in the original product, formulation 3) as buffer solutions on the stability of adalimumab drug compositions. Adalimumab antigen solution was subjected to ultrafiltration replacement, replacing the original buffer solution with a low concentration of phosphate / citric acid (1.75 mM / 0.925 mM pH 5.2). The protein content of the concentrate after ultrafiltration replacement was measured, and formulations 1-3 as listed in Table 2 were prepared based on the protein content of the original solution.

[0092] Table 2. Composition of the formulation

[0093]

[0094] Formulations 1-3 were inspected for appearance and visible foreign matter; all were colorless and free of visible foreign matter. The polymer content was determined by SEC-HPLC, and the purity of the charge isomers was determined by IEC-HPLC. The results are shown in Table 3.

[0095] Table 3. Protein purity by SEC-HPLC and IEC-HPLC

[0096]

[0097] Data are expressed as mean ± standard deviation, repeated 3 times, the same applies below.

[0098] Table 3 shows that the monomer content detected by SEC-HPLC in formulations 1-3 was 99.71%-99.75%, which was very close. This indicates that the preparation process of the formulations was relatively accurate. The protein content was calculated as 100%. The data at 0 time of each formulation can be used for comparison to detect and analyze the accelerated test and evaluate each formulation.

[0099] Thermal stability test

[0100] Differential scanning calorimetry (DSC) was performed on samples of the three formulations at time 0 to determine the denaturation temperature (T). onset and T m1 The results were used to evaluate its thermal stability, and the specific results are shown in Figure 1-2.

[0101] Figure 1 shows the temperature (T) at which the three prescription formulations began to denature. onset The trend of T values ​​for formulations 1-3 onset The values ​​were 66.7℃, 60.2℃, and 62.9℃, respectively. From T... onset In terms of value, the T of formulation 1 onset It has the highest T value, the best effect, and the highest thermal stability; Formulation 2 has the highest T value. onsetThe lowest value indicates that it has the lowest temperature at which it begins to denature and the worst thermal stability.

[0102] Figure 2 shows the temperature (T) at which 50% denaturation occurred in the three formulations. m1 The trend of T values ​​for formulations 1-3 m1 The values ​​were 75.0, 66.3, and 73.9 (°C), respectively. From T m1 For example, the T of formulation 1 m1 Formulation 1 has the highest T value, the best effect, and the highest thermal stability, followed by Formulation 3, while Formulation 2 has the highest T value. m1 The value is the lowest.

[0103] In summary, formulation 1 exhibits better thermal stability than formulations 3 and 2.

[0104] Stability test

[0105] Vibration test

[0106] The liquid formulation samples 1-3, which had been vibrated for 3 days, were compared and analyzed by SEC-HPLC and IEC-HPLC. The results are shown in Table 4.

[0107] Table 4 shows that there were no significant changes in the SEC-HPLC purity, IEC-HPLC purity, and protein content of formulations 1-3. This indicates that formulations 1-3 all exhibited good stability under vibration conditions.

[0108] Table 4. SEC-HPLC and IEC-HPLC results of formulations 1-3 after 3 days of vibration.

[0109]

[0110] freeze-thaw test

[0111] The liquid formulation samples 1-3 that underwent freeze-thaw cycles were compared using SEC-HPLC and IEC-HPLC, and the results are shown in Table 5.

[0112] Table 5 shows that after three freeze-thaw cycles, the SEC-HPLC purity, polymer content, and protein content of formulations 1-3 remained unchanged. The main peak content of formulation 3 was lower than that of other formulations in IEC-HPLC, indicating that formulations 1 (aspartic acid) and 2 (histidine) were superior to formulation 3 (phosphate / citric acid buffer).

[0113] Table 5. Protein purity of the formulation after three freeze-thaw cycles using SEC-HPLC and IEC-HPLC.

[0114]

[0115] Note: The Δ value represents the difference between the parameter detected after processing under different conditions and the corresponding parameter when it is 0, reflecting the change of the corresponding parameter under different processing conditions.

[0116] High temperature test

[0117] Formulations 1-3, after being subjected to high temperature for 7 days and 14 days (40℃), were analyzed by SEC-HPLC and IEC-HPLC, respectively. The results are shown in Table 6.

[0118] From Tables 6-1 and 6-2, we can conclude that: ① After 7 and 14 days of high-temperature degradation, the SEC-HPLC protein purity and polymer content of formulations 1 and 3 showed varying degrees of decrease and increase. The polymer content of formulations 1 and 2 was lower than that of formulation 3, while the protein content remained largely unchanged except for a slight decrease (98.9%) in formulation 3 after 14 days of high-temperature degradation. This indicates that formulations 1 and 2 have better physical stability under high-temperature conditions than formulation 3. ② The CE-SDS purity and IEC-HPLC purity of formulations 1 and 2 after high-temperature degradation were higher than those of formulation 3, indicating that the chemical stability of formulations 1 and 2 was also higher than that of formulation 3.

[0119] Table 6-1 Purity of Formulations after 7 and 14 Days of High Temperature (SEC-HPLC and IEC-HPLC)

[0120]

[0121] Table 6-2 Changes in SEC-HPLC and IEC-HPLC of the formulation after 7 and 14 days of high temperature.

[0122]

[0123] In summary, formulations prepared using aspartic acid as a buffer are superior to those using the phosphate / citric acid buffer in the original marketed product, especially under high-temperature conditions; and formulation 1 containing aspartic acid as a buffer exhibits higher thermal stability (higher TL). monset and T m1 ).

[0124] Example 2

[0125] This embodiment compares the effects of buffer solution and pH value on the stability of adalimumab drug compositions. The adalimumab antigen solution was subjected to ultrafiltration to replace the original buffer solution before concentration. The concentrated ultrafiltrate was then analyzed for protein content, and formulations 4-9 listed in Table 7 were prepared based on the protein content (formulation 4 uses a phosphate / citric acid buffer system).

[0126] Table 7. Formulation Composition of Pharmaceutical Preparations

[0127]

[0128] After stability tests were conducted on each formulation sample (vibration for 3 days; freeze-thaw cycles for 3 days; high temperature for 7 / 14 days; light exposure for 10 days; accelerated testing for 1 month), its appearance, visible foreign matter, protein content (results are shown in Table 8), and SEC-HPLC purity (results are shown in Table 9).

[0129] No particles were found in the appearance or visible foreign matter of any of the formulations, indicating that they were all relatively stable.

[0130] As can be seen from Table 8 below, there are no significant changes in the protein content of each formulation.

[0131] As shown in Table 9 below, ① the polymer content of formulations 5-9 is lower than that of formulation 4, while the monomer purity is higher, especially after 10 days of light exposure. ② From the SEC-HPLC purity data, different contents of aspartic acid (5-20 mM) and different pH values ​​(4.8-5.6) all showed good protective effects against adalimumab.

[0132] Table 8. Protein content (mg / mL) of formulations 4-9

[0133]

[0134] Table 9. SEC-HPLC changes of formulations 4-9 (compared to 0).

[0135]

[0136] Example 3

[0137] This embodiment compares the effects of protein content and stabilizer type on the stability of adalimumab drug compositions. The adalimumab antigen solution was subjected to ultrafiltration to replace the original buffer solution before concentration. The concentrated ultrafiltrate was then analyzed for protein content, and formulations 4, 11, 13, 14, and 15 (formulation 4 uses a phosphate / citric acid buffer system) were calculated and prepared based on the protein content and listed in Table 10.

[0138] Table 10 Formulation Composition

[0139]

[0140] After stability tests (vibration for 3 days; freeze-thaw cycles for 3 days; high temperature for 14 days; light exposure for 10 days; accelerated testing for one month) were conducted on the formulation samples, their appearance, visible foreign matter, protein content, SEC-HPLC purity, and IEC-HPLC purity were then measured. No particles were found in the appearance or visible foreign matter of any of the formulations, indicating that they were all relatively stable.

[0141] As can be seen from the protein content in Table 11 below, there were no significant changes in the formulations of each preparation.

[0142] From Table 12 below, based on SEC-HPLC purity, we can see that: ① Formulations 11-15 remained stable under all stability test conditions. Compared to formulation 4, formulations 11-15 showed better protective effects after 10 days of light exposure. ② Based on SEC-HPLC purity, different protein contents (5-100 mg / mL) and different stabilizers (sucrose, trehalose dihydrate, mannitol) all showed good protective effects against adalimumab.

[0143] From Table 13 below, based on IEC-HPLC purity, we can see that: ① Formulations 11-15 remained stable under all stability test conditions. Compared to formulation 4, formulations 11-15 showed better protective effects under high temperature for 14 days and light exposure for 10 days. ② Based on IEC-HPLC purity, different protein contents (5-100 mg / mL) and different stabilizers (sucrose, trehalose dihydrate, arginine hydrochloride, mannitol) all showed good protective effects against adalimumab.

[0144] Table 11 Protein content results (mg / mL)

[0145]

[0146] Table 12 Changes in SEC-HPLC

[0147]

[0148] Table 13 Changes in IEC-HPLC Purity

[0149]

Claims

1. An adalimumab composition comprising adalimumab, a buffer, a stabilizer, and a surfactant, wherein, The concentration of adalimumab is 5 mg / mL to 100 mg / mL; the buffer is aspartic acid, and the concentration of aspartic acid is 5 mM to 20 mM; the stabilizer is 90.7 mg / mL trehalose dihydrate, 83 mg / mL sucrose, or 43.7 mg / mL mannitol; the surfactant is polysorbate 80, and its concentration is 1 mg / mL; the pH value of the composition is 4.8 to 5.

6.

2. The composition according to claim 1, wherein, The concentration of adalimumab is 5 mg / mL, 50 mg / mL or 100 mg / mL.

3. The composition according to claim 1, wherein, The concentration of aspartic acid is 5 mM, 10 mM or 20 mM.

4. The composition according to claim 1, wherein, The composition has a pH value of 4.8, 5.2, or 5.

6.

5. The composition according to any one of claims 1 to 4, wherein, The composition is any one of the following: (1) 50 mg / mL adalimumab, 83 mg / mL sucrose, 1 mg / mL polysorbate 80 and 20 mM aspartic acid, pH 5.2; (2) 5 mg / mL adalimumab, 83 mg / mL sucrose, 1 mg / mL polysorbate 80 and 20 mM aspartic acid, pH 5.2; (3) 5 mg / mL adalimumab, 83 mg / mL sucrose, 1 mg / mL polysorbate 80 and 20 mM aspartic acid, pH 4.8; (4) 5 mg / mL adalimumab, 83 mg / mL sucrose, 1 mg / mL polysorbate 80 and 20 mM aspartic acid, pH 5.6; (5) 5 mg / mL adalimumab, 83 mg / mL sucrose, (6) 5 mg / mL adalimumab, 83 mg / mL sucrose, 1 mg / mL polysorbate 80 and 10 mM aspartic acid, pH 5.2; (7) 100 mg / mL adalimumab, 83 mg / mL sucrose, 1 mg / mL polysorbate 80 and 20 mM aspartic acid, pH 5.2; (8) 5 mg / mL adalimumab, 90.7 mg / mL trehalose dihydrate, 1 mg / mL polysorbate 80 and 20 mM aspartic acid, pH 5.2; and (9) 5 mg / mL adalimumab, 43.7 mg / mL mannitol, 1 mg / mL polysorbate 80 and 20 mM aspartic acid, pH 5.

2.

6. A lyophilized formulation containing adalimumab, wherein the lyophilized formulation is obtained by freeze-drying the composition according to any one of claims 1 to 5.

7. Use of the adalimumab composition according to any one of claims 1 to 5 or the lyophilized formulation containing adalimumab according to claim 6 in the preparation of a medicament for treating inflammatory diseases; wherein the inflammatory diseases are selected from rheumatoid arthritis, ankylosing spondylitis, psoriasis, psoriatic arthritis, juvenile idiopathic arthritis, Crohn's disease, ulcerative colitis, hidradenitis suppurativa, and uveitis.

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