HK1204921B - Stable liquid formulation of etanercept - Google Patents

Description

Stable liquid formulation of etanercept

The application is a divisional application of an invention patent application with the application date of 2012, 06, 01, and the application number of 201280026919.7, and the name of the invention is 'etanercept stable liquid preparation'.

Technical Field

The present invention relates to a stable liquid formulation of etanercept (recombinant p75 sTNFR: Fc fusion protein), in particular, to a liquid formulation comprising one or more stabilizers selected from the group consisting of: methionine, lysine, histidine and pharmaceutically acceptable salts thereof, in an amount sufficient to reduce the formation of etanercept by-products during storage.

Background

Etanercept is a biological modulator of inflammation that acts as a competitive inhibitor of TNF- α, binds to cell surface TNF- α receptors, and inhibits TNF- α mediated immune responses. Etanercept is a macromolecule with a molecular weight of about 150kDa and is a homodimer of two Fc fusion proteins linked by disulfide bonds, where each Fc fusion protein is composed of a human soluble p75TNF receptor coupled to the Fc portion of human immunoglobulin G subclass 1 (Goldenberg, Clinical Therapeutics, 21 (1): 75-87, 1999; Moreland et al, ann. lnn. med., 130 (6): 478-.

The prototype fusion protein was synthesized by Bruce A. Beutler (southwest Medical Center of Texas University, University of Texas) at the earliest in the 90 s of the 20 th century and was marketed by the Ann corporation (Amgen) under the trade name of Enbrel (Enbrel) in 2002. Etanercept is a TNF-alpha inhibitor used in the treatment of rheumatoid arthritis, psoriasis and ankylosing spondylitis and has been clinically tested for the treatment of vasculitis, alzheimer's disease and crohn's disease.

Similar to protein drugs, antibody drugs have a very short half-life, and are prone to chemical and physical denaturation by adverse temperature, shear stress, vibration, freeze-thaw, ultraviolet irradiation, excessive pH change, organic solvents, and microbial contamination. Chemical denaturation, including dimer dissociation, oxidation, deamidation, isomerization, and polymerization, is affected by the amino acids that make up the antibody and the solvent conditions (salt, pH, and temperature) in which the antibody is contained. Physical denaturation, which includes loss of tertiary structure, covalent/non-covalent aggregation and adhesion of monomers, is affected by hydrophobic regions (patches) on the protein surface that are altered by the surrounding environment containing the antibody, such as solvents, complex protein structure, such as charge distribution, and thermal stabilization. Physical or chemical denaturation of an antibody results in loss of its physiological activity. Since denaturation is an irreversible process, once denatured, an antibody may not be able to restore its natural properties to the original state, resulting in a reduction in its therapeutic efficacy. It has also been suggested that monomeric aggregation causes an immune response. Thus, many studies have been conducted on formulations containing physiologically effective amounts of Antibody but without aggregates (Ishikawa et al, biol. pharm. Bull., 33(8): 1413-.

There are many methods available to prevent denaturation of proteins in liquid formulations. In certain protein drugs, the stability problem is solved by lyophilization. For example, U.S. patent No. 7,592,004 discloses a lyophilized formulation of dallizumab (daclizumab) stabilized with 5 to 25mM histidine buffer (pH 5.5 to 6.5), 0.005 to 0.03% nonionic surfactant (polysorbate), and 100 to 300mM non-reducing sugar (sucrose). U.S. patent publication No. 2010-0158925 discloses lyophilized formulations of cetuximab stabilized with histidine buffer and lactobionic acid (lactobionic acid). However, the lyophilization process creates freezing and drying stresses (stress), such as ice crystal formation, pH changes, and high concentrations of solutes, which can lead to antibody denaturation. In addition, since the freeze-drying process requires a large-capacity freeze dryer in production, the production cost is increased in mass production. Reconstitution of the lyophilized product prior to use is also inconvenient by dissolving it in a sterile aqueous medium.

As an alternative approach to address these limitations, stabilizers are added to liquid formulations to improve antibody stability. Surfactants, serum albumin, polysaccharides, amino acids, polymers, salts, etc. are known stabilizers for proteins, including antibodies (Wang, int. J. pharm., 185: 129-.

U.S. patent publication No. 2011-.

U.S. patent publication No. 2011-0020328 discloses a therapeutically effective amount of an anti-CD 20 antibody formulation, wherein the pharmaceutically stable composition comprises 10 to 100mM sodium acetate, 25 to 100mM sodium chloride, 0.5% to 5% arginine free base, 0.02 to 0.2mM EDTA and 0.01 to 0.2% polysorbate 80 at a pH of 5.0 to 7.0.

U.S. patent No. 7,785,592 discloses a stable formulation in which palivizumab at 75mg/mL or more is stabilized by using histidine buffer and glycine without ionic salts and surfactants, and which is stable at 2 to 8 ℃ for at least 15 months.

U.S. patent No. 6,991,790 discloses a stable aqueous pharmaceutical formulation comprising a therapeutically effective amount of an antibody, an acetate buffer having a pH of about 4.8 to 5.5, a surfactant, and a polyol, without an isotonic agent (e.g., sodium chloride).

U.S. Pat. No. 7,648,702 discloses a liquid formulation in which a fusion protein of human p75 tumor necrosis factor receptor linked to the Fc portion of human immunoglobulin G1(IgG1) is stabilized with 10 to 200mM L-arginine as an anticoagulant. Only this patent uses etanercept as the active ingredient.

However, in consideration of the physicochemical properties of the respective active ingredients, an appropriate stabilizer should be used to prepare a stable formulation. When the stabilizers are used in combination, competition between them and adverse effects may result in adverse effects. In addition, the concentration of the antibody should be within a range suitable for stabilization, and its concentration should be higher than that of the protein drug. Therefore, more effort and care is required to stabilize antibodies in solution (fire et al, J.pharm.Sci., 93 (6): 1390-.

There has been little research on stable liquid formulations of etanercept, and the only method known to the inventors for stabilizing etanercept is to use L-arginine in liquid formulations. Therefore, there is an urgent need to develop a novel liquid preparation which can stably maintain the activity of etanercept for a long period of time and can stabilize etanercept more effectively than the known preparation containing L-arginine.

Disclosure of Invention

Technical problem

The present inventors have made many efforts to develop a method for preparing a liquid formulation capable of stably maintaining the activity of etanercept. As a result, it was found that one or more stabilizers selected from the group consisting of: methionine, lysine and histidine were remarkably effective in stabilizing etanercept in a solution, thereby completing the present invention.

Technical scheme

It is an object of the present invention to provide an aqueous formulation comprising a stabilizer in solution for reducing the formation of etanercept by-products during storage and maintaining the activity of etanercept over an extended period of time.

The invention has the advantages of

The liquid formulation of the present invention can effectively prevent the formation of etanercept by-products and stably maintain its pharmaceutical efficacy for long-term storage. Thus, sterile formulations can be administered to patients to ensure patient safety without the need for reconstitution prior to administration. Therefore, it is expected to be applicable to various fields requiring etanercept.

Best mode for carrying out the invention

To achieve the above object, in one aspect, the present invention provides a liquid formulation comprising one or more stabilizers selected from the group consisting of: methionine, lysine, histidine and pharmaceutically acceptable salts thereof.

The formulation of the present invention may be a liquid formulation of etanercept comprising etanercept, one or more stabilizers selected from the group consisting of: methionine, lysine, histidine and pharmaceutically acceptable salts thereof.

The preparation reduces the generation of etanercept by-product due to denaturation during storage, and improves the storage stability.

As stabilizers, methionine, lysine, histidine and pharmaceutically acceptable salts thereof may be present in the formulation in an amount of 0.1 to 250 mM.

The etanercept may be present in the formulation in an amount of 1 to 100 mg/mL.

The formulation may further comprise one or more materials selected from the group consisting of: buffer, isotonic agent, excipient and preservative.

The buffer may be selected from the group consisting of: citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate, histidine and Tris, the buffer may be present in an amount of 0.1 to 100 mM.

The isotonic agent may be selected from the group consisting of: sodium chloride, potassium chloride, boric acid, sodium borate, mannitol, glycerol, propylene glycol, polyethylene glycol, maltose, sucrose, erythritol, arabitol, xylitol, sorbitol, and glucose, and the content of isotonic agent may be 1 to 1000 mM.

The etanercept formulation of the present invention may be a liquid formulation comprising 1 to 100mg/mL of etanercept, 0.1 to 250mM of methionine or a pharmaceutically acceptable salt thereof, 0.1 to 100mM of phosphate buffer, and 1 to 1000mM of sodium chloride.

The etanercept formulation of the present invention may be a liquid formulation comprising 1 to 100mg/mL of etanercept, 0.1 to 250mM of lysine or a pharmaceutically acceptable salt thereof, 0.1 to 100mM of phosphate buffer, and 1 to 1000mM of sodium chloride.

The etanercept formulation of the present invention may be a liquid formulation comprising 1 to 100mg/mL of etanercept, 0.1 to 250mM of histidine or a pharmaceutically acceptable salt, 0.1 to 100mM of phosphate buffer, and 1 to 1000mM of sodium chloride.

The etanercept formulation of the present invention may be a liquid formulation comprising 1 to 100mg/mL of etanercept, 0.1 to 250mM of histidine or a pharmaceutically acceptable salt thereof, 0.1 to 250mM of lysine or a pharmaceutically acceptable salt thereof, 0.1 to 100mM of phosphate buffer, and 1 to 1000mM of sodium chloride.

The etanercept formulation of the present invention may be a liquid formulation comprising 1 to 100mg/mL of etanercept, 0.1 to 250mM of histidine or a pharmaceutically acceptable salt thereof, 0.1 to 250mM of methionine or a pharmaceutically acceptable salt thereof, 0.1 to 100mM of phosphate buffer, and 1 to 1000mM of sodium chloride.

The etanercept formulation of the present invention may be a liquid formulation comprising 1 to 100mg/mL of etanercept, 0.1 to 250mM of methionine or a pharmaceutically acceptable salt thereof, 0.1 to 250mM of lysine or a pharmaceutically acceptable salt thereof, 0.1 to 100mM of phosphate buffer, and 1 to 1000mM of sodium chloride.

The present invention is explained in detail as follows.

The invention proves for the first time that: methionine, lysine, histidine and pharmaceutically acceptable salts thereof have the effects of reducing the formation of etanercept by-products in solution and stably maintaining the activity thereof with respect to long-term storage, thereby proposing their novel use as stabilizers for a pharmaceutically effective amount of etanercept.

In order to prepare stable formulations, suitable stabilizers should be employed in view of the physicochemical properties of the respective active ingredients. Depending on the kind, concentration and combination of materials contained in the formulation, competition between these materials in the formulation may lead to adverse effects. Therefore, it is difficult to prepare a stable specific pharmaceutical preparation.

In view of the above background, the present inventors have found that a liquid formulation comprising one or more stabilizers selected from the group consisting of the following improves the storage stability of etanercept by reducing etanercept by-products in solution during storage, as compared to a formulation without the stabilizers: methionine, lysine, histidine and pharmaceutically acceptable salts thereof.

Specifically, in one embodiment of the present invention, in order to investigate the optimum stabilizer for preparing a stable liquid formulation of etanercept, various amino acids, surfactants and polymers were added to test their stabilizing effects. As a result, it was found that lysine, histidine, methionine or a combination thereof is remarkably effective in stabilizing etanercept, which prevents denaturation of etanercept in a solution at high temperature upon storage.

It was further found that the etanercept stabilizing effect of the formulation of the present invention was comparable to or better than that of the known etanercept stabilizing formulation comprising L-arginine (U.S. patent No. 7,648,702). In particular, methionine was found to have a more excellent etanercept stabilizing effect than L-arginine.

Specifically, the etanercept by-product formed during storage was detected by size exclusion-HPLC (SE-HPLC). As a result, the formulations prepared using lysine as a stabilizer showed similar impurity levels and the formulations prepared using histidine or methionine as a stabilizer showed less impurity levels compared to the total amount of impurities in etanercept formulations prepared using L-arginine as a stabilizer (e.g., U.S. patent No. 7,648,702) (see table 2). Hydrophobic interaction-high performance liquid chromatography (HI-HPLC) analysis also showed similar results. These results indicate that histidine, lysine, methionine prevent denaturation of etanercept to inhibit its byproduct formation (see table 3).

Therefore, the liquid preparation of the present invention can stably maintain the drug effect of etanercept for a long period of time by effectively reducing the formation of etanercept by-products.

As used herein, the term "etanercept (recombinant p75 sTNFR: Fc fusion protein)" refers to a homodimeric protein of two Fc fusion proteins linked by disulfide bonds, each Fc fusion protein consisting of a human soluble 75 kilodalton (p75) TNF (tumor necrosis factor) receptor coupled to the Fc portion of human IgG 1.

Specifically, etanercept is a homodimeric form of two Fc fusion proteins linked by 3 disulfide bonds, wherein each Fc fusion protein is composed of the extracellular ligand binding portion of the human soluble p75TNF receptor linked to the Fc portion of human IgG 1. The Fc portion of etanercept contains a CH2 domain, a CH3 domain, and a hinge region, but lacks the CH1 domain of IgG 1. Etanercept may have a molecular weight of about 150 kilodaltons (kDa). The etanercept is currently under the trade name(Anchen, Kitakura, Calif.) under CAS number 185243-69-0.

Etanercept can be prepared by recombinant DNA techniques, but is not limited thereto.

Etanercept of the present invention is a biological inflammation modulator that acts as a competitive inhibitor of TNF-alpha, binds to cell surface TNF-alpha receptors, inhibits TNF-alpha mediated immune responses, and is used in the treatment of rheumatoid arthritis, psoriasis and ankylosing spondylitis, and clinical trials for the treatment of vasculitis, alzheimer's disease and crohn's disease.

The liquid formulation of the present invention comprises a therapeutically effective amount of etanercept. Preferably, the amount of etanercept is 1-100 mg/mL.

As used herein, the term "stabilized liquid formulation" or "stabilized liquid formulation" refers to a formulation that retains the physical and chemical identity and integrity of the therapeutically active ingredient, etanercept, in solution during storage. Analytical determination of etanercept stability can be performed according to protein stability assays well known in the art. Stability can be measured at a predetermined temperature over a predetermined time. For rapid assay, the formulations may be stored at higher or "elevated" temperatures, e.g. 40 ℃, for 2 weeks to 1 month or more, at which time dependent stability is tested.

The term "stabilizer" as used herein refers to a specific chemical substance that interacts with biomolecules and/or general pharmaceutical excipients in a formulation to improve its stability. Stabilizers generally protect proteins from air/solution interface-induced stress and solution/surface-induced stress that leads to protein aggregation. In the present invention, the stabilizer is a component that reduces the formation of etanercept by-products in solution during storage, thereby maintaining its activity for a long period of time; preferably one or more stabilizers selected from the group consisting of: methionine, lysine, histidine and pharmaceutically acceptable salts thereof.

In the case of amino acids, both the L-form and the D-form are within the scope of the present invention. Not only the amino acids themselves (e.g., methionine, lysine and histidine), their analogs, solvates, hydrates and stereoisomers, and pharmaceutically acceptable salts thereof are within the scope of the invention, so long as they exhibit substantially the same effect.

The term "pharmaceutically acceptable salt" as used herein refers to a compound prepared as a salt of an amino acid, such as methionine, lysine and histidine, as long as its function is maintained as a stabilizer of the present invention. Specifically, salts may be formed by acid addition, for example, with the following acids: inorganic acids (e.g., hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, and the like), organic carboxylic acids (e.g., acetic acid, haloacetic acids (e.g., trifluoroacetic acid), propionic acid, maleic acid, succinic acid, malic acid, citric acid, tartaric acid, salicylic acid), sugar acids (e.g., glucuronic acid, galacturonic acid, gluconic acid, ascorbic acid), acidic polysaccharides (e.g., hyaluronic acid, chondroitin sulfate, arginine), organic sulfonic acids, including sugar sulfonate esters, such as chondroitin sulfate (e.g., methane sulfonic acid, p-toluene sulfonic acid).

The term "analog" as used herein refers to a compound having the same function as an amino acid such as methionine, lysine and histidine, which is used as a stabilizer in the present invention. In the present invention, amino acids such as methionine, lysine and histidine include their analogs. In the present invention, examples of the analogs may include methionine analogs, i.e., selenomethionine, hydroxymethylbutyric acid, ethionine, and trifiuoromethionine, but are not limited thereto.

The term "by-product" as used herein refers to an undesirable product that reduces the proportion of the therapeutically active ingredient etanercept in the formulation. Typical by-products include "low molecular weight products" resulting from the denaturation of etanercept by deamination or hydrolysis, as well as "high molecular weight products" such as oligomers and aggregates, or combinations thereof.

The term "high molecular weight product" as used herein includes fragments of etanercept that subsequently aggregate by denaturation (e.g., due to degradation of the polypeptide by deamination or hydrolysis), or mixtures thereof. Typically, a high molecular weight product is a complex having a molecular weight higher than the therapeutic monomer etanercept, which may be greater than about 150 kDa.

The term "low molecular weight product" as used herein includes, for example, a therapeutic polypeptide produced by deamination or hydrolysis, i.e., an etanercept fragment. Typically, a low molecular weight product is a complex having a molecular weight lower than the therapeutic monomer etanercept, which may be less than about 150 kDa.

The liquid formulation of the present invention comprises one or more stabilizers selected from the group consisting of: methionine, lysine, histidine and pharmaceutically acceptable salts thereof. In particular, the liquid formulation comprises methionine, lysine or histidine as a stabilizer, or two stabilizers, such as methionine and lysine, methionine and histidine, or lysine and histidine, or three stabilizers, such as methionine, lysine and histidine. In the above-mentioned formulation comprising a combination of amino acids, a pharmaceutically acceptable salt of methionine, lysine, histidine may also be added, or methionine, lysine and histidine may each be a modified form of a pharmaceutically acceptable salt thereof. Preferably, the liquid formulation comprises methionine, lysine, histidine, methionine and lysine, methionine and histidine, or lysine and histidine as a stabilizer; more preferably, methionine is included as a stabilizer.

Methionine is an essential amino acid having the chemical formula C₅H₁₁NO₂S and a molecular weight of 149.21. Methionine is a nonpolar amino acid comprising a thioether group (-S-CH) in its side chain₃). The pK1 value was 2.28, the pK2 value was 9.21, and the isoelectric Point (PI) value was 5.74. Methionine stabilizes antibodies by competing with methionine residues in the antibody for reaction with free hydroxyl radicals to prevent oxidation of the antibody in liquid formulations (Lam et al, J.Pharm.Sci., 86 (11): 1250-1255, 1997; Wang, Inc.J.Pharm., 185: 129-188, 1999).

Lysine is an essential amino acid having the chemical formula C₆H₁₄N₂O₂And a molecular weight of 146.19. Lysine is a basic amino acid with a pK1 value of 2.18, a pK2 value of 8.95, a pK3 value of 10.53 and a PI value of 9.74.

Histidine is an essential amino acid having a positively charged imidazole function in its side chain and has the formula C₆H₉N₃O₂And a molecular weight of 155.15. Histidine is a basic amino acid with a pK1 value of 1.82, a pK2 value of 9.17, a pK3 value of 6.0 and a PI value of 7.59.

In contrast, L-arginine is alreadyThe essential amino acid in commercial etanercept formulations is known to be of formula C₆H₁₄N₄O₂And a molecular weight of 174.2. Arginine is a basic amino acid with a pK1 value of 2.17, a pK2 value of 9.04, a pK3 value of 12.48 and a PI value of 10.76. That is, methionine, lysine and histidine as stabilizers in the present invention are completely different from arginine, a known etanercept stabilizer, in structure, chemical formula, physicochemical properties and ionization tendency. The present invention demonstrates for the first time that amino acids such as methionine, lysine and histidine are suitable as stabilizers for etanercept.

The content of the stabilizer in the liquid formulation of the present invention is 0.1 to 250mM, preferably 1 to 100mM, more preferably 5 to 50 mM.

According to one or more stabilizers selected from the group consisting of: methionine, lysine, histidine and pharmaceutically acceptable salts thereof, the liquid formulation of the present invention may further comprise any of the materials conventionally contained in the formulation of protein drugs or antibody drugs to improve the stability of etanercept, except those materials which impair the function of the present invention. For example, the known etanercept stabilizer L-arginine may be added to the liquid formulation of the present invention.

In addition to the stabilizer, the liquid formulation of the present invention may further comprise a buffer. As used herein, the term "buffer" refers to a component that improves the isotonicity and chemical stability of the formulation for maintaining a physiologically suitable pH. The buffer prevents rapid changes in the pH of the liquid formulation to maintain the pH of the solution to stabilize etanercept. Preferred examples of the buffer include citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate, histidine and Tris, but are not limited thereto. In a specific embodiment, the buffer is a phosphate buffer. The buffer may be used alone or in combination of two or more.

In various embodiments of the invention, the formulation pH should be about 5 to 7.5, or about 5.8 to 6.8. In a specific embodiment, the formulation pH is about 6.0 to 6.6. Ranges from intermediate values to the above pH levels, for example, about pH 5.2 to about pH 6.4 (e.g., pH 6.2), are also part of the invention. For example, ranges utilizing any combination of the above values as upper and/or lower limits are included. If necessary, the pH can be adjusted by techniques known in the art. For example, the pH can be adjusted to any desired range by adding HCl or by adding histidine, if necessary.

In another embodiment, the buffer concentration is 0.1 to 100mM, preferably 1 to 50mM, more preferably 5 to 25 mM. Ranges from intermediate values to the above concentrations are also intended to be part of the present invention. For example, ranges utilizing combinations of the above concentrations as upper and/or lower limits are included. In particular embodiments, the buffer should be present in an amount sufficient to maintain a physiologically sufficient pH.

The liquid formulation of the present invention may further comprise an isotonic agent in addition to the stabilizer. The term "isotonicity agent" as used herein refers to a component which functions to partially maintain the isotonicity and protein level of the formulation and partially maintain the level, ratio or proportion of therapeutically active polypeptide in the formulation. The osmolality of the isotonicity agent is the same as plasma and thus can be administered to a subject by intravenous infusion without altering the osmolality of the subject's plasma. Indeed, in one embodiment of the invention, the isotonic agent is present in an amount sufficient to render the formulation suitable for intravenous infusion. Usually, isotonic agents also act as bulking agents. Thus, the isotonic agent may allow the protein to overcome various stresses, such as freezing and shearing.

The role of the isotonic agent is to maintain a suitable isotonic pressure in the body when the etanercept solution is administered into the body. Examples of isotonic agents may include, but are not limited to, sodium chloride, potassium chloride, boric acid, sodium borate, mannitol, glycerol, propylene glycol, polyethylene glycol, maltose, sucrose, erythritol, arabitol, xylitol, sorbitol, and glucose, which are commonly used. In a specific embodiment, the isotonic agent is sodium chloride. These isotonic agents may be used alone or in combination of two or more.

In another embodiment, the concentration of the isotonic agent (e.g., sodium chloride) is 1 to 1000mM, preferably 10 to 500mM, more preferably 50 to 250 mM. Ranges from intermediate to the above concentrations are also part of the present invention. For example, ranges utilizing any combination of the above concentrations as upper and/or lower limits are included. The amount of isotonic agent should be sufficient to maintain the permeability of the formulation.

The liquid formulation of the present invention may further comprise pharmaceutically acceptable excipients, and examples of the excipients may include sugars and polyols, surfactants, polymers, and the like. Examples of sugars and polyols may include sucrose, trehalose, lactose, maltose, galactose, mannitol, sorbitol, glycerol; examples of the surfactant may include nonionic surfactants such as polysorbate 20, polysorbate 80, and poloxamer; examples of polymers may include dextran, polyethylene glycol, carboxymethyl cellulose, hyaluronic acid, and cyclodextrin.

The liquid formulation of the present invention may further comprise a preservative. By preservative is meant a chemical substance added to a pharmaceutical preparation as an antimicrobial agent. Examples of preservatives can include benzalkonium chloride, benzethol, chlorhexidine, phenol, m-cresol, benzyl alcohol, methyl paraben, propyl paraben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, and benzoic acid, but are not so limited. These preservatives may be used alone or in combination of two or more.

In a specific embodiment, the formulation of the invention is a stable liquid formulation comprising 1 to 100mg/mL of etanercept, 0.1 to 250mM of methionine or a pharmaceutically acceptable salt thereof, 0.1 to 100mM of phosphate buffer and 1 to 1000mM of sodium chloride. More specifically, the formulation of the present invention is a stable liquid formulation comprising 1 to 100mg/mL of etanercept, 0.1 to 100mM of methionine or a pharmaceutically acceptable salt thereof, 0.1 to 50mM of phosphate buffer and 1 to 500mM of sodium chloride, at a pH of 6.0 to 6.6.

In a specific embodiment, the formulation of the invention is a stable liquid formulation comprising 1 to 100mg/mL of etanercept, 0.1 to 250mM of histidine or a pharmaceutically acceptable salt thereof, 0.1 to 100mM of phosphate buffer and 1 to 1000mM of sodium chloride. More specifically, the formulation of the present invention is a stable liquid formulation comprising etanercept in an amount of 1 to 100mg/mL, histidine or a pharmaceutically acceptable salt thereof in an amount of 0.1 to 100mM, a phosphate buffer in an amount of 0.1 to 50mM, and sodium chloride in an amount of 1 to 500mM, at a pH of 6.0 to 6.6.

In a specific embodiment, the formulation of the invention is a stable liquid formulation comprising 1 to 100mg/mL of etanercept, 0.1 to 250mM of lysine or a pharmaceutically acceptable salt thereof, 0.1 to 100mM of phosphate buffer, and 1 to 1000mM of sodium chloride. More specifically, the formulation of the present invention is a stable liquid formulation comprising etanercept in an amount of 1 to 100mg/mL, lysine or a pharmaceutically acceptable salt thereof in an amount of 0.1 to 100mM, a phosphate buffer in an amount of 0.1 to 50mM and sodium chloride in an amount of 1 to 500mM, at a pH of 6.0 to 6.6.

In a specific embodiment, the formulation of the invention is a stable liquid formulation comprising 1 to 100mg/mL of etanercept, 0.1 to 250mM of histidine or a pharmaceutically acceptable salt thereof, 0.1 to 250mM of lysine or a pharmaceutically acceptable salt thereof, 0.1 to 100mM of phosphate buffer and 1 to 1000mM of sodium chloride. More specifically, the formulation of the present invention is a stable liquid formulation comprising etanercept in an amount of 1 to 100mg/mL, histidine in an amount of 1 to 50mM or a pharmaceutically acceptable salt thereof, lysine in an amount of 1 to 100mM or a pharmaceutically acceptable salt thereof, a phosphate buffer in an amount of 0.1 to 50mM, and sodium chloride in an amount of 1 to 500mM, at a pH of 6.0 to 6.6.

In a specific embodiment, the formulation of the invention is a stable liquid formulation comprising 1 to 100mg/mL of etanercept, 0.1 to 250mM of histidine or a pharmaceutically acceptable salt thereof, 0.1 to 250mM of methionine or a pharmaceutically acceptable salt thereof, 0.1 to 100mM of phosphate buffer and 1 to 1000mM of sodium chloride. More specifically, the formulation of the present invention is a stable liquid formulation comprising 1 to 100mg/mL of etanercept, 1 to 50mM of histidine or a pharmaceutically acceptable salt thereof, 1 to 100mM of methionine or a pharmaceutically acceptable salt thereof, 0.1 to 50mM of phosphate buffer and 1 to 500mM of sodium chloride, at a pH of 6.0 to 6.6.

In a specific embodiment, the formulation of the invention is a stable liquid formulation comprising 1 to 100mg/mL of etanercept, 0.1 to 250mM of methionine or a pharmaceutically acceptable salt thereof, 0.1 to 250mM of lysine or a pharmaceutically acceptable salt thereof, 0.1 to 100mM of phosphate buffer and 1 to 1000mM of sodium chloride. More specifically, the formulation of the present invention is a stable liquid formulation comprising 1 to 100mg/mL of etanercept, 1 to 50mM of methionine or a pharmaceutically acceptable salt thereof, 1 to 50mM of lysine or a pharmaceutically acceptable salt thereof, 0.1 to 50mM of phosphate buffer and 1 to 500mM of sodium chloride, at a pH of 6.0 to 6.6.

The formulations of the invention are useful in the treatment of diseases for which etanercept is therapeutically effective. Etanercept is a biological inflammation modulator to inhibit TNF-alpha mediated immune responses, and the formulation of the present invention is useful for treating rheumatoid arthritis, psoriasis, ankylosing spondylitis, vasculitis, alzheimer's disease or crohn's disease, but is not limited thereto. The formulations of the present invention may be administered orally or parenterally, i.e., subcutaneously, intramuscularly, intraperitoneally, transdermally and/or intravenously, but are not limited thereto.

In another aspect, the present invention provides a method of improving the stability of etanercept using a liquid formulation comprising one or more stabilizers selected from the group consisting of: methionine, lysine, histidine and pharmaceutically acceptable salts thereof.

The liquid formulation can improve the storage stability of etanercept by reducing the production of etanercept by-products due to denaturation during storage.

Modes for carrying out the invention

The present invention will be described in more detail below with reference to examples. However, these examples are for illustrative purposes only, and the present invention is not limited by these examples.

< example 1> stability of etanercept aqueous formulation was tested according to addition of stabilizer

To investigate the optimum stabilizer for preparing a stable aqueous preparation of etanercept, histidine was added as a stabilizer to a 5mM phosphate solution, etanercept was added thereto to a concentration of 50mg/mL, and sodium chloride was added as an isotonic agent to prepare preparation 1.

Furthermore, etanercept was added to a 10mM phosphate solution to a concentration of 50mg/mL, and sodium chloride was added as an isotonic agent. Then, lysine, arginine, methionine, glycine, polysorbate 20, polysorbate 80, poloxamer 188, propylene glycol, protamine sulfate, sucrose and guanidine hydrochloride were added, respectively, to prepare formulations 2 to 12.

Finally, sodium chloride was added as an isotonic agent to 10mM phosphate solution, to which etanercept was added to a concentration of 50mg/mL, to prepare a stabilizer-free formulation. The stabilizer composition in the formulation is shown in table 1 below. 0.5mL of each formulation was placed in a 1.0mL glass syringe, sealed, and stored at 50 ℃.

TABLE 1

[ Table 1]

	Stabilizer composition
		Preparation 1	Histidine 10mM
Preparation 2	Lysine 25mM
		Preparation 3	Arginine 25mM
Preparation 4	Methionine 25mM
		Preparation 5	1 percent of glycine
Preparation 6	Tween 200.1%
		Preparation 7	Tween 800.1%
Preparation 8	F680.1％
		Preparation 9	0.1 percent of propylene glycol
Preparation 10	Protamine sulfate 0.03%
		Preparation 11	2 percent of sucrose
Preparation 12	0.2 percent of guanidine hydrochloride
		Stabilizer-free formulations	-

After 7 days of storage at 50 ℃, formulations 1-12 of table 1 and formulations without stabilizers were analyzed by SE-HPLC to detect the types of low molecular weight products and high molecular weight products, such as oligomers and aggregates resulting from the denaturation of etanercept, the total amount of these products representing the sum of impurities. In addition, the structural change of etanercept was detected by HI-HPLC. In HI-HPLC, etanercept-related material was divided into 4 peaks, including pre-peak, peak 1, peak 2, and peak 3. Pre-peak and peak 1 represent low molecular weight products, peak 2 represents etanercept, and peak 3 represents low agglomeration or low activity dimer. Thus, the total of pre-peak, peak 1 and peak 3 represent the total of impurities. The results are shown in tables 2 and 3, respectively.

TABLE 2

[ Table 2]

TABLE 3

[ Table 3]

As shown by the SE-HPLC results of table 2, after 7 days of storage at 50 ℃, formulation 1 containing histidine as a stabilizer showed a total of 22.2% of impurities, formulation 2 containing lysine as a stabilizer showed a total of 24.8% of impurities, formulation 3 containing arginine as a stabilizer showed a total of 24.2% of impurities, and formulation 4 containing methionine as a stabilizer showed a total of 20.8% of impurities. Formulations without stabilizers showed a total of 30.2% of impurities, in contrast to each of them showing a considerably lower total of impurities. Compared to formulation 3, which utilized L-arginine as a stabilizer (U.S. patent No. 7648702), formulation 2, which contained lysine, showed a similar total of 24.8% impurities, while formulation 1, which contained histidine, and formulation 4, which contained methionine, showed a lower total of impurities, 22.2% and 20.8%, respectively.

The HI-HPLC results shown in Table 3 are similar to the SE-HPLC results. The formulation without stabilizer showed a total of 41.9% impurities. In contrast, the total of impurities of each of formulations 1, 2, 3 and 4 were 33.7%, 34.9%, 34.3% and 30.3%, respectively, indicating that histidine, lysine, L-arginine and methionine added to each formulation had the effect of preventing denaturation of etanercept. In particular, methionine-containing formulation 4 showed the lowest sum of impurities, indicating that methionine is most effective for the stabilization of etanercept.

Thus, the present invention demonstrates: lysine, histidine and methionine have the effect of stabilizing etanercept by preventing denaturation of etanercept, which is equivalent to or better than that of L-arginine, and particularly methionine stabilizes etanercept better than L-arginine.

< example 2> stability of aqueous preparation of etanercept according to concentration of methionine

To investigate the optimal methionine concentration for stabilizing etanercept, formulations 13 and 14 were prepared by adding 120mM sodium chloride to 10mM phosphoric acid solution, followed by the addition of 25mM and 12.5mM methionine, respectively, followed by the addition of 50mg/mL etanercept. According to a commercially available preparation method of an aqueous preparation of etanercept, a control group was prepared by adding 25mM L-arginine to a 10mM phosphate solution containing 100mM sodium chloride and 1% sucrose, followed by adding etanercept to 50 mg/mL. 0.5mL of each preparation was put into a 1.0mL glass syringe, sealed, and stored at 40 ℃, 25 ℃ and 4 ℃. The composition of the resulting aqueous formulation is shown in table 4 below.

TABLE 4

[ Table 4]

After 1 and 3 weeks of storage at 40 ℃ and 4 weeks and 4 and 8 weeks of storage at 25 ℃ and 4 ℃, the preparations 13, 14 and the control group were tested for the total sum of impurities by SE-HPLC and HI-HPLC (as in example 1). The results are shown in tables 5 and 6, respectively.

TABLE 5

[ Table 5]

TABLE 6

[ Table 6]

As shown by the results in tables 5 and 6, both formulation 13 and 14, which contained methionine as a stabilizer, exhibited low impurity formation under all of the 40 ℃, 25 ℃ and 4 ℃ storage conditions, as compared to the control prepared according to the preparation method of a commercially available aqueous formulation of etanercept. Specifically, the SE-HPLC and HI-HPLC results showed that both formulation 13 containing 25mM methionine and formulation 14 containing 12.5mM methionine showed lower impurity formation after 3 weeks storage at 40 ℃ and 8 weeks storage at 25 ℃ and similar impurity formation after 8 weeks storage at 4 ℃ compared to the control group. These results indicate that the methionine-containing formulation of the present invention is a more stable formulation than the commercially available formulation in order to maintain the activity of etanercept for a long period of time.

In summary, the present invention shows that an aqueous etanercept formulation containing methionine prevents denaturation of etanercept to maintain its activity for a long period of time, and methionine is a very effective stabilizer for the aqueous etanercept formulation.

< example 3> stability test of Etanercept aqueous formulation containing histidine and lysine or histidine and methionine

Histidine 5mM and lysine 25mM were added as stabilizers to a 10mM phosphate solution, to which etanercept was added to a concentration of 50mg/mL and sodium chloride as an isotonic agent, to prepare a formulation 15.

In addition, histidine 5mM and methionine 25mM were added as stabilizers to a 10mM phosphate solution, to which etanercept was added to a concentration of 50mg/mL and sodium chloride as an isotonic agent, to prepare formulation 16.

Finally, sodium chloride was added as an isotonic agent to 10mM phosphate solution, to which etanercept was added to a concentration of 50mg/mL, to prepare a stabilizer-free formulation.

0.5mL of each of the prepared formulations was put into a 1.0mL glass syringe, sealed, and stored at 50 ℃.

The stabilizer composition of the formulation is shown in table 7 below.

TABLE 7

[ Table 7]

	Stabilizer composition
		Formulation 15	Histidine 5mM + lysine 25mM
Preparation 16	Histidine 5mM + methionine 25mM
		Stabilizer-free formulations	-

After 7 days of storage at 50 ℃, formulations 15, 16 in table 7 and the formulation without stabilizer were analyzed by SE-HPLC as described in example 1 to examine their sum of impurities. The results are shown in Table 8.

TABLE 8

[ Table 8]

As shown by the SE-HPLC results of table 8, formulation 16 containing histidine as a stabilizer showed a total of 21.5% of impurities after 7 days of storage at 50 ℃, which was very low compared to 30.2% of the total of impurities of the formulation without stabilizer. Formulation 16 showed a very low sum of impurities compared to formulation 1 containing 10mM histidine at 22.2% and formulation 2 containing 25mM lysine at 24.8%.

That is, the present invention shows that the histidine and lysine-containing formulation has an excellent effect of preventing denaturation of etanercept by reducing the total amount of impurities upon storage, as compared to the stabilizer-free formulation and each of the histidine, lysine and methionine-containing formulations, respectively.

In addition, as shown by the SE-HPLC results of table 8, formulation 16 containing histidine as a stabilizer showed a total of 20.9% of impurities after 7 days of storage at 50 ℃, which was very low compared to the formulation without stabilizer showing a total of 30.2% of impurities. Specifically, formulation 16 exhibited a lower sum of impurities than formulation 15, which contained histidine and lysine as stabilizers.

That is, the present invention shows that the histidine and methionine-containing formulation has an excellent effect of preventing denaturation of etanercept by reducing the total amount of impurities upon storage, compared to the formulation without a stabilizer.

< example 4> stability test of methionine and lysine-containing etanercept aqueous formulation

Methionine 12.5mM and lysine 12.5mM as stabilizers were added to a 10mM phosphate solution, to which etanercept was added to a concentration of 50mg/mL and sodium chloride as an isotonic agent, to prepare formulation 17.

In addition, sodium chloride was added as an isotonic agent to 10mM phosphate solution, and etanercept was added to a concentration of 50mg/mL to prepare a formulation containing no stabilizer.

0.5mL of each of the prepared formulations was put into a 1.0mL glass syringe, sealed, and stored at 50 ℃. The composition of the stabilizers in the formulation is shown in table 9 below.

TABLE 9

[ Table 9]

	Stabilizer composition
		Preparation 17	Methionine 12.5mM + lysine 12.5mM
Stabilizer-free formulations	-

After 7 days of storage at 50 ℃, formulations 17 of table 9 and formulations without stabilizers were analyzed by SE-HPLC as described in example 1 to examine their sum of impurities. The results are shown in Table 10.

Watch 10

[ Table 10]

As shown by the SE-HPLC results of table 10, formulation 17 containing methionine and lysine as stabilizers showed a total of 27% of impurities after 7 days of storage at 50 ℃, which was very low compared to the formulation without stabilizers, which showed a total of 32.8% of impurities.

That is, the present invention shows that the methionine and lysine-containing formulation has an excellent effect of preventing denaturation of etanercept by reducing the total amount of impurities upon storage, compared to the formulation without a stabilizer.

Claims (16)

1. A liquid formulation of etanercept comprising etanercept, and one or more stabilizers selected from the group consisting of: methionine, lysine, histidine and pharmaceutically acceptable salts thereof.

2. The liquid formulation of claim 1, wherein the stabilizing agent is methionine and/or a pharmaceutically acceptable salt thereof.

3. The liquid formulation of claim 1, wherein the stabilizer is present in an amount of 0.1 to 250 mM.

4. The liquid formulation of claim 1, wherein etanercept is present in an amount of 1 to 100 mM.

5. The liquid formulation of claim 1, wherein the liquid formulation has improved storage stability of etanercept by reducing etanercept by-products produced by denaturation during storage as compared to a formulation without a stabilizer.

6. The liquid formulation of claim 1, further comprising one or more materials selected from the group consisting of: buffer, isotonic agent, excipient or preservative.

7. The liquid formulation of claim 6, wherein the buffer is selected from the group consisting of: citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate, histidine and Tris.

8. The liquid formulation of claim 6, wherein the buffer is present in an amount of 0.1 to 100 mM.

9. The liquid formulation of claim 6, wherein the isotonic agent is selected from the group consisting of: sodium chloride, potassium chloride, boric acid, sodium borate, mannitol, glycerol, propylene glycol, polyethylene glycol, maltose, sucrose, erythritol, arabitol, xylitol, sorbitol, and glucose.

10. The liquid formulation of claim 6, wherein the isotonic agent is present in an amount of 1 to 1000 mM.

11. The liquid formulation of claim 1, wherein the liquid formulation comprises 1 to 100mg/mL etanercept, 0.1 to 250mM methionine or a pharmaceutically acceptable salt thereof, 0.1 to 100mM phosphate buffer, and 1 to 1000mM sodium chloride.

12. The liquid formulation of claim 1, wherein the liquid formulation comprises 1 to 100mg/mL etanercept, 0.1 to 250mM lysine or a pharmaceutically acceptable salt thereof, 0.1 to 100mM phosphate buffer, and 1 to 1000mM sodium chloride.

13. The liquid formulation of claim 1, wherein the liquid formulation comprises 1 to 100mg/mL etanercept, 0.1 to 250mM histidine or a pharmaceutically acceptable salt thereof, 0.1 to 100mM phosphate buffer, and 1 to 1000mM sodium chloride.

14. The liquid formulation of claim 1, wherein the liquid formulation comprises 1 to 100mg/mL etanercept, 0.1 to 250mM histidine or a pharmaceutically acceptable salt thereof, 0.1 to 250mM lysine or a pharmaceutically acceptable salt thereof, 0.1 to 100mM phosphate buffer, and 1 to 1000mM sodium chloride.

15. The liquid formulation of claim 1, wherein the liquid formulation comprises 1 to 100mg/mL etanercept, 0.1 to 250mM histidine or a pharmaceutically acceptable salt thereof, 0.1 to 250mM methionine or a pharmaceutically acceptable salt thereof, 0.1 to 100mM phosphate buffer, and 1 to 1000mM sodium chloride.

16. The liquid formulation of claim 1, wherein the liquid formulation comprises 1 to 100mg/mL etanercept, 0.1 to 250mM methionine or a pharmaceutically acceptable salt thereof, 0.1 to 250mM lysine or a pharmaceutically acceptable salt thereof, 0.1 to 100mM phosphate buffer, and 1 to 1000mM sodium chloride.