NZ618054B2

NZ618054B2 - Stable liquid formulation of etanercept

Info

Publication number: NZ618054B2
Application number: NZ618054A
Authority: NZ
Inventors: Suk Young Choi; Youn Kyung Ko; Jin Eon So
Original assignee: Lg Chem Ltd
Priority date: 2011-06-03
Filing date: 2012-06-01
Publication date: 2016-09-27

Abstract

Disclosed is a liquid formulation of etanercept, comprising etanercept; and methionine or pharmaceutically acceptable salts thereof as a stabiliser, wherein the formulation has increased storage stability of etanercept compared to the formulation comprising arginine as a stabiliser, by reducing etanercept by-products that are produced due to denaturation during storage. ercept by-products that are produced due to denaturation during storage.

Description

Description Title of Invention: STABLE LIQUID FORMULATION OF CEPT Technical Field The present ion relates to a liquid formulation of etanercept (recombinant p75 sTNFR:Fc fusion protein), and more particularly, to a liquid formulation comprising one or more stabilizers selected from the group consisting of nine, lysine, histidine, and pharmaceutically acceptable salts thereof in an amount sufficient to reduce by—products of etanercept during storage.

Background Art Etanercept is a biological inﬂammation modulator that functions as a competitive inhibitor of TNF—(1, binding to cell surface TNF-(1 receptor, to inhibit TNF-(1 mediated immune responses. Etanercept is a macromolecule with a molecular weight of ap— proximately 150 kDa, and is a homodimer of two Fc fusion proteins linked by a disulfide bond, each PC fusion protein consisting of a human soluble p75 TNF or coupled to the Fc portion of human immunoglobulin G subclass 1 (Goldenberg, Clinical eutics, 21(1): 75—87, 1999; Moreland et a1., Ann. Intern. Med., 130(6): 478-486, 1999).

This prototypic fusion protein was first synthesized in the early 1990s by Bruce A.

Beutler at the University of Texas Southwestern Medical Center, and marketed by Amgen under the trade name of Enbrel in 2002. Etanercept is a TNF-(1 inhibitor used to treat rheumatoid arthritis, psoriasis, and ankylosing spondylitis, and under clinical trials for the treatment of itis, Alzheimer's disease, and Crohn's disease.

Like protein drugs, dy drugs have a very short half life, and chemical and physical denaturation can be easily caused by unfavorable temperature, shear stress, ion, freeze—thawing, UV exposure, excessive pH change, organic solvents, and ial contamination. Chemical denaturation includes dimer dissociation, oxidation, deamidation, isomerization, and polymerization, which are inﬂuenced by the amino acids constituting the antibody and ions of the solvent containing the antibody (salt, pH and temperature). Physical denaturation includes loss of tertiary structure, covalent/non—covalent aggregation and adhesion of monomers, which are in— ﬂuenced by hydrophobic patches on the protein surface changed by dy— containing surrounding nments such as ts, complex protein structures such as charge distribution, and l stability. The al or chemical denaturation of an antibody causes loss of its physiological activities. Since the denaturation is an irre— versible process, antibodies, once denatured, may not recover their native properties to the initial state, leading to a reduction in their therapeutic cies. It has been also ted that aggregation of monomers causes immune responses. Therefore, many studies have been conducted on antibody formulations ning a physiologically effective amount without aggregates (Ishikawa et al., Biol. Pharm. Bull., 33(8): 1413— 1417, 2010; Levin et al., The Development of Therapeutic Monoclonal Antibody Products, editors. Boston (MA): BioProcess Technology Consultants, Inc, Chapter 9).

There are many methods available for preventing protein denaturation in liquid for— mulations. In some protein drugs, the stability problems are addressed via lyophilization. For instance, U.S. Patent No. 7,592,004 discloses a lyophilized for— mulation of daclizumab that is stabilized using 5 to 25 mM of a histidine buffer solution (pH 5.5 to 6.5), 0.005 to 0.03% of a non—ionic tant, polysorbate and 100 to 300 mM of a non—reducing sugar, sucrose. U.S. Patent ation No. 2010—0158925 discloses a lyophilized formulation of cetuXimab that is stabilized using a histidine buffer solution and ionic acid. However, the lyophilization process generates ng and drying stresses such as formation of ice crystals, pH change, and high concentration of solute, and these stresses may cause antibody denaturation.

In addition, since a large—capacity lyophilizer is needed for the lyophilization process during the production, high production costs arise during a large scale of production.

Dissolving the lyophilized product in sterile s media for reconstitution before use also poses an enience.

As an alternative to solve these limitations, a stabilizer is added in liquid for— mulations for the improvement of antibody stability. Surfactants, serum albumins, ccharides, amino acids, polymers, salts or the like are known as stabilizers for ns including antibodies (Wang, Int. J. Pharm., 185: 129—188, 1999; Wang et al., J. Pharm. Sci., 96(1): 1—26, 2007).

US Patent Publication No. 2011—0070231 discloses a stable liquid composition of IgG antibody, in which the stable pharmaceutical liquid formulation includes 50 mg/ mL or more of daclizumab in 20 to 60 mM of a succinate buffer solution (pH 5.5 to 6.5), 0.02 to 0.04% of polysorbate, and 75 to 150 mM of sodium chloride.

US Patent Publication No. 2011—0020328 discloses a therapeutically effective amount of anti—CD20 antibody formulation, in which the ceutically stable com— position includes 10 to 100 mM of sodium acetate, 25 to 100 mM of sodium chloride, 0.5 to 5% of arginine free base, 0.02 to 0.2 mM of EDTA and 0.01 to 0.2% of polysorbate 80, and its pH is 5.0 to 7.0.

US Patent No. 7,785,592 discloses a stable formulation, in which 75 mg/mL or more of zumab is stabilized by using a histidine buffer solution and glycine without ionic salts and a surfactant, and its ity is maintained at 2 to 8°C for at least 15 months.

US Patent No. 6,991,790 discloses a stable aqueous ceutical formulation, including a therapeutically effective amount of an antibody, an acetate buffer solution of approximately pH 4.8 to 5.5, a tant, and a polyol without an isotonic agent such as sodium chloride.

US Patent No. 702 discloses a liquid formulation, in which a fusion protein of the human p75 tumor necrosis factor receptor linked to the Fc n of the human immunoglobulin Gl (IgGl) is stabilized by using approximately 10 to 200 mM of L— arginine as an aggregation preventing agent. This patent is the only technique of using etanercept as an active ingredient.

In order to prepare stable formulations, however, appropriate stabilizers should be used considering the physicochemical properties of each active ingredient. When the stabilizers are used in combination, competition etween and adverse effects may lead to undesirable effects. In addition, the concentrations of antibodies should be within the range suitable for the stabilization, and their concentrations are relatively higher than those of n drugs. Thus, much effort and caution are required to stabilize antibodies in solutions (Shire et al., J. Pharm. Sci., 93(6): 1390-1402, 2004).

There are few s on the stable liquid formulations of etanercept, and the only method for stabilizing etanercept that the inventors are aware of is to use L—arginine in the liquid formulation. Therefore, there is an urgent need to develop a new liquid for— mulation which is able to stably maintain the activity of etanercept for a long period and which is more effective in etancercept stabilization than the known formulation comprising nine.

Disclosure of Invention Technical Problem The present inventors have made many efforts to develop a method for preparing a liquid formulation capable of stably ining the ty of etanercept. As a result, they found that one or more stabilizers selected from the group consisting of me— thionine, lysine, and histidine show remarkable effects on the stabilization of etanercept in a solution, thereby completing the present ion.

Solution to Problem An object of the present invention is to provide an s formulation, comprising stabilizers for reducing formation of etanercept by—products during storage in a solution and maintaining its activity for a long period of time.

Advantageous Effects of Invention The liquid formulation according to the present invention can effectively prevent formation of cept by—products and stably maintain its pharmaceutical efficacies WO 65917 for erm storage. Therefore, the titution procedure is not required before administration, and the sterile formulation can be administered to patients to ensure patient safety. Thus, it is expected to be applied to the fields in need of etanercept ent.

Best Mode for Carrying out the Invention In one aspect to achieve the above object, the present invention provides a liquid for— mulation, comprising one or more stabilizers selected from the group consisting of me— thionine, lysine, histidine, and pharmaceutically able salts thereof.

The formulation of the present ion may be a liquid ation of etanercept comprising, etanercept; and one or more stabilizers ed from the group consisting of methionine, lysine, histidine, and ceutically acceptable salts thereof.

The formulation shows increased storage stability by reducing etanercept by—products that are produced due to denaturation during storage.

As a stabilizer, methionine, lysine, histidine, or ceutically acceptable salts thereof may be present in the formulation in an amount of 0.1 to 250 mM.

Etanercept in the formulation may be present in an amount of l to 100 mg/mL.

The formulation may further include one or more materials seleted from the group consisting of a buffer, an isotonic agent, an excipient, and a preservative.

The buffer may be selected from the group consisting of citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate, histidine, and Tris, and 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, glycerin, propylene glycol, hylene glycol, maltose, sucrose, erythritol, arabitol, xylitol, sorbitol, and glucose, and may be present in an amount of l to 1000 mM.

The formulation of etanercept according to the present invention may be a liquid for— mulation, comprising 1 to 100 mg/mL of cept, 0.1 to 250 mM of methionine or pharmaceutically acceptable salts thereof, 01 to 100 mM of phosphate buffer, and l to 1000 mM of sodium chloride.

The formulation of cept according to the present invention may be a liquid for— mulation, comprising 1 to 100 mg/mL of etanercept, 0.1 to 250 mM of lysine or phar— maceutically able salts thereof, 01 to 100 mM of phosphate buffer, and l to 1000 mM of sodium chloride.

The formulation of etanercept according to the present invention may be a liquid for— on, comprising 1 to 100 mg/mL of etanercept, 0.1 to 250 mM of histidine or pharmaceutically acceptable salts thereof, 01 to 100 mM of phosphate buffer, and l to 1000 mM of sodium chloride.

The formulation of etanercept according to the present invention may be a liquid for— mulation, comprising 1 to 100 mg/mL of etanercept, 0.1 to 250 mM of histidine or pharmaceutically acceptable salts thereof, 01 to 250 mM of lysine or pharmaceutically acceptable salts thereof, 01 to 100 mM of phosphate buffer, and l to 1000 mM of sodium chloride.

The formulation of etanercept according to the present invention may be a liquid for— mulation, sing 1 to 100 mg/mL of etanercept, 0.1 to 250 mM of histidine or pharmaceutically acceptable salts thereof, 01 to 250 mM of methionine or pharma— ceutically acceptable salts f, 01 to 100 mM of phosphate buffer, and l to 1000 mM of sodium chloride.

The formulation of cept according to the present invention may be a liquid for— mulation, comprising 1 to 100 mg/mL of etanercept, 0.1 to 250 mM of methionine or pharmaceutically acceptable salts thereof, 01 to 250 mM of lysine or pharmaceutically acceptable salts thereof, 01 to 100 mM of phosphate buffer, and l to 1000 mM of sodium chloride.

Hereinafter, the present invention will be described in detail.

The present invention has demonstrated for the first time that methionine, lysine, histidine and pharmaceutically acceptable salts f have the effects of reducing formation of etanercept ducts in solution and stably maintaining its activity for long—term e, and thus suggests a novel use thereof as stabilizers of a — ceutically effective amount of etanercept.

In order to prepare stable ations, appropriate stabilizers should be used con— sidering the physicochemical properties of each active ient. According to the difference in types, concentration, and combinations of materials included in a for— mulation, competition between the materials in the formulation can lead to undesirable effects. Thus, it is difficult to prepare a stable drug specific formulation.

In the light of the above background, the present inventors have found that the liquid formulation comprising one or more izers selected from the group consisting of nine, lysine, and histidine, and pharmaceutically acceptable salts thereof increases storage stability of etanercept compared to the formulation having no sta— bilizers, by reducing etanercept by—products during e in a solution.

Specifically, in one embodiment of the present invention, in order to investigate the l izer for the preparation of the stable liquid formulation of etanercept, a variety of amino acids, surfactants, and polymers are added to examine their stabi— lization effects. As a result, , histidine, methionine, or the combination f was found to show remarkable effects on stabilizing etanercept by preventing its de— naturation during storage in a solution at high temperature.

Further, the formulation according to the present invention was found to show cept stabilizing effects which were lent to or higher than that of the for— mulation comprising nine which is the known stable formulation of etanercept (US Patent No. 7,648,702). In particular, methionine was found to show the very excellent effect of stabilizing etanercept better than L—arginine.

Specifically, etanercept by—products formed during storage were ed by Size Exclusion—HPLC (SE—HPLC). As a result, compared to the total amount of impurities of the etanercept formulation that was prepared by using nine as the stabilizer in US Patent No. 7,648,702, the formulation prepared using lysine as the stabilizer showed a similar amount of impurities, and the formulation prepared using histidine or methionine as the stabilizer showed a lower amount of impurities (see Table 2). Hy— drophobic Interaction—HPLC LC) analysis also showed r results. These results indicate that histidine, lysine, and methionine prevent etanercept denaturation to inhibit formation of by—products thereof (see Table 3).

Therefore, the liquid formulation according to the present invention can stably maintain pharmaceutical efficacies of etanercept for a long period of time by ef— fectively reducing formation of etanercept by—products.

As used , the term "etanercept (recombinant p75 sTNFR:Fc fusion protein)" refers to a protein which is a homodimer form of two Fc fusion proteins linked by ide bonds, each PC fusion n consisting of a human soluble 75 kilodalton (p75) TNF(tumor necrosis factor) receptor coupled to the Fc portion of human IgGl.

Specifically, etanercept is a homodimer form of two Fc fusion ns linked by 3 disulfide bonds, each PC fusion protein consisting of the extracellular ligand—binding portion of the human soluble p75 TNF receptor linked to the Fc n of human IgGl. The PC component of etanercept contains the CH2 domain, the CH3 domain and hinge region, but not the CH1 domain of IgGl. The etanercept may have a molecular weight of approximately 150 kilodaltons(kDa). This etanercept may be currently sold under the trade name ENBREL® (Amgen Inc., Thousand Oaks, CA.), and have CAS number 185243—69—0.

Etanercept may be produced by recombinant DNA technology, but is not limited thereto.

The etanercept of the t invention is a ical inflammation modulator that functions as a competitive inhibitor of TNF-0t, binding to cell surface TNF-0t receptor, to inhibit TNF-0t mediated immune responses, and is used to treat rheumatoid arthritis, psoriasis, and ankylosing spondylitis, and is under clinical trials for the treatment of vasculitis, Alzheimer's disease, and Crohn's disease.

In the liquid formulation according to the present invention, the etanercept is contained in a therapeutically effective amount. Preferably, etanercept is present in an amount of l to 100 mg/ml.

As used herein, the term "stabilized liquid formulation" or "stable liquid formulation" refers to a formulation that retains the physical and al identity and integrity of the therapeutically active ingredient, etanercept, during storage in a solution. An an— alytical measurement of the etanercep ity may be performed by protein stability assay widely known in the art. The stability may be measured at a predetermined tem— perature for a predetermined time. For rapid assay, the formulation may be stored at a higher or "elevated" temperature, for example, 40°C for 2 weeks to 1 month or longer, and its time—dependent stability measured at this time.

As used herein, the term "stabilizer" refers to a specific chemical that interacts with a ical molecule and/or a l pharmaceutical ent in a ation to improve its stability. The stabilizer generally protects proteins from air/solution interface—induced stress and solution/surface—induced stress which cause protein ag— gregation. In the present invention, the izer is a component that reduces formation of etanercept by—products during e in a solution to maintain its activity for a long period of time, and is preferably one or more ed from the group consisting of me— thionine, lysine, histidine and pharmaceutically acceptable salts thereof.

As for the amino acids, both L—forms and D—forms are included in the scope of the present ion. Not only the amino acids themselves such as methionine, lysine, and histidine, but also anlogues, solvates, hydrates and stereoisomers, and the pharma— ceutically acceptable salts thereof are within the scope of the present invention as long as they show ntially the same effect.

As used herein, the term "pharmaceutically acceptable salts" refers to compounds that are prepared in the form of salts of the amino acids, such as methionine, lysine, and histidine, within the range retaining their functions as the stabilizers of the present invention. Specifically, it may form a salt by acid on, and for example, it may form a salt with an inorganic acid (e.g., hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, etc.), organic carboxylic acid (e.g., acetic acid, halo acetic acid such as triﬂuoroacetic acid, propionic acid, maleic acid, succinic acid, malic acid, citric acid, tartaric acid, salicylic acid), sugar acid (glucuronic acid, galacturonic acid, gluconic acid, ascorbic acid), acidic polysaccharide (e.g., hyaluronic acid, chondroitin sulfate, arginine acid), organic sulfonic acid including sulfonic acid sugar ester such as chondroitin sulfate (e.g., methane sulfonic acid, p—toluene sulfonic acid).

As used herein, the term "analogues" refers to compounds that show the same functions as the amino acids, such as methionine, , and ine, used as sta— bilizers of the present invention. In the t invention, the amino acids such as me— thionine, lysine, and histidine include the analogues of thereof. In the present ion, examples of the analogues may include methionine analogues, namely, se— lenomethionine, hydroxy methyl butanoic acid, ethionine, and oromethionine, but are not limited thereto.

As used herein, the term "by—products" refers to the undesirable products that reduce the ratio of the therapeutically active ingredient, etanercept, in the formulation. The typical by—products e "low molecular weight products" resulting from etanercept denaturation by deamination or hydrolysis, "high molecular weight products" such as oligomers and aggregates, or mixtures thereof.

As used herein, the term "high lar weight products" includes etanercept fragments that are subsequently aggregated by denaturation (e.g., ed by polypeptide degradation due to deamination or hydrolysis) or mixtures thereof.

Typically, the high molecular weight products are complexes having higher molecular weights than the therapeutic monomer etanercept, and may have a molecular weight of more than approximately 150 kDa.

As used herein, the term "low molecular weight products" includes, for e, therapeutic polypeptides produced by deamination or hydrolysis, namely, etanercept fragments. Typically, the low lar weight products are complexes having lower molecular weights than the therapeutic monomer etanercept, and may have a molecular weight of less than approximately 150 kDa.

The liquid formulation of the present invention includes one or more stabilizers selected from the group consisting of methionine, lysine, histidine, and pharma— ceutically able salts thereof. Specifically, the liquid formulation comprises me— thionine, lysine, or histidine as a stabilizer, or two stabilizers such as methionine and lysine, nine and histidine, or lysine and histidine, or three stabilizers such as me— thionine, lysine, and histidine. In the above formulation comprising combinations of the amino acids, the pharmaceutically acceptable salts of methionine, lysine, histidine respectively may be added further, or methionine, lysine, and histidine may be the modified forms of the pharmaceutically acceptable salts respectively. The liquid for— mulation ses preferably methionine, lysine, histidine, methionine and lysine, methionine and histidine, or lysine and ine, and more preferably methionine as a izer.

Methionine is an essential amino acid, and has a chemical formula of C5H11NOZS and a molecular weight of 149.21. Methionine is a non—polar amino acid, and contains a thioether group(—S—CH3) in its side chain. It has a pKl value of 2.28, pKz value of 9.21, and isoelectronic point (P1) value of 5.74. nine prevents oxidation of antibodies in liquid ations to stabilize antibodies by competing with the methionine WO 65917 residues in antibodies for reaction with the free hydroxyl radicals (Lam et al., J. Pharm.

Sci., 86(11): 1250-1255, 1997; Wang, Int. J. Pharm., 185: 129—188, 1999).

Lysine is an essential amino acid, and has a chemical formula of C6H14N202 and a molecular weight of 146.19. Lysine is a basic amino acid, and has a pKl value of 2.18, pKz value of 8.95, pK3 value of 10.53 and PI value of 9.74.

Histidine is an ial amino acid with an imidazole functional group having positive charge in its side chain, and has a chemical formula of C6H9N302 and a molecular weight of . Histidine is a basic amino acid, and has a pKl value of 1.82, pKz value of 9.17, pK3 value of 6.0 and PI value of 7.59.

In contrast, L—arginine contained in the known commercially available formulation of etanercept is an essential amino acid, and has a chemical formula of C6H14N402 and a molecular weight of 174.2. Arginine is a basic amino acid, and has a pKl value of 2.17, pKz value of 9.04, pK3 value of 12.48 and PI value of 10.76. That is, methionine, lysine, and histidine used as a izer in the present ion are totally different from arginine as a known stabilizer for etanercept in construct, chemical formula, physicochemical properties, and ionization tendency. The present invention has demonstrated for the first time that amino acids such methionine, lysine, and histidine are the appropriate stabilizers for etanercept.

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

The liquid formulation of the present invention may further include any material which is generally contained in formulations of protein drugs or antibody drugs to se etanercept stability according to one or more stabilizers selected from the group consisting of methionine, lysine, histidine, and pharmaceutically acceptable salts thereof except for those deteriorating the function of the present invention. For example, L—arginine known as a stabilizer for etanercept may be added to the liquid formulation of the present invention.

The liquid formulation according to the t invention may r include a buffer in addition to the izer. As used herein, the term "buffer" refers to the component that improves isotonicity and chemical stability of the formulation, and functions to maintain physiologically suitable pH. The buffer prevents a rapid pH change of the liquid formulation to maintain pH of the solution for the stabilization of cept. Preferred examples of the buffer e citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, e, ine, and Tris, but are not d thereto. In the specific embodiment, the buffer is a phosphate buffer. The buffer may be used either alone or in combinations of two or more thereof.

In the s embodiments of the present invention, the ation should have pH of approximately 5 to 7.5 or pH of approximately 5.8 to 6.8. In the specific em— WO 65917 nt, the formulation has pH of approximately 6.0 to 6.6. The ranges from the in— termediate to the above mentioned pH levels, for example, imately pH 5.2 to ap— proximately pH 6.4 (e.g., pH 6.2) are also intended to be part of this invention. For example, ranges of values using a ation of any of the above mentioned values as upper and/or lower limits are intended to be included. If necessary, the pH may be adjusted by techniques known in the art. For example, the pH may be adjusted to any desirable range by addition of HCl or by addition of histidine, if necessary.

In another embodiment, the buffer is present at a concentration of 0.1 to 100 mM, preferably 1 to 50 mM, and more ably 5 to 25 mM. The ranges from the in— termediate to the above mentioned concentrations are also intended to be part of this invention. For example, ranges of concentrations using a combination of any of the above mentioned concentrations as upper and/or lower limits are intended to be included. In the specific embodiment, the buffer should be present in an amount ient to maintain the physiologically sufficient pH.

The liquid formulation according to the present invention may further include an isotonic agent in addition to the stabilizer. As used herein, the term "isotonic agent" refers to a component that functions to partially maintain isotonicity of the formulation and the protein level, and partially maintain the level, ratio, or proportion of the thera— peutically active polypeptide present in the formulation. The isotonic agent ses the same c pressure as blood plasma, and so can be intravenously infused into a subject without ng the osmotic pressure of the subject's blood plasma. Indeed, in one embodiment according to the present invention, isotonic agent is present in an amount sufficient to render the formulation suitable for intravenous infusion. Often, the isotonic agent serves as a bulking agent as well. As such, the ic agent may allow the protein to me s stresses such as freezing and shear.

The isotonic agent serves to maintain the proper osmotic pressure in the body, when etanercept in the solution is administered into the body. Examples of the isotonic agent may include the commonly used sodium chloride, potassium chloride, boric acid, sodium borate, mannitol, in, ene glycol, polyethylene glycol, maltose, sucrose, erythritol, arabitol, xylitol, sorbitol, and glucose, but is not limited thereto. In the specific embodiment, the isotonic agent is NaCl. These isotonic agents may be used either alone or in combinations of two or more thereof.

In still another embodiment, the isotonic agent (e.g., NaCl) is present at a con— centration of l to 1000 mM, preferably 10 to 500 mM, and more preferably 50 to 250 mM. The ranges from the intermediate to the above mentioned concentrations are also intended to be part of this invention. For example, ranges of concentrations using a combination of any of the above mentioned concentrations as upper and/or lower limits are intended to be included. The isotonic agent should be present in an amount WO 65917 sufficient to maintain osmosis of the ation.

The liquid formulation according to the t invention may r include a phar— maceutically acceptable excipient, and examples of the excipient may include sugars and polyols, surfactants, rs or the like. es of the sugars and polyols may include sucrose, trehalose, lactose, maltose, galactose, mannitol, sorbitol, glycerol, examples of the tants may include non—ionic surfactants such as polysorbate 20, polysorbate 80, and poloxamer, and examples of the polymers may e dextran, polyethylene glycol, carboxyl methylcellulose, hyaluronic acid, and cyclodextrin.

The liquid formulation according to the present invention may further include a vative. The preservative means a chemical that is added to pharmaceutical for— mulations as an antimicrobial agent. Examples of the preservative may include ben— zalkonium chloride, benzethonium, exidine, phenol, m—cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, ol, p—cresol, chloro—cresol, phenylmercuric nitrate, osal, and benzoic acid, but are not limited o. These preservatives may be used either alone or in combinations of two or more thereof.

In the specific embodiment, the formulation of the present invention is a stable liquid formulation comprising 1 to 100 mg/mL of etanercept, 0.1 to 250 mM of methionine or pharmaceutically acceptable salts thereof, 01 to 100 mM of phosphate , and l to 1000 mM of sodium chloride. More specifically, the formulation of the present invention is the stable liquid formulation comprising 1 to 100 mg/mL of etanercept, 0.1 to 100 mM of methionine or pharmaceutically acceptable salts thereof, 01 to 50 mM of phosphate buffer, and l to 500 mM of sodium chloride, at pH 6.0 to 6.6.

In the specific embodiment, the formulation of the present invention is a stable liquid formulation comprising 1 to 100 mg/mL of etanercept, 0.1 to 250 mM of histidine or pharmaceutically acceptable salts f, 01 to 100 mM of phosphate buffer, and l to 1000 mM of sodium chloride. More specifically, the formulation of the t invention is the stable liquid formulation comprising 1 to 100 mg/mL of etanercept, 0.1 to 100 mM of histidine or pharmaceutically acceptable salts thereof, 01 to 50 mM of phosphate buffer, and l to 500 mM of sodium chloride, at pH 6.0 to 6.6.

In the specific ment, the formulation of the present invention is a stable liquid formulation comprising 1 to 100 mg/mL of etanercept, 0.1 to 250 mM of lysine or pharmaceutically acceptable salts thereof, 01 to 100 mM of phosphate buffer, and l to 1000 mM of sodium chloride. More specifically, the formulation of the present invention is the stable liquid formulation comprising 1 to 100 mg/mL of etanercept, 0.1 to 100 mM of lysine or pharmaceutically acceptable salts thereof, 01 to 50 mM of phosphate buffer, and l to 500 mM of sodium chloride, at pH 6.0 to 6.6.

In the specific embodiment, the formulation of the present invention is a stable liquid formulation comprising 1 to 100 mg/mL of etanercept, 0.1 to 250 mM of histidine or pharmaceutically acceptable salts f, 0.1 to 250 mM of lysine or pharmaceutically acceptable salts f, 01 to 100 mM of phosphate buffer, and l to 1000 mM of sodium chloride. More specifically, the formulation of the present invention is the stable liquid formulation comprising 1 to 100 mg/mL of etanercept, l to 50 mM of histidine or pharmaceutically acceptable salts thereof, 1 to 100 mM of lysine or phar— maceutically acceptable salts thereof, 01 to 50 mM of phosphate buffer, and l to 500 mM of sodium chloride, at pH 6.0 to 6.6.

In the ic embodiment, the formulation of the present invention is a stable liquid formulation comprising 1 to 100 mg/mL of etanercept, 0.1 to 250 mM of ine or pharmaceutically acceptable salts thereof, 01 to 250 mM of methionine or pharma— ceutically able salts thereof, 01 to 100 mM of phosphate buffer, and l to 1000 mM of sodium chloride. More specifically, the formulation of the present invention is the stable liquid formulation comprising 1 to 100 mg/mL of etanercept, l to 50 mM of histidine or pharmaceutically acceptable salts thereof, 1 to 100 mM of methionine or pharmaceutically able salts thereof, 01 to 50 mM of phosphate buffer, and l to 500 mM of sodium chloride, at pH 6.0 to 6.6.

In the specific embodiment, the ation of the present invention is a stable liquid formulation comprising 1 to 100 mg/mL of etanercept, 0.1 to 250 mM of methionine or pharmaceutically acceptable salts thereof, 01 to 250 mM of lysine or ceutically able salts thereof, 01 to 100 mM of phosphate buffer, and l to 1000 mM of sodium chloride. More specifically, the formulation of the present invention is the stable liquid formulation sing 1 to 100 mg/mL of etanercept, l to 50 mM of me— thionine or pharmaceutically acceptable salts thereof, 1 to 50 mM of lysinee or phar— maceutically acceptable salts thereof, 01 to 50 mM of phosphate buffer, and l to 500 mM of sodium chloride, at pH 6.0 to 6.6.

The formulation of the present invention can be used for treatment of a disease in which etanercept is therapeutically effective. Etanercept is a biological inflammation modulator to inhibit TNF—(x mediated immune responses, and the formulation of the present invention is used to treat rheumatoid arthritis, psoriasis, ankylosing spondylitis, vasculitis, Alzheimer's e, or Crohn's disease, not limited thereto. The formulation of the present invention may be administered by oral or parenteral, i.e., anelusly, intramuscularly, ntraperitoneail, intra—abdominal, transdermal route, and/or intra— ly, but is not limited thereto.

In another aspect, the present invention provides a method for incresing ity of etanercept using a liquid formulation comprising one or more stabilizers selected from the group ting of methionine, , histidine, and pharmaceutically acceptable salts thereof.

The liquid formulation can increase storage stability of etanercept by reducing etanercept ducts that are produced due to denaturation during storage.

Mode for the ion Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are for rative purposes only, and the invention is not intended to be limited by these Examples. <Example 1> Stability test of etanercept aqueous formulation ing to addition of stabilizer In order to investigate the l izer for the preparation of the stable aqueous formulation of etanercept, histidine as a stabilizer was added to a 5 mM ate solution to which etanercept was added to a concentration of 50 mg/mL, and sodium chloride was added thereto as an isotonic agentto prepare Formulation 1.

In addition, etanercept was added to a 10 mM phosphate solution to a tration of 50 mg/mL, and sodium chloride was added thereto as an isotonic agent. Then, each of lysine, arginine, methionine, glycine, polysorbate 20, polysorbate 80, poloxamer 188, propylene glycol, protamine sulfate, sucrose, and guanidine HCl was added to prepare Formulations 2 to 12. y, sodium chloride as the isotonic agent was added to a 10 mM phosphate solution to which etanercept was added to a concentration of 50 mg/mL to prepare a stabilizer—free formulation. The stabilizer compositions of the formulations are shown in the following Table 1. Each 0.5 mL of the prepared formulation was put in 1.0 mL glass syringes, sealed, and stored at 50°C.

Table l [Table l] stabilizer—free formulation — At 7 days after storage at 50°C, Formulations l to 12 and the stabilizer—free for— mulation of Table l were analyzed by SE—HPLC to examine the types of low molecular weight products and high molecular weight products such as oligomers and aggregates resulting from etanercept denaturation, and the total amount of these ts was represented as Total Impurity. In on, structural changes of etanercept were ed by HI—HPLC. In HI—HPLC, cept—related substances were separated 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 dimers with low aggregation or actiVity. Thus, the total amount of pre—Peak, Peak 1, and Peak 3 was represented as Total Impurity. The results are shown in Tables 2 and 3, respectively.

Table 2 WO 65917 [Table 2] Table 3 [Table 3] As shown in the SE—HPLC results of Table 2, when stored at 50°C for 7 days, For— mulation 1 containing histidine as a stabilizer showed total impurity of 22.2%, For— mulation 2 containing lysine as a stabilizer showed total impurity of 24.8%, For— mulation 3 containing arginine as a stabilizer showed total impurity of 24.2%, For— mulation 4 ning methionine as a stabilizer showed total impurity of 20.8%. All of them showed remarkably low Total Impurity, compared to stabilizer—free for— mulation showing Total ty of 30.2%. When these ations were compared to Formulation 3 using L—arginine as a stabilizer (US Patent No. 7,648,702), For— mulation 2 containing lysine showed similar total impurity of 24.8%, and Formulation 1 containing histidine and Formulation 4 containing methionine showed reduced Total Impurity of 22.2% and 20.8%, respectively.

The HI—HPLC results of Table 3 are similar to the SE—HPLC results. The stabilizer— free formulation showed Total Impurity of 41.9%. On the ry, each of For— mulations l, 2, 3, and 4 showed Total Impurity of 33.7%, 34.9%, 34.3%, and 30.3% respectively, indicating that histidine, lysine, nine, and methionine added in each formulation have the effects of ting etanercept denaturation. In particular, For— mulation 4 containing methionine showed the lowest Total ty, indicating that methionine is most ive in etanercept ization.

Consequently, the present invention demonstrated that lysine, histidine, and me— thionine have effects of stabilizing etanercept by preventing its denaturation, which are equivalent to or higher than that of L—arginine, and in particular, methionine is more effective in etanercept stabilization than L—arginine. <Example 2> Stability test of etanercept s formulation according to me- thionine concentration In order to investigate the optimal concentration of methionine for the stabilization of etanercept, 120 mM sodium chloride was added to 10 mM phosphate solutions, and each of 25 mM and 12.5 mM methionine was added thereto, after which etanercept in a concentration of 50 mg/mL so was added as to prepare Formulations 13 and 14, re— spectively. According to the preparation of a commercially available etanercept aqueous formulation, 25 mM L—arginine was added to 100 mM sodium chloride and 1% sucrose in a 10 mM phosphate solution, and etanercept was added to a con— centration of 50 mg/mL so as to e a control. Each 0.5 mL of the formulation was put in 1.0 mL glass syringes, sealed, and stored at 40°C, 25°C and 4°C. The com— positions of the prepared aqueous formulations are shown in the ing Table 4.

Table 4 [Table 4] Formulation 13 Etanercept 50 mg/mL, phosphate solution 10 mM, NaCl 120 mM, methionine 25 mM (pH 6.3) ation 14 Etanercept 50 mg/mL, phosphate solution 10 mM, NaCl 120 mM, methionine 12.5 mM (pH 6.3) Control Etanercept 50 mg/mL, phosphate on 25 mM, NaCl 100 mM, sucrose 1%, L—arginine 25 mM (pH 6.3) After Formulation 13, ation 14, and the control were stored at 40°C for 1 and 3 weeks, and at 25°C and 4°C for 4 and 8 weeks each, their Total ty was measured by SE—HPLC and HI—HPLC, as in Example 1. The s are shown in Tables 5 and 6, respectively.

Table 5 [Table 5] If5TotalImpurityby SE—HPLC (%)_0°C25°C0week 1week w—0eek4 weeks weeks weeks week weeks weeks Formulatio 5.5 11. 20. 5.5 14.8 18.8 5.5 8.8 10.2 .55 125 20 55 160 199 55II1 Table 6 [Table 6] Total Impurity by HI—HPLC (%) _0°C_5°C Formulatio 10.0 15.9 2. 10. l5. 18. 10.0 ll. 11. n 13 Formulatio 17.0 2. 16. 19. 8 11. n 14 As shown in the results of Tables 5 and 6, Formulation 13 and Formulation 14 containing methionine as the stabilizer showed low impurity production under all of the storage conditions of 40°C, 25°C and 4°C, compared to the control group ed according to the preparation method of a commercially available etanercept aqueous formulation. Specifically, the SE—HPLC and HI—HPLC results showed that For— mulation 13 containing 25 mM methionine and Formulation 14 containing 12.5 mM methionine showed lower impurity production at 40°C and 3 weeks, and at 25°C and 8 weeks, and r impurity tion at 4°C and 8 weeks, compared to the control.

These results indicate that the nine—containing formulation of the present invention is a more stable formulation for ining the etanercept actiVity for a long period of time than the commercially available formulation.

In conclusion, the present invention demonstrated that the cept aqueous for— mulation containing methionine prevents denaturation of etanercept to in its activity for a long period of time, and methionine is very effective as a izer in the etanercept aqueous formulation. <Example 3> Stability test of etanercept aqueous ation comprising histidine and , or histidine and methionine Histidine 5mM and lysine 25mM as a stabilizer were added to a 10 mM phosphate solution to which etanercept was added to a concentration of 50 mg/mL, and sodium chloride was added thereto as an isotonic agent to prepare Formulation 15.

In addition, Histidine 5mM and methionine 25mM as a stabilizer were added to a 10 mM phosphate solution to which etanercept was added to a concentration of 50 mg/ mL, and sodium chloride was added thereto as an isotonic agentto e Formulation Finally, sodium chloride as the isotonic agent was added to a 10 mM ate solution and etanerceptwas added thereto to a concentration of 50 mg/mL to e a stabilizer—free formulation.

Each 0.5 mL of the prepared formulation was put in 1.0 mL glass syringes, sealed, and stored at 50°C.

The stabilizer compositions of the formulations are shown in the following Table 7.

Table 7 [Table 7] Formulation 15 Histidine 5mM + Lysine 25mM Formulation 16 Histidine 5mM + Methionine 25mM stabilizer—free formulation At 7 days after storage at 50°C, Formulations 15, 16 and stabilizer—free ation of Table 7 were analyzed by SE—HPLC as example 1 to measure their Total Impurity.

The results are shown in Tables 8.

Table 8 [Table 8] SE—HPLC Total Impurity (%) izer—free ation [ 1 19] As shown in the SE—HPLC results of Table 8, when stored at 50°C for 7 days, For— mulation 16 containing ine as a stabilizer showed total impurity of 21.5%, and showed remarkably low Total Impurity, compared to the stabilizer—free formulation showing Total Impurity of 30.2%. And Formulation 16 showed remarkably low Total Impurity, compared to Formulation 1 containing 10mM histidine showing Total Impurity of 22.2%, and Formulation 2 containing lysine 25mM showing Total Impurity of 24.8%.

That is, the present invention demonstrated that the formulation comprising histidine and lysine has the remarkably excellent effects of preventing etanercept denaturation by reducing the amount of total impurity for storage, compared to the stabilizer—free formulation and each formulation comprising histidine, lysine, and methionine re— vely.

In on, as shown in the SE—HPLC results of Table 8, when stored at 50°C for 7 days, Formulation 16 containing histidine as a stabilizer showed Total ty of .9%, which is remarkably low, compared to the stabilizer—free formulation g Total Impurity of 30.2%. In particular, Formulation 16 showed low Total Impurity compared to Formulation 15 containing histidine and lysine as a stabilizer.

That is, the present invention demonstrated that the formulation comprising histidine and nine has remarkably effects on the preventing of etanercept denaturation by reducing the amount of Total ty for e, compared to the stabilizer—free for— <Example 4> Stability test of etanercept aqueous formulation comprising me- thionine and lysine Methionine 12.5mM and lysine 12.5mM as a stabilizer were added to a 10 mM ate solution to which etanercept was added to a concentration of 50 mg/mL, and sodium chloride was added thereto as an isotonic agent to prepare Formulation 17.

In addition, sodium chloride as the isotonic agent was added to a 10 mM phosphate solution and etanercept was added thereto to a concentration of 50 mg/mL to prepare a stabilizer—free formulation.

Each 0.5 mL of the prepared formulation was put in 1.0 mL glass syringes, sealed, and stored at 50°C. The izer compositions of the ations are shown in the following Table 9.

Table 9 [Table 9] Formulation 17 Methionine 12.5mM +Lysine 12.5mM At 7 days after storage at 50°C, Formulations 17, and stabilizer—free formulation of Table 9 were ed by SE—HPLC as example 1 to measure their Total Impurity. The results are shown in Table 10.

Table 10 [Table 10] SE—HPLC Total Impurity (%) As shown in the SE—HPLC s of Table 10, when stored at 50°C for 7 days, For— mulation 17 containing methionine and lysine as a stabilizer showed Total Impurity of 27%, which is remarkably low, compared to the stabilizer—free formulation showing Total Impurity of 32.8%.

That is, the present invention demonstrated that the formulation comprising me— thionine and lysine has remarkably effects on the stabilizing of cept by reducing the amount of Total Impurity, compared to the stabilizer—free formulation.

Claims

Claims 1.

1. A liquid formulation of etanercept, comprising cept; and methionine or pharmaceutically acceptable salts thereof as a stabilizer, wherein the formulation has increased storage ity of cept compared to the formulation comprising arginine as a stabilizer, by reducing cept by-products that are ed due to denaturation during storage.

2. The liquid formulation according to claim 1, which further comprises one or more stabilizers selected from the group consisting of lysine, histidine, and pharmaceutically acceptable salts thereof.

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

4. The liquid formulation ing to claim 1, wherein the etanercept is present in an amount of 1 to 100 mg/mL.

5. The liquid formulation according to claim 1, which further comprises one or more materials selected from the group consisting of a buffer, an ic agent, an excipient, or a preservative.

6. The liquid formulation according to claim 5, wherein the buffer is selected from the group consisting of citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate, histidine, and Tris.

7. The liquid formulation according to claim 5, wherein the buffer is present in an amount of 0.1 to 100 mM.

8. The liquid formulation according to claim 5, n the isotonic agent is selected from the group consisting of sodium chloride, potassium chloride, boric acid, sodium borate, mannitol, glycerin, propylene , polyethylene glycol, maltose, sucrose, erythritol, arabitol, xylitol, sorbitol, and glucose.

9. The liquid ation according to claim 5, wherein the isotonic agent is present in an amount of 1 to 1000 mM.

10. The liquid formulation according to claim 1, comprising 1 to 100 mg/mL of etanercept, 0.1 to 250 mM of methionine or pharmaceutically acceptable salts thereof, 0.1 to 100 mM of phosphate buffer, and 1 to 1000 mM of sodium de.

11. The liquid formulation according to claim 1, comprising etanercept; methionine and lysine as a stabilizer.

12. The liquid formulation according to claim 1, comprising etanercept; nine and histidine as a stabilizer.

13. The liquid formulation according to claim 2, comprising 1 to 100 mg/mL of etanercept, 0.1 to 250 mM of histidine or pharmaceutically acceptable salts thereof, 0.1 to 250 mM of methionine or ceutically acceptable salts thereof, 0.1 to 100 mM of phosphate buffer, and 1 to 1000 mM of sodium chloride.

14. The liquid formulation according to claim 2, comprising 1 to 100 mg/mL of etanercept, 0.1 to 250 mM of methionine or pharmaceutically acceptable salts thereof, 0.1 to 250 mM of lysine or pharmaceutically acceptable salts thereof, 0.1 to 100 mM of ate buffer, and 1 to 1000 mM of sodium chloride.