STABILIZED Fc FUSION PROTEIN SOLUTION

Abstract

To provide a stable aqueous solution formulation of an Fc fusion protein.SOLUTION: There is provided an aqueous solution formulation comprising: (i) an Fc fusion protein; and (ii) sulfate ions at a concentration of 5-200 mM to stabilise the Fc fusion protein, wherein the formulation is free of amino acids selected from arginine, lysine and proline, and salts thereof, and the formulation is also free of magnesium ions.SELECTED DRAWING: None

Description

[Background technology]

[0001] BACKGROUND OF THEINVENTION Fc fusion proteins formulated in aqueous solutions are prone to structural degradation during storage. The processes involved in protein degradation include physical processes (e.g., quaternary, tertiary, or secondary) loss of structure, aggregation, particle formation) and chemical processes (i.e., deamidation, asparagine (processes involving changes in covalent bonds, such as acid isomerization, oxidation, and hydrolysis) Each decomposition product (e.g., soluble aggregates, insoluble aggregates, and chemically modified variants) can be analyzed. ) may affect the biological activity, toxicity, or immunogenicity of the Fc fusion protein.

[0002] Therefore, all degradation product levels are set for each Fc fusion protein product. It must be maintained within strict specifications. The rate of the decomposition process is temperature dependent and Fc fusion proteins are generally more stable at low temperatures. As a result, commercially available Fc fusion proteins These products typically must be kept refrigerated. However, they are available in a variety of sizes and can be self-administered by patients. With the increasing trend towards subcutaneously administered products, Use outside the cold chain for a period of time, e.g. 12 weeks or longer. There is a strong demand for the development of Fc fusion protein products that can be used in cold chain Being able to store a product outside of the fountain often provides a great deal of convenience to the patient during use. In addition, if excursion outside the cold chain becomes possible, the quality of the shipped goods will be improved. The flow can be significantly improved.

[0003] The present invention addresses the problem of instability of Fc fusion proteins, in particular the degradation of Fc fusion proteins, Address the issue of aggregation.

[0004] WO2003 / 072060 (Immunex) describes L-arginine as an aggregation inhibitor containing an Fc domain. A protein formulation comprising:

[0005] WO2012 / 143418 (Sandoz) discloses a compound that uses lysine and proline as stability enhancing agents. Formulations of etanercept are disclosed that include an amino acid selected from the group consisting of:

[0006] WO2013 / 006454 (Biogen) discloses a method for treating a pulmonary arthritis with L-arginine less than 1 mM and etanercept. Formulations of etanercept are disclosed that contain a sufficient amount of salt to prevent aggregation of the drug. Summary of the Invention

[0007] (Summary of the invention) The present invention addresses the problem of instability of Fc fusion proteins. Ming is, (i) an Fc fusion protein; and (ii) sulfate ions at a concentration of 5 to 200 mM for stabilizing the Fc fusion protein; Including, Does not contain amino acids selected from arginine, lysine and proline, and salts thereof and does not contain magnesium ions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] Detailed Description of the Invention The present invention relates to a method for stabilizing an aqueous solution of an Fc fusion protein with sulfate ions at a concentration of 5 to 200 mM. This relates to the discovery that it is possible to

[0009] As used herein, the term "aqueous formulation" refers to a formulation containing water, preferably distilled water or deionized water. The aqueous solution formulation of the present invention refers to a solution of water for injection, sterile water for injection, or bacteriostatic water for injection. a digested Fc fusion protein, sulfate ions, and optionally one or more additives and / or excipients, and comprising an amino acid selected from arginine, lysine, and proline, and salts thereof. First, it does not contain magnesium ions. Also, the aqueous formulation may contain one or more ingredients, e.g. It may contain partially soluble or insoluble additives or excipients. The presence of the active ingredient results in a multi-phase formulation, such as a suspension or emulsion. is a homogeneous solution as determined visually or by light scattering.

[0010] As used herein, the term "Fc fusion protein" refers to a fusion protein that is a fusion protein that is derived from the Fc portion of an immunoglobulin. "Heterologous polypeptide" refers to a protein containing a genetically linked heterologous polypeptide or its derivative. "Lipeptides" are peptides that are not naturally found genetically linked to the Fc portion and are typical It means a polypeptide that is not an antibody-derived polypeptide. "Genetically linked" with respect to peptides means that two proteins / polypeptides are linked together by a peptide This means that the sequences are linked by a tethered bond and can be encoded by a single nucleic acid molecule. For example, the heterologous polypeptide can bind to a ligand, preferably a specific ligand. A heterologous polypeptide is a polypeptide that is synthesized by synthesis from another protein, e.g., a protein that has a role in the human body. More generally, the heterologous The polypeptide may be therapeutically useful. Exemplary such heterologous polypeptides include, but are not limited to, Without being subjected to any of the above-mentioned procedures, the cytokines, blood clotting factors, or growth factors, or their functional properties, A further example is a GLP-1 agonist. Examples include conjugates, such as Fc fusion proteins conjugated to another moiety. Derivatives are available. Such moieties include chemically inert multimers such as PEG.

[0011] In certain embodiments, the two Fc fusion proteins are linked together, e.g., via a disulfide bond. They can associate to form dimeric proteins.

[0012] In certain embodiments, the Fc region of the immunoglobulin comprises IgG1, IgG2, IgG3, or IgG4. The Fc region of an immunoglobulin is an IgA, IgD, IgE, or IgY, such as IgG, IgM, IgA1, or IgA2. The most preferred Fc region of the antibody is the Fc region of IgG, in particular IgG1.

[0013] In some embodiments, the Fc fusion protein comprises one or more properties of the Fc fusion protein. , such as serum half-life, complement fixation, Fc receptor binding, and / or effector functions (e.g., The antibody contains one or more modifications in the Fc region that alter the activity of the antibody (antigen-dependent cellular cytotoxicity).

[0014] In one embodiment, the Fc fusion protein is capable of binding to a ligand, i.e., a ligand-binding The peptides include heterologous peptides of the same or similar structure.

[0015] In one embodiment, the heterologous polypeptide binds to tumor necrosis factor (TNF), e.g., TNFα. and may include, for example, TNF receptors, such as TNF receptor 2, particularly soluble forms thereof. .

[0016] In one embodiment, the heterologous polypeptide can bind to CD80 or CD86, e.g. It comprises the extracellular domain of CLTA-4 or a part thereof.

[0017] In one embodiment, the heterologous polypeptide can bind to VEGF, e.g., VEGFR1 and and / or the extracellular domain of VEGR2 or a portion thereof.

[0018] In one embodiment, the heterologous polypeptide is capable of binding to IL-1, e.g., IL-1R11 and / or the extracellular domain of IL-1RAcP, or a portion thereof.

[0019] In one embodiment, the heterologous polypeptide binds to a thrombopoietin receptor, such as c-Mpl. It can be combined.

[0020] In one embodiment, the heterologous polypeptide is a blood coagulation factor such as Factor VIII or Factor IX. It is a fixed factor or a part thereof.

[0021] In one embodiment, the heterologous polypeptide is a hActRIIb protein or a derivative thereof. .

[0022] In one embodiment, the heterologous polypeptide is a GLP-1 agonist, such as dulaglutide. It is.

[0023] Examples of Fc fusion proteins include etanercept, abatacept, and belatacept. Etanercept is of particular interest.

[0024] Further examples include aflibercept, rilonacept, romiplostim, eloctate, These include luspatercept and Alprolix.

[0025] In certain embodiments, the Fc fusion protein is substantially pure. The Fc fusion protein preferably contains a single Fc fusion protein and does not contain substantial amounts of any additional proteins. In an embodiment, the Fc fusion protein comprises at least 99% of the total protein content of the formulation, preferably or at least 99.5%, and more preferably at least about 99.9%. In this case, the Fc fusion protein is sufficiently pure for use in a pharmaceutical formulation.

[0026] The Fc fusion protein is preferably a therapeutic Fc fusion protein. The protein has a desired therapeutic or prophylactic activity and is useful for treating, inhibiting, or preventing a disease or medical disorder. or for prophylactic purposes.

[0027] The Fc fusion protein is preferably about 1 to 400 mg / ml, more preferably 20 to 100 mg / ml, and even more preferably is present at a concentration of about 50 mg / ml.

[0028] As described above, the present inventors have demonstrated that the stability of Fc fusion proteins in aqueous formulations is improved by the addition of sulfate ions. It has been found that the presence of sulfate ions improves the solubility of the enzyme. Preferably, the sulfate ions are present in the range of 25 to 150 mM, for example 25 to 150 mM. 100 mM, or 25 to 90 mM, more preferably 50 to 100 mM, or 50 to 90 mM, for example 50 to 75 mM Another range of interest is 50-125 mM, e.g., 75-125 mM. It is mM.

[0029] The source of sulfate ions is preferably, for example, sodium sulfate, potassium sulfate, and ammonium sulfate. The source of sulfate ions should be magnesium sulfate. isn't it.

[0030] The present inventors also found that the stability of Fc fusion proteins in aqueous formulations was improved by the addition of polyanionic surfactants other than sulfate. It has been discovered that the presence of ions (or two or more multivalent ions) can further improve Therefore, the aqueous solution formulation of the present invention preferably contains a polyanion other than sulfuric acid as a further stabilizing component. Contains an ion (or two or more).

[0031] A polyanion (other than sulfate) has a charge of at least minus two (similarly, "-2 For example, a polyanion may be written as minus two ("divalent anion" or "minus two"). anion), negative three (a trivalent anion), or negative four (a tetravalent anion) In one embodiment, the polyanion has a negative two charge and the dianion In another embodiment, the polyanion has a charge of minus three and the trianion In a further embodiment, the polyanion is an anion having a negative two charge. and a mixture of anions with a negative charge of minus three, i.e. divalent anions and trivalent In another embodiment, the polyanion is a mixture of divalent anions, trivalent anions, ions, or mixtures thereof. Polyanions are A species that does not contain any groups capable of forming a positive charge (by protonation). The on does not contain a basic nitrogen center, i.e., a nitrogen center that can be protonated. In particular, multivalent The anion does not include a quaternary ammonium group (ie, a positively charged tetra-substituted nitrogen atom). Polyanions have pK at 25°C. a 5-8 or 3-9 and does not contain a nitrogen center that can be protonated. The polyanion is not an amino acid, specifically one of the 20 naturally occurring amino acids in either the L or D isomeric form. nor any mixture thereof (including racemic mixtures). Polyanions are not peptides, glutamic acids, aspartic acids, or mixtures thereof. or a protein (i.e., a molecule containing two or more amino acid residues). In the present invention, the polyanion does not contain a nitrogen atom. The polyanion is not a nitrogen-containing chelating agent. Preferably, the polyanion is less than 500 Da, and especially not ethylenediaminetetraacetic acid (EDTA). For example, the molecular weight is less than 400 Da, less than 300 Da, or less than 200 Da.

[0032] For example, the polyanion is an organic polyanion. An example of an organic polyanion is citric acid. Further examples are succinic acid, malic acid, and maleic acid, especially maleic acid. The polyanion can also be an inorganic polyanion other than sulfate, for example, phosphate. Examples of counterions for polyvalent anions include sodium, potassium, and ammonium ions. In particular, sodium and potassium ions. The counter ion should not be magnesium. For example, polyanions are sodium citrate, sodium maleate, or sodium phosphate. It can be used as sodium.

[0033] In one embodiment, the polyanion(s) comprises an organic polyanion: In one embodiment, the formulation contains no inorganic polyanions. Does not include.

[0034] Mixtures of polyanions (e.g., organic and inorganic polyanions or two organic polyanions) Thus, for example, a mixture of two or more (e.g., two or three) sulfuric acid salts may be used. Different polyanions can be utilized. In a preferred embodiment, the polyanion In one embodiment, citric acid and maleic acid are used as polyanions. The enzyme utilizes carboxylic acids such as carboxylic acid, citric acid, and maleic acid.

[0035] The concentration of the (or each) polyvalent anion other than sulfate in the solution formulation is, for example, about 1 to 100 mM; Preferably, the concentration is 1 to 60 mM or 1 to 50 mM, more preferably about 5 to 100 mM, 5 to 60, or 5 to 50 mM, more preferably about Suitably, it may be about 10 to 60, 10 to 50 mM, 10 to 30 mM, 10 to 20 mM, or 30 to 50 mM.

[0036] When using two or more polyvalent ions (other than sulfuric acid), the total concentration of the polyvalent ions is preferably about 1 to 20%. 100 mM, preferably about 1 to 60 mM or 1 to 50 mM, more preferably about 5 to 100 mM, or 5 to 60, or 5 to 50 mM, more preferably about 10 to 60, or 10 to 50 mM, or 10 to 30 mM, or 10 to 20 mM, or is 30-50 mM.

[0037] The aqueous solution formulations of the present invention can cover a wide range of osmolarity, including hypotonic, isotonic, and hypertonic aqueous solutions. Preferably, the aqueous formulation of the present invention is substantially isotonic. The aqueous solution formulation is isotonic. Preferably, the osmolarity of the aqueous solution formulation is selected to be in accordance with the route of administration. For example, to minimize pain during injection. A suitable aqueous solution formulation has an average pH of about 200 mOsm / L to about 500 mOsm / L. Preferably, the osmolality is in the range of about 250 mOsm / L to about 350 mOsm / L. More preferably, the osmolality is about 300 mOsm / L.

[0038] The osmolarity of the aqueous solution formulation can be adjusted using an osmotic adjuster. can be charged or uncharged.

[0039] Preferably, the aqueous solution formulations of the present invention are isotonic.

[0040] In one embodiment, the osmolarity is adjusted by the presence of a non-charged osmotic modifier in the formulation. The concentration of the osmolality adjuster is adjusted to the sulfate ion and any polyanion ions present in the formulation. For example, these may be adapted depending on the osmolyzing effect of the If so, no further osmolarity adjuster is required.

[0041] Examples of uncharged osmotic modifiers include sugars, sugar alcohols, and other polyhydric alcohols, e.g. For example, sucrose, trehalose, mannitol, raffinose, lactose, and dextro. glycerol, propylene glycol or polyethylene glycol, e.g. PEG300 or PEG400. In the present invention, the uncharged osmotic adjusters are sucrose, trehalose, mannitol, sorbitol, etc. Such uncharged osmotic modifiers, when utilized, are selected from the group consisting of PEG, PEG 300, and PEG 400. For example, about 10 to 1000 mM, for example, about 50 to 500 mM, for example, about 50 to 300 mM, for example, about 10 to 300 mM , for example at a concentration of about 10 to 100 mM, for example about 10 to 50 mM.

[0042] In one embodiment, the aqueous formulation does not contain xylitol.

[0043] Alternatively, osmolality may be adjusted by the presence of a charged osmotic modifier in the formulation. A charged osmotic modifier is a charged substance other than sulfate or a polyvalent anion. Examples include salts, such as sodium or potassium ions with chloride, nitrate, or acetate ions. The charged osmolality modifiers are used in combination with, for example, about 25 to 40% sodium chloride. 500 mM, preferably about 50 to 250 mM, for example about 150 mM; or about 10 to 250 mM, for example about 10 to 100 It may be present in the formulation at a concentration of about 10 to 50 mM, for example.

[0044] In one embodiment of the invention, the aqueous formulation does not contain a charged osmotic agent, in particular sodium chloride. The aqueous formulation contains less than 5 mM sodium, e.g., less than 1 mM sodium chloride. .

[0045] In one embodiment of the invention, the aqueous solution formulation does not contain a non-charged osmotic agent.

[0046] Preferably, the pH of the aqueous formulation is about pH 4.0 to about pH 7.0. In one embodiment, the pH is About pH 4.0 to about pH 6.0, for example about pH 5.0 to about pH 6.0, for example about pH 5.0 to about pH 5.7, for example In a more preferred embodiment, the pH is about pH 6.0 to about pH 7.0, for example about pH 6.0 to about pH 6.5, for example about pH 6.3.

[0047] In one embodiment, the aqueous formulation of the present invention is provided with a pH stabilizer for the formulation. The buffer may be selected to enhance the stability of the Fc fusion protein. Typically, the pKa is within 2 pH units (e.g., 1.5) of the formulation pH (particularly as determined at 25° C.). at least one ionizable group within 1 pH unit of the Suitably, the buffer has a concentration of about 0.5 mM to about 50 mM, for example about 1 mM to about 20 mM, for example It is present at about 1 mM to about 10 mM, such as 1 mM to about 5 mM, for example, about 2 mM to about 5 mM. An exemplary buffering agent is acetic acid. An exemplary buffering agent is a polyanion. When the buffer is a polyanion (e.g., phosphate or citrate), it is treated as a polyanion and should be considered in determining the composition of the formulation. It is not treated as a buffer for purposes of determining the amount of each component.

[0048] In one embodiment, the formulation does not contain any buffering agents that are not polyanionic.

[0049] The aqueous solution formulation of the present invention may optionally contain a surfactant. The agent may be a non-ionic surfactant, such as an alkyl glycoside, e.g., dodecyl maltoside; Resorbate surfactants, such as polysorbate 80 or polysorbate 20; Triethylene glycol (2) dodecyl ether, polyethylene glycol (2) oleyl ether and polyethylene glycol (2) hexadecyl ether. Alkyl ethers of glycols; polyethylene glycols and polypropylene glycols block copolymers, such as poloxamer 188, poloxamer 407, poloxamer 171 or poloxamer 185; or alkyl phenyl ether of polyethylene glycol , for example 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol. 27 Preferably, the surfactant has a concentration of about 10 μg / mL to about 2000 μg / mL, for example about 50 μg / mL to about 1 000 μg / mL, for example, from about 100 μg / mL to about 500 μg / mL.

[0050] The aqueous formulation of the present invention may optionally contain phenol, m-cresol, chlorocresol, benzene Alcohol, propylparaben, methylparaben, benzalkonium chloride, and benzyl chloride The composition may contain a preservative, suitably selected from the group consisting of azethorium and carbamide. When present, the preservative is present in a concentration of about 0. 01 mM to about 100 mM. Phenol, m-cresol, chlorocresol, benzyl alcohol The preservative selected from chol, propylparaben, and methylparaben may be, for example, at a concentration of about 10 ml. Benzochloride may be present at a concentration of from about 100 mM to about 100 mM, for example from about 20 mM to about 80 mM, for example from 25 mM to about 50 mM. The preservative selected from zirconium and benzethonium chloride may be, for example, at a concentration of about 0.01 mM to It may be present at about 1 mM, such as from about 0.05 mM to about 0.5 mM, for example from about 0.05 mM to about 0.2 mM.

[0051] In one embodiment, the aqueous formulation of the present invention contains all amino acids (i.e., peptide bonds). free amino acids that do not contain any isomers, in particular their L-isomer forms or the corresponding D-isomers Any of the 20 naturally occurring amino acids in their isomeric form or any mixture of their isomers (racemic (including mixtures).

[0052] As used herein, the term "high molecular weight species" refers to a molecule of the parent activated Fc fusion protein. Any component of the Fc fusion protein content having an apparent molecular weight at least about twice that of the That is, the high molecular weight species are multimeric aggregates of the parent Fc fusion protein. The product may contain a parent Fc fusion protein molecule that has been significantly altered in conformation. or multimeric aggregates are composed of parent proteins in native or near-native conformations. The high molecular weight species may be aggregates of protein units. , electrophoresis, analytical ultracentrifugation / sedimentation velocity, light scattering, dynamic light scattering, and field-effect spectroscopy. This can be done using methods known in the art, including low fractionation. Cut.

[0053] As used herein, the term "low molecular weight species" refers to a molecule of the parent activated Fc fusion protein. This refers to any component of the Fc fusion protein content that has an apparent molecular weight less than the amount of The low molecular weight species are fragments of the parent Fc fusion protein. The high molecular weight species was determined by size exclusion spectrometry. Chromatography, electrophoresis, analytical ultracentrifugation / sedimentation velocity, light scattering, dynamic light scattering, and This was carried out using methods known in the art, including field-flow fractionation. This can be done.

[0054] As used herein, the term "related species" refers to a species that has been modified by chemical modification of the parent Fc fusion protein. Refers to any component of the Fc fusion protein content that is formed, e.g., deamidated or oxidized species Related species are preferably purified by cation exchange chromatography, anion exchange chromatography, etc. The antibody is detected by reverse phase chromatography or capillary electrophoresis.

[0055] Suitably, the aqueous formulations of the present invention can be stored at 25° C., 30° C., or stable enough to remain substantially free of visible particles after storage at 40°C. Visible particles are preferably determined using 2.9.20. European Pharmacopoeia monograph (Particulate contamination: visible particles). It is detected using

[0056] Advantageously, the aqueous formulations of the present invention have low concentrations of relevant species that remain low over extended storage periods. It is stable enough to

[0057] In one embodiment, the aqueous formulation of the invention is stored at 25° C., 30° C., or at least 94% (by weight of total Fc fusion protein) after storage at 40°C. at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example Retains at least 99% of the parent Fc fusion protein. Fc (by weight of total Fc fusion protein) The percentage of fusion protein was determined by size exclusion chromatography, cation exchange chromatography, and chromatography, anion exchange chromatography, reverse phase chromatography, or It can be determined by column electrophoresis.

[0058] Suitably, the aqueous formulations of the present invention can be stored at 25° C., 30° C., or after storage at 40° C., it contains less than 6% high molecular weight species (by weight of total protein). In embodiments, the amount of high molecular weight species is at least 1, 2, or 3 months at 25° C., 30° C., or After storage at 40° C., the total Fc fusion protein weight is 5% or less, preferably 4% or less, Preferably, the increase is 3% or less, more preferably 2% or less, more preferably 1% or less. Quantification of high molecular weight species is , expressed as weight percent of total Fc-fusion protein in the aqueous formulation.

[0059] Suitably, the aqueous formulations of the present invention can be stored at 25° C., 30° C., or 40° C., there should be an increase in high molecular weight species, but the increase is not observed under the same conditions. and the corresponding identical but otherwise lacking 5-200 mM sulfate ions after storage over time. At least 10% lower, preferably at least 25% lower, more preferably at least 10% lower than the aqueous formulation. At least 50% lower.

[0060] Suitably, the aqueous formulations of the present invention can be stored at 25° C., 30° C., or 40° C., there should be an increase in high molecular weight species, but the increase is not observed under the same conditions. and after storage for 5-200 mM sulfate ion and 1-100 mM polyanion other than sulfate and preferably at least 10% lower than the corresponding aqueous formulation lacking the agonist but otherwise identical. Preferably at least 25% lower, more preferably at least 50% lower.

[0061] In one embodiment, the aqueous formulation of the present invention is administered to a subject in need thereof. Such a formulation is suitable for administering an Fc fusion protein to a subject. The aqueous solution formulation can be used in a method for administering a substance to the body. The formulation is administered by intramuscular injection or by intravenous injection or infusion. More preferably, the formulation is administered by dermally. The drug is administered by subcutaneous injection.

[0062] When the Fc fusion protein is etanercept, the aqueous solution formulation of the present invention is Arthritis such as rheumatoid arthritis, ankylosing spondylitis, granulomatous disease, psoriasis, Crohn's disease, inflammatory bowel disease, atopic dermatitis The compounds are useful in the treatment of autoimmune and inflammatory diseases, including allergic dermatitis, asthma, and COPD.

[0063] In another embodiment, the present invention provides a packaged, The pharmaceutical formulation includes the aqueous solution formulation of the present invention. The pharmaceutical formulation is preferably The solution is packaged in a vial suitable for the introduction of a needle to transfer the solution. The vials are either single dose or The pharmaceutical preparation for multi-dose vials preferably contains a preservative. In an embodiment, the pharmaceutical formulation is packaged in a glass vial with a rubber stopper. The formulation may be provided in a kit, including instructions for use and, optionally, a syringe suitable for intramuscular or subcutaneous administration. Alternatively, the packaged pharmaceutical formulation may be a pre-filled, single-dose formulation suitable for intramuscular or subcutaneous administration. The autoinjector may be provided in the form of a single-use disposable syringe. A further aspect of the present invention is an injectable composition comprising the aqueous solution formulation of the present invention. It is a prefilled, single-use syringe.

[0064] Alternatively, the packaged pharmaceutical formulation may be a pre-filled, multi-dose pen suitable for intramuscular or subcutaneous administration. (e.g., a disposable pen). Thus, a further aspect of the present invention is A pre-filled, multi-dose pen for injection containing a clear aqueous formulation. Pharmaceutical preparations for use in the treatment of chronic conditions preferably contain a preservative.

[0065] The components of the aqueous formulations of the present invention are preferably pharma- ceutically acceptable. The term "pharmaceutical acceptable" means a compound that is free from adverse adverse consequences, such as toxicity, hypersensitivity, and allergies. The intended use and It refers to a component of a pharmaceutical formulation suitable for administration to the human or animal, e.g. mammalian, body.

[0066] A further aspect of the invention is a method for stabilizing an Fc fusion protein in an aqueous formulation. The fusion protein was stabilized by adding sulfate ions to the formulation at concentrations of 5–200 mM. A further aspect of the present invention is a method for the preparation of an Fc fusion protein in an aqueous solution. A method for stabilizing a protein, comprising adding sulfate ions at a concentration of 5 to 200 mM and 1 to 10 mM Stabilizing the fusion protein by adding 0 mM polyanions other than sulfate. The above-mentioned method includes, for example, a method for detecting high molecular weight of an Fc fusion protein during storage. Methods for inhibiting the formation of low molecular weight species, methods for inhibiting the formation of low molecular weight species, or ... The present invention can be a method for inhibiting the formation of protein-related species.

[0067] The present invention also provides a method for stabilizing an Fc fusion protein for storage in an aqueous formulation, comprising: Provide for the use of sulfate ions at concentrations between 5 and 200 mM.

[0068] The present invention also provides a method for stabilizing an Fc fusion protein for storage in an aqueous formulation, comprising: Use of mixtures of sulfate ions at concentrations between 5 and 200 mM and polyvalent anions other than sulfate at concentrations between 1 and 100 mM to provide.

[0069] Further specific aspects of the present invention include the following. (A)(i) Fc fusion proteins, such as etanercept; (i) A concentration of 5 to 200 mM, for example 50 to 100 mM, or 50 to stabilize the fusion protein. ~90 mM sulfate ions; (ii) a polyanion other than sulfate, such as chlorine, at a concentration of 1 to 100 mM, e.g., 10 to 50 or 10 to 60 mM; a polyanion selected from enoate, malate, and phosphate; (iii) optionally one or more preservatives; and (iv) optionally one or more uncharged osmolality modifiers; The aqueous formulation comprises an amino acid selected from the group consisting of arginine, lysine and proline, and salts thereof. The aqueous solution formulation does not contain the selected amino acid and does not contain magnesium ions.

[0070] (B)(i) Fc fusion proteins, such as etanercept; (i) A concentration of 5 to 200 mM, for example 50 to 100 mM, or 50 to stabilize the fusion protein. ~90 mM sulfate ions; (ii) citrate ions at a concentration of 5-60 mM, e.g., 10-60 mM; (iii) phosphate ions at a concentration of 1-10 mM, e.g., 1-5 mM, e.g., 3-5 mM; (iv) optionally one or more preservatives; and (v) optionally one or more uncharged osmolality modifiers; The aqueous formulation comprises an amino acid selected from the group consisting of arginine, lysine and proline, and salts thereof. The aqueous solution formulation does not contain the selected amino acid and does not contain magnesium ions.

[0071] (C)(i) Fc fusion proteins, such as etanercept; (i) A concentration of 5 to 200 mM, for example 50 to 100 mM, or 50 to stabilize the fusion protein. ~90 mM sulfate ions; (ii) maleate ions at a concentration of 5 to 60 mM, e.g., 10 to 60 mM; (iii) phosphate ions at a concentration of 1-10 mM, e.g., 1-5 mM, e.g., 3-5 mM; (iv) optionally one or more preservatives; and (v) optionally one or more uncharged osmolality modifiers; The aqueous formulation comprises an amino acid selected from the group consisting of arginine, lysine and proline, and salts thereof. The aqueous solution formulation does not contain the selected amino acid and does not contain magnesium ions.

[0072] (D)(i) Fc fusion proteins, such as etanercept; (i) A concentration of 5 to 200 mM, for example 50 to 100 mM, or 50 to stabilize the fusion protein. ~90 mM sulfate ions; (ii) maleate ions at a concentration of 5-60 mM, e.g., 10-30, e.g., 10-20 mM; (iii) citrate ions at a concentration of 5-60 mM, e.g., 10-30, e.g., 10-20 mM; (iv) optionally one or more preservatives; and (v) optionally one or more uncharged osmolality modifiers; The aqueous formulation comprises an amino acid selected from the group consisting of arginine, lysine and proline, and salts thereof. The aqueous solution formulation does not contain the selected amino acid and does not contain magnesium ions.

[0073] (E) Essentially (i) Fc fusion proteins, such as etanercept; (i) A concentration of 5 to 200 mM, for example 50 to 100 mM, or 50 to stabilize the fusion protein. ~90 mM sulfate ions; (ii) a polyanion other than sulfate, such as chlorine, at a concentration of 1 to 100 mM, e.g., 10 to 50 or 10 to 60 mM; a polyanion selected from enoate, malate, and phosphate; (iii) optionally one or more preservatives; and (iv) optionally one or more uncharged osmolality modifiers; The aqueous solution formulation comprises arginine, lysine and proline, and salts thereof. The aqueous solution formulation does not contain a selected amino acid and does not contain magnesium ions.

[0074] (F) In essence (i) Fc fusion proteins, such as etanercept; (i) A concentration of 5 to 200 mM, for example 50 to 100 mM, or 50 to stabilize the fusion protein. ~90 mM sulfate ions; (ii) citrate ions at a concentration of 5-60 mM, e.g., 10-60 mM; (iii) phosphate ions at a concentration of 1-10 mM, e.g., 1-5 mM, e.g., 3-5 mM; (iv) optionally one or more preservatives; and (v) optionally one or more uncharged osmotic modifiers; The aqueous solution formulation comprises arginine, lysine and proline, and salts thereof. The aqueous solution formulation does not contain a selected amino acid and does not contain magnesium ions.

[0075] (G) Essentially (i) Fc fusion proteins, such as etanercept; (i) A concentration of 5 to 200 mM, for example 50 to 100 mM, or 50 to stabilize the fusion protein. ~90 mM sulfate ions; (ii) maleate ions at a concentration of 5 to 60 mM, e.g., 10 to 60 mM; (iii) phosphate ions at a concentration of 1-10 mM, e.g., 1-5 mM, e.g., 3-5 mM; (iv) optionally one or more preservatives; and (v) optionally one or more uncharged osmotic modifiers; The aqueous solution formulation comprises arginine, lysine and proline, and salts thereof. The aqueous solution formulation does not contain a selected amino acid and does not contain magnesium ions.

[0076] (H) Essentially (i) Fc fusion proteins, such as etanercept; (i) A concentration of 5 to 200 mM, for example 50 to 100 mM, or 50 to stabilize the fusion protein. ~90 mM sulfate ions; (ii) maleate ions at a concentration of 5-60 mM, e.g., 10-30, e.g., 10-20 mM; (iii) citrate ions at a concentration of 5-60 mM, e.g., 10-30, e.g., 10-20 mM; (iv) optionally one or more preservatives; and (v) optionally one or more uncharged osmotic modifiers; The aqueous solution formulation comprises arginine, lysine and proline, and salts thereof. The aqueous solution formulation does not contain a selected amino acid and does not contain magnesium ions.

[0077] (I) an aqueous solution in which the uncharged osmotic agent is present at a concentration of about 50 to 500 mM, e.g., 50 to 300 mM; Any of agents (A) to (H).

[0078] (J)(i) Fc fusion proteins, such as etanercept; (i) A concentration of 5 to 200 mM, for example 50 to 100 mM, or 50 to stabilize the fusion protein. ~90 mM sulfate ions; (ii) a polyanion other than sulfate, such as chlorine, at a concentration of 1 to 100 mM, e.g., 10 to 50 or 10 to 60 mM; a polyanion selected from enoate, malate, and phosphate; (iii) optionally one or more preservatives; and (iv) optionally one or more charged osmolality modifiers; The aqueous formulation comprises an amino acid selected from the group consisting of arginine, lysine and proline, and salts thereof. The aqueous solution formulation does not contain the selected amino acid and does not contain magnesium ions.

[0079] (K)(i) Fc fusion proteins, such as etanercept; (i) A concentration of 5 to 200 mM, for example 50 to 100 mM, or 50 to stabilize the fusion protein. ~90 mM sulfate ions; (ii) citrate ions at a concentration of 5-60 mM, e.g., 10-60 mM; (iii) phosphate ions at a concentration of 1-10 mM, e.g., 1-5 mM, e.g., 3-5 mM; (iv) optionally one or more preservatives; and (v) optionally one or more charged osmolality modifiers; The aqueous formulation comprises an amino acid selected from the group consisting of arginine, lysine and proline, and salts thereof. The aqueous solution formulation does not contain the selected amino acid and does not contain magnesium ions.

[0080] (L)(i) Fc fusion proteins, such as etanercept; (i) A concentration of 5 to 200 mM, for example 50 to 100 mM, or 50 to stabilize the fusion protein. ~90 mM sulfate ions; (ii) maleate ions at a concentration of 5 to 60 mM, e.g., 10 to 60 mM; (iii) phosphate ions at a concentration of 1-10 mM, e.g., 1-5 mM, e.g., 3-5 mM; (iv) optionally one or more preservatives; and (v) optionally one or more charged osmolality modifiers; The aqueous formulation comprises an amino acid selected from the group consisting of arginine, lysine and proline, and salts thereof. The aqueous solution formulation does not contain the selected amino acid and does not contain magnesium ions.

[0081] (M) Essentially (i) Fc fusion proteins, such as etanercept; (i) A concentration of 5 to 200 mM, for example 50 to 100 mM, or 50 to stabilize the fusion protein. ~90 mM sulfate ions; (ii) a polyanion other than sulfate, such as chlorine, at a concentration of 1 to 100 mM, e.g., 10 to 50 or 10 to 60 mM; a polyanion selected from enoate, malate, and phosphate; (iii) optionally one or more preservatives; and (iv) optionally one or more charged osmolality modifiers; The aqueous solution formulation comprises arginine, lysine and proline, and salts thereof. The aqueous solution formulation does not contain a selected amino acid and does not contain magnesium ions.

[0082] (N) Essentially (i) Fc fusion proteins, such as etanercept; (i) A concentration of 5 to 200 mM, for example 50 to 100 mM, or 50 to stabilize the fusion protein. ~90 mM sulfate ions; (ii) citrate ions at a concentration of 5-60 mM, e.g., 10-60 mM; (iii) phosphate ions at a concentration of 1-10 mM, e.g., 1-5 mM, e.g., 3-5 mM; (iv) optionally one or more preservatives; and (v) optionally one or more charged osmolality modifiers; The aqueous solution formulation comprises arginine, lysine and proline, and salts thereof. The aqueous solution formulation does not contain a selected amino acid and does not contain magnesium ions.

[0083] (O) In essence (i) Fc fusion proteins, such as etanercept; (i) A concentration of 5 to 200 mM, for example 50 to 100 mM, or 50 to stabilize the fusion protein. ~90 mM sulfate ions; (ii) maleate ions at a concentration of 5 to 60 mM, e.g., 10 to 60 mM; (iii) phosphate ions at a concentration of 1-10 mM, e.g., 1-5 mM, e.g., 3-5 mM; (iv) optionally one or more preservatives; and (v) optionally one or more charged osmolality modifiers; The aqueous solution formulation comprises arginine, lysine and proline, and salts thereof. The aqueous solution formulation does not contain a selected amino acid and does not contain magnesium ions.

[0084] (P)(i) Fc fusion proteins, e.g., etanercept; (i) A concentration of 5 to 200 mM, for example 50 to 100 mM, or 50 to stabilize the fusion protein. ~90 mM sulfate ions; (ii) maleate ions at a concentration of 5-60 mM, e.g., 10-30, e.g., 10-20 mM; (iii) citrate ions at a concentration of 5-60 mM, e.g., 10-30, e.g., 10-20 mM; (iv) optionally one or more preservatives; and (v) optionally one or more charged osmolality modifiers; The aqueous formulation comprises an amino acid selected from the group consisting of arginine, lysine and proline, and salts thereof. The aqueous solution formulation does not contain the selected amino acid and does not contain magnesium ions.

[0085] (Q) Essentially (i) Fc fusion proteins, such as etanercept; (i) A concentration of 5 to 200 mM, for example 50 to 100 mM, or 50 to stabilize the fusion protein. ~90 mM sulfate ions; (ii) maleate ions at a concentration of 5-60 mM, e.g., 10-30, e.g., 10-20 mM; (iii) citrate ions at a concentration of 5-60 mM, e.g., 10-30, e.g., 10-20 mM; (iv) optionally one or more preservatives; and (v) optionally one or more charged osmolality modifiers; The aqueous solution formulation comprises arginine, lysine and proline, and salts thereof. The aqueous solution formulation does not contain a selected amino acid and does not contain magnesium ions.

[0086] (R) Aqueous preparation containing a charged osmotic modifier such as sodium chloride at a concentration of approximately 50-250 mM One of the agents (J) to (Q). (S) Any of the aqueous solution formulations (A) to (R) at a pH of about 5.0 to about 6.0, for example, about 5.0 to about 5.7, for example, about 5.3. Either way. (T) Any of the aqueous solution formulations (A) to (R) at a pH of about 6.0 to about 7.0, for example, about 6.0 to about 6.5, for example, about 6.3 Either way.

[0087] (abbreviation) PEG Polyethylene glycol HMWS high molecular weight species SEC Size Exclusion Chromatography CEX Cation Exchange Chromatography EXAMPLES

[0088] (Example) (material) EDTA disodium salt (Mw 372 Da), 1,2-propanediol (Mw 76 Da), glycerol (M 92 Da), mannitol (Mw 182 Da), NaCl (Mw 58 Da), and trehalose (Mw 342 Da) are Si gma was obtained from Aldrich.

[0089] (Methods for assessing the stability of Fc fusion proteins) (a) Visual evaluation Visible particles are preferably measured using 2.9.20. European Pharmacopoeia monograph (Particulate Contamination: Visible Particles) The equipment required consists of a viewing station containing: R: A suitable sized matte black panel held in a vertical position A suitably sized anti-reflective white panel held in a vertical position next to the black panel A suitable shaded white light source and an adjustable lamp holder fitted with a suitable diffuser (each A viewing illuminator containing two 13W fluorescent tubes, 525mm long, is suitable. The lighting intensity at the switching points will be maintained at 2000 lux to 3750 lux.

[0090] Remove any adhesive labels from the container and clean and dry the exterior, making sure there are no air bubbles. While avoiding contact with the container, gently swirl or invert the container and observe it in front of the white panel for approximately 5 seconds. This procedure is repeated in front of the black panel. The presence of any particles is noted. Rank the visual scores as follows: Visual score 1: A clear solution with few particles Visual score of 2: ~5 very small particles Visual score of 3: ~10 to 20 very small particles Visual score 4: 20-50 particles, including large particles Visual score of 5: >50 particles including large particles Particles in samples with visual scores of 4 and 5 were evident upon occasional visual evaluation under normal light. Although detectable in the visual range, samples with visual scores of 1-3 usually yield clear solutions with the same evaluation. Samples with a visual score of 1 to 3 were considered "passing"; Samples that do not meet the criteria are considered to be “failed.”

[0091] (b) Size Exclusion Chromatography (SEC) The amounts of high molecular weight species (HMWS) and low molecular weight species (LMWS) were determined using a 300 × 7.8 mm column with a guard column. The measurement is performed using an S3000 (or equivalent) size exclusion column. The mobile phase is potassium phosphate pH 6.5, flow rate 0.4 ml / min, injection volume 1 μl, detection at 210 and 280 nm. It is expressed as % of the main peak (ie, native protein), % of HMWS, and % of LMWS.

[0092] (c) Cation exchange chromatography (CEX) The amount of related species is measured using a Protein-Pak Hi Res SP column. Mobile phase A contains 20 mM LiCl. Mobile phase B was 20 mM sodium phosphate + 0.5 M NaCl (pH 6.5), and mobile phase B was 20 mM sodium phosphate + 0.5 M NaCl (pH 6 The following gradient elution is used: 0 min - 100% A, 4 min - 80% A, 10 min - 55% A, 12 min - 0% A. Flow rate 1.0 ml / min. Injection volume was 3 μl and UV detection was at 214 nm. Results is the % of the main peak (i.e., native protein), the % of acidic species, and the % of basic species. % of relevant species = % of acidic species + % of basic species.

[0093] Example 1 (Stability of etanercept formulations) The SEC method was used to determine the amount of etanercept (50%) in the formulations shown in the table below (formulations F1-1 to F1-6). The stability of the formulations was tested at 10 mg / mL. The pH of all the formulations tested was adjusted to 6.3. The solutions were prepared to be approximately isotonic. As a result, the concentrations of the key excipients tested were The relative contribution to osmotic pressure was varied. Formulations F1-4 to F1-6 were combinations of excipients. , hypertonic.

[0094] [Table 1]

[0095] The stability of formulations F1-1 to F1-6 is shown in the following table. The table shows the SEC stability after storage at 25° C. and 40° C. The chromatogram shows that the main peak is retained.

[0096] [Table 2]

[0097] The results showed that when sucrose was used as an osmotic adjuster, the The results show that the stability is slightly improved when compared to when the Using sodium sulfate compared to using sodium chloride or sucrose As a result, remarkably excellent stability was obtained.

[0098] Sodium sulfate in combination with sodium chloride or sucrose does not produce the same results as sodium sulfate alone. The stability was not significantly improved when using the German (F1-4 vs. F1-3). However, by combining sodium sulfate with sodium citrate, A significant increase in stability was found (F1-6 vs. F1-3).

[0099] Example 2 (Stability of etanercept formulations) The presence of sodium sulfate in the formulations shown in the table below (formulations F2-1 to F2-9) using the SEC method The effect of polyanions on the stability of etanercept (50 mg / mL) under low temperature conditions was further investigated. The pH of all tested formulations was adjusted to 6.3.

[0100] [Table 3]

[0101] The stability of formulations F2-1 to F2-9 is shown in the following table. The table shows the SEC stability after storage at 25° C. and 40° C. The chromatogram shows that the main peak is retained.

[0102] [Table 4]

[0103] The results showed that a low concentration of sulfate (50 mM) was more effective than a high concentration of sulfate (100 mM). It is also shown that the stabilizing effect is smaller than that of F2-1 (F2-1 vs. F2-2).

[0104] In both cases, the addition of sodium citrate further improved stability (F2-4 vs. F2-2 The effect of sodium citrate was also examined in the presence of 100 mM sodium sulfate. The effect was found to be concentration-dependent (F2-5 vs. F2-4).

[0105] In addition, sodium maleate (50 mM or 10 mM) was added to sodium sulfate (100 mM). Addition of F2-6 and F2-7 to F2-2 further improved stability in a concentration-dependent manner (F2-6 and F2-7 vs. F2-2).

[0106] Similarly, by adding a mixture of sodium citrate and sodium maleate, the concentration Stability was further improved in a gene-dependent manner (F2-8 vs. F2-6 and F2-9 vs. F2-7).

[0107] Throughout this specification and the claims that follow, the context requires otherwise. Except where necessary, the words "comprise" and "comprises" and "comprising" and variations thereof are intended to mean the inclusion of the recited integer, step, group of integers, or group of steps. is not to be construed as implying the exclusion of any other integer, step, group of integers, or group of steps. It will be understood.

[0108] While the present invention has been particularly shown and described with reference to preferred embodiments thereof, the form and Various modifications of detail may be made without departing from the scope of the invention as encompassed by the appended claims. It will be understood by those skilled in the art that the present invention may be practiced without the need for any particular method or device. It should be understood that the embodiments described are not mutually exclusive and that features from various embodiments may be combined in accordance with the present invention. It should also be understood that the above-mentioned methods may be combined in whole or in part.

[0109] Cited publications, patents, patent applications, internet sites, and accession numbers All database sequences (including both polynucleotide and polypeptide sequences) are Notwithstanding any provision of this Agreement, each individual publication, patent, patent application, internet site, or accession number may be modified without notice. It is specifically and specifically stated that the accession number / database sequence is so incorporated by reference. and to the same extent as if set forth herein, and for all purposes are hereby fully incorporated by reference. Be absorbed.

Claims

1. 1. An aqueous formulation comprising: (i) an Fc fusion protein; and (ii) sulfate ions at a concentration of 5-200 mM to stabilize the Fc fusion protein; Including, does not contain any amino acid selected from arginine, lysine and proline, and salts thereof; The aqueous solution preparation is free of magnesium ions.

2. The Fc fusion protein comprises a heterologous polypeptide that is ligand-binding, and an immunoglobulin.

2. The aqueous solution formulation of claim 1, wherein the antibody is a fusion protein comprising the Fc portion of an IgG1 antibody, in particular an IgG1 antibody.

3. The Fc fusion protein is selected from etanercept, abatacept, or belatacept. The aqueous solution formulation according to claim 1,

4. The aqueous solution formulation of claim 3, wherein the Fc fusion protein is etanercept.

5. The sulfate ions are formulated at a concentration of 25 to 150 mM, for example 25 to 100 mM, more preferably 50 to 100 mM. The aqueous solution formulation according to any one of claims 1 to 4, wherein the aqueous solution formulation is present in

6. The source of sulfate ions may be, for example, sodium sulfate, potassium sulfate, and ammonium sulfate. The aqueous solution formulation according to any one of claims 1 to 5, wherein the sulfate is selected from the group consisting of sulfates.

7. The fusion protein is at a concentration of 1-400 mg / ml, preferably 20-100 mg / ml, more preferably about 5 7. The aqueous formulation of claim 1, wherein the aqueous formulation is present in the formulation at 0 mg / ml.

8. Any one of claims 1 to 7, further comprising polyvalent anions other than sulfate as further stabilizing components. The aqueous solution formulation described above.

9. 9. The aqueous solution formulation of claim 8, wherein the polyanion is an organic polyanion.

10. 10. The aqueous solution formulation of claim 9, wherein the organic polyanion is citrate.

11. The organic polyanion is selected from succinic acid, malic acid, and maleic acid, particularly maleic acid. The aqueous solution formulation according to claim 9, wherein

12. 10. The aqueous solution formulation according to claim 9, comprising citric acid and maleic acid as organic polyanions.

13. 9. The aqueous solution of claim 8, wherein the polyanion is an inorganic polyanion, such as phosphate. Agent.

14. The concentration of the polyanion other than sulfate is 1 to 100 mM, preferably 1 to 60 mM or 1 to 50 mM, more preferably 1 to 100 mM. More preferably, it is 5 to 60 or 5 to 50 mM, more preferably 10 to 60 or 10 to 50 mM, for example 30 to 50 mM. The aqueous solution formulation according to any one of claims 8 to 13.

15. The method according to any one of claims 1 to 14, wherein the formulation has an osmolarity of 200 to 500 mOsm / l, for example about 300 mOsm / l. The aqueous solution formulation according to any one of claims 1 to 10.

16. The aqueous solution formulation of any one of claims 1 to 15, wherein the formulation is isotonic.

17. 17. The aqueous solution formulation of any one of claims 1 to 16, comprising an uncharged osmotic agent.

18. The uncharged osmotic modifier is sucrose, trehalose, mannitol, raffinose, Lactose, dextrose, sorbitol, lactitol, glycerol, propylene 18. The aqueous solution formulation of claim 17, wherein the surfactant is selected from the group consisting of glycol and polyethylene glycol.

19. The uncharged osmotic agent is present in the formulation at a concentration of 10 to 1000 mM, preferably 50 to 300 mM.

19. The aqueous solution formulation according to claim 17 or 18.

20. 20. The aqueous solution formulation of any one of claims 1 to 19, comprising a charged osmotic modifier.

21. 21. The aqueous solution formulation of claim 20, wherein the charged osmotic modifier is sodium chloride.

22. The charged osmotic modifier is present in the formulation at a concentration of 25 to 500 mM, preferably 50 to 250 mM.

22. The aqueous solution formulation according to claim 20 or 21.

23. The aqueous solution preparation according to any one of claims 1 to 22, wherein the pH of the preparation is from about pH 4.0 to about pH 7.

0. Agent.

24. The pH of the formulation is from about pH 6.0 to about pH 7.0, such as from about pH 6.0 to pH 6.5, such as about pH 6.

3.

24. The aqueous solution formulation according to claim 23.

25. The aqueous solution formulation of any one of claims 1 to 24, further comprising a buffering agent.

26. The buffer has a concentration of about 0.5 mM to about 50 mM, for example about 1 mM to about 20 mM, for example about 1 mM to about 10 mM 26. The aqueous formulation of claim 25, wherein M is present at, for example, 1 mM to about 5 mM, for example, about 2 mM to about 5 mM. 。

27. The aqueous solution formulation according to any one of claims 1 to 26, further comprising a non-ionic surfactant.

28. The non-ionic surfactant has a concentration of about 10 μg / mL to about 2000 μg / mL, for example about 50 μg / mL 28. The aqueous solution of claim 27, present at about 1000 μg / mL, for example about 100 μg / mL to about 500 μg / mL. Liquid preparations.

29. 29. The aqueous solution formulation of any one of claims 1 to 28, further comprising a preservative.

30. The preservative is phenol, m-cresol, chlorocresol, benzyl alcohol, From propylparaben, methylparaben, benzalkonium chloride, and benzethonium chloride 30. The aqueous solution formulation of claim 29, selected from the group consisting of:

31. 31. The aqueous formulation of claim 29 or 30, wherein the preservative is present at a concentration of about 0.01 to about 100 mM.

32. Does not include all amino acids, particularly both the L and D isomeric forms of the 20 naturally occurring amino acids The aqueous solution formulation according to any one of claims 1 to 31, which does not contain

33. (i) Fc fusion proteins, such as etanercept; (i) a concentration of 5 to 200 mM, for example 50 to 100 mM, to stabilize the fusion protein; or 50-90 mM sulfate ion; (ii) maleate ions at a concentration of 5 to 60 mM, e.g., 10 to 30, e.g., 10 to 20 mM; (iii) citrate ions at a concentration of 5 to 60 mM, e.g., 10 to 30, e.g., 10 to 20 mM; (iv) optionally one or more preservatives; and (v) optionally one or more tonicity adjusting agents; and an amino acid selected from arginine, lysine and proline, and salts thereof. The aqueous solution formulation does not contain any niacin and does not contain magnesium ions.

34. The pH of the formulation is from about pH 6.0 to about pH 7.0, such as from about pH 6.0 to pH 6.5, such as about pH 6.

3.

34. The aqueous solution formulation of claim 33.

35. The aqueous solution formulation according to any one of claims 1 to 34, which is a pharmaceutical composition.

36. 36. The aqueous formulation of any one of claims 1 to 35 for use in therapy.

37. Administering the formulation by subcutaneous or intramuscular injection, or by intravenous injection or infusion. The aqueous solution formulation according to any one of claims 1 to 36, for use in an

38. A pre-filled syringe for injection comprising the aqueous solution formulation of any one of claims 1 to 37.

39. A pre-filled, multi-dose pen for injection comprising the aqueous formulation according to any one of claims 1 to 37. 。

40. A method for stabilizing an Fc fusion protein, comprising adding sulfate ions to a formulation at a concentration of 5 to 200 mM stabilizing the fusion protein by adding

41. A method for inhibiting the formation of high molecular weight species of said Fc fusion protein during storage. Item 41. The method according to item 40.

42. A method for inhibiting the formation of low molecular weight species of said Fc fusion protein during storage. Item 41. The method according to item 40.

43. 40. A method for inhibiting the formation of related species of said Fc-fusion protein during storage. The method described.

44. Sulfuric acid at concentrations between 5 and 200 mM to stabilize Fc fusion proteins for storage in aqueous formulations Use of ions.

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