HK40057504A - Arginine-free tnfr:fc-fusion polypeptide compositions and methods of use - Google Patents

Description

Arginine-free TNFR-FC-fusion polypeptide compositions and methods of use

The application is a divisional application of Chinese patent application with application date of 2012/29/6, application number of 201280032584.X and invention name of 'TNFR without arginine: FC-fusion polypeptide composition and use method', and the original application is a Chinese national phase application of International application PCT/US 2012/044988.

RELATED APPLICATIONS

This application claims the benefit of U.S. provisional patent application serial No. 61/504,110, filed 2011, 7/1, § 119(e), the contents of which are hereby incorporated by reference in their entirety.

Technical Field

Certain aspects of the invention relate to therapeutic polypeptide-based compositions.

Background

Therapeutic polypeptide formulations are typically stored prior to use. However, if stored in aqueous form for a prolonged period of time, especially in the absence of a stabilizing agent such as arginine, the polypeptide will be unstable. An alternative approach that relies on aqueous storage is to prepare a lyophilized form of the polypeptide, however, reconstitution of the dried polypeptide typically results in aggregation or denaturation.

Disclosure of Invention

In some embodiments, arginine-free polypeptide compositions are provided. Formulations for the preparation of arginine-free polypeptide solutions that are stable over long periods of time have been identified. These formulations have several advantages over arginine-stable solutions, including reduced cost and reduced incidence of side effects associated with the presence of arginine. Surprisingly, aqueous polypeptide formulations can be stabilized in the absence of arginine or other stabilizing amino acids (e.g., lysine or glycine or other stabilizing amino acids, such as amino acids having a positive charge) by using relatively high salt concentrations.

In some embodiments, provided herein are compositions comprising (or consisting of or consisting essentially of): an isolated polypeptide (e.g., a therapeutic polypeptide, such as a therapeutic polypeptide comprising an immunoglobulin domain); and a salt in an amount sufficient to prevent aggregation of the polypeptide, thereby stabilizing the composition (e.g., in the absence of arginine or other added amino acids). In some embodiments, the compositions provided herein are aqueous compositions (e.g., aqueous solutions). In some embodiments, the polypeptide and salt are provided in water without a buffer. In some embodiments, the composition comprises an aqueous buffer or other solvent (e.g., organic solvent). In some embodiments, one or more excipients are included.

In some embodiments, aspects of the invention relate to arginine-free polypeptide compositions comprising an isolated polypeptide comprising an Fc region of a human immunoglobulin (e.g., IgG 1). In some embodiments, aspects of the invention relate to arginine-free polypeptide compositions comprising an isolated polypeptide comprising an extracellular ligand-binding portion of human p75 Tumor Necrosis Factor (TNF). In some embodiments, aspects of the invention relate to arginine-free polypeptide compositions comprising an isolated polypeptide comprising an extracellular ligand-binding portion of human p75 Tumor Necrosis Factor (TNF) fused to the Fc region of human IgG 1.

In some aspects, provided herein are compositions comprising (or consisting of or consisting essentially of): an isolated polypeptide which is an extracellular ligand-binding portion of human p75 tumor necrosis factor receptor fused to the Fc region of human IgG 1; and a salt in an amount sufficient to prevent aggregation of the polypeptide, thereby stabilizing the composition (e.g., in the absence of arginine or other added amino acids). In some embodiments, the compositions provided herein are aqueous compositions (e.g., aqueous solutions). In some embodiments, the protein and salt are provided in water without a buffer. In some embodiments, the composition comprises an aqueous buffer or other solvent (e.g., organic solvent). In some embodiments, one or more excipients are included.

In other aspects, provided herein are methods comprising combining: an isolated polypeptide which is an extracellular ligand-binding portion of human p75 tumor necrosis factor receptor fused to the Fc region of human IgG 1; an aqueous buffer; and a salt in an amount sufficient to prevent aggregation of the polypeptide, thereby formulating a stable composition.

In still other aspects, provided herein are methods comprising administering to an individual a composition comprising: an isolated polypeptide which is an extracellular ligand-binding portion of human p75 tumor necrosis factor receptor fused to the Fc region of human IgG 1; an aqueous buffer; and a salt in an amount sufficient to prevent aggregation of the isolated polypeptide, thereby stabilizing the composition.

In some embodiments, the composition comprises less than 10mM free amino acids (e.g., arginine, lysine, and/or glycine). In some embodiments, the composition comprises less than 1mM free amino acids. In some embodiments, the composition comprises less than 1mM arginine. In some embodiments, the composition comprises less than 0.5mM arginine. In some embodiments, the composition comprises less than 0.1mM, less than 0.05mM, less than 0.01mM, less than 0.005mM, or less than 0.001mM arginine. In some embodiments, the composition is free of free amino acids. In some embodiments, the composition is substantially free of arginine. The isolated polypeptide of any of the compositions described herein can comprise an arginine amino acid residue as part of its amino acid sequence. Arginine residues, which together with other amino acid residues form the amino acid sequence of a protein, are not considered "free" amino acids. Thus, a composition that is "free of amino acids" refers to a composition that does not contain free amino acids but can contain an isolated polypeptide having an arginine amino acid residue as part of its amino acid sequence.

In certain embodiments, the compositions described herein comprise from about 10mg/ml to about 100mg/ml of the isolated polypeptide. In some embodiments, the isolated polypeptide is etanercept.

In some embodiments, the concentration of the aqueous buffer is less than 100mM, less than 50mM, or less than 25 mM. In certain embodiments, the concentration of the aqueous buffer is from about 1mM to about 15 mM. In some embodiments, the concentration of the aqueous buffer is about 1 mM. In some embodiments, the concentration of the aqueous buffer is less than 1mM, less than 0.5mM, less than 0.25mM, less than 0.1mM, less than 0.05mM, or less than 0.01 mM. In some embodiments, the aqueous buffer is sodium, histidine, potassium phosphate, sodium or potassium citrate, maleic acid, ammonium acetate, tris (hydroxymethyl) aminomethane (tris), acetate, diethanolamine, or a combination thereof. However, other buffers may also be used (e.g., in lower amounts), as the aspects of the invention are not limited in this regard. In some embodiments, the compositions described herein do not contain an aqueous buffer. In such embodiments, the proteins in the composition are self-buffering, e.g., a moderately concentrated protein may be self-buffering (e.g., in an aqueous solution without an added buffer).

In some embodiments, the salt is present at a concentration of greater than 50mM or greater than 100 mM. In some embodiments, the salt is present at a concentration of 120mM to about 150 mM. In some embodiments, the salt is present at a concentration greater than 150mM, depending on the amount of aqueous buffer present in the solution. Generally, if the amount of aqueous buffer is reduced in the composition, the amount of salt (e.g., NaCl) is increased in order to maintain the osmotic pressure and thermal stability of the composition. For example, if the aqueous buffer is present in the composition at a concentration of less than 15mM, the salt may be present in the composition at a concentration of greater than 150 mM. In some embodiments, the salt is sodium chloride. The salt component of the composition refers to salts other than those present in the aqueous buffer.

In other embodiments, any of the compositions described herein may comprise an excipient. The excipient may be sucrose, lactose, glycerol, xylitol, sorbitol, mannitol, maltose, inositol, trehalose, glucose, Bovine Serum Albumin (BSA), human SA or recombinant HA, dextran, PVA, hydroxypropyl methylcellulose (HPMC), polyethyleneimine, gelatin, polyvinylpyrrolidone (PVP), Hydroxyethylcellulose (HEC), polyethylene glycol, ethylene glycol, glycerol, dimethyl sulfoxide (DMSO), Dimethylformamide (DMF), proline, L-serine, sodium glutamate, alanine, glycine, lysine hydrochloride, sarcosine, gamma-aminobutyric acid, sodium glutamate, glycine, lysine hydrochloride, and mixtures thereof,SDS, polysorbate, polyoxyethylene copolymer, potassium phosphate, sodium acetate, ammonium sulfate, magnesium sulfate, sulfurSodium, trimethylamine N-oxide, betaine, zinc ion, copper ion, calcium ion, manganese ion, magnesium ion, CHAPS, sucrose monolaurate, 2-O-beta-mannoglycerate, or a combination thereof. Other excipients may be used, as the aspects of the invention are not limited in this respect. In a particular embodiment, the excipient is sucrose. In such embodiments, the concentration of sucrose may be from about 0.5% to about 1.5%. In certain embodiments, any of the compositions described herein can have a concentration of sucrose of about 1% by weight.

In some embodiments, any of the compositions described herein can have a pH of about 5.5 to about 7.8. In some embodiments, any of the compositions described herein can have a pH of about 5.8 to about 6.5. In some embodiments, the compositions described herein may have a pH of 5.8 to 6.5. In some embodiments, the composition may have a pH of 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5.

In one embodiment, the composition comprises (consists of or consists essentially of): 50mg/ml of etanercept, about 10mM sodium phosphate, about 140mM sodium chloride, and about 1% sucrose, wherein the pH of the composition is from about pH 6.0 to about pH 7.0.

In each of the embodiments described herein, the composition is free of additional L-arginine (arginine-free). That is, in any of the compositions described herein, no L-arginine is added or combined with a polypeptide. However, the polypeptide itself may contain arginine amino acid residues, as described elsewhere herein.

Any of the compositions described herein can have a commercially viable shelf life of at least 24 months.

Any of the compositions described herein may also be suitable for subcutaneous administration (e.g., non-toxic, purified, sterile, and/or suitably isotonic).

Further, in any of the compositions described herein, the isolated polypeptide can be purified.

In certain embodiments, the compositions described herein can be sterilized.

Any of the compositions described herein can be used to treat rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, wegener's disease (granuloma), crohn's disease (or inflammatory bowel disease), Chronic Obstructive Pulmonary Disease (COPD), hepatitis c, endometriosis, asthma, cachexia, psoriasis or atopic dermatitis, or other inflammatory or autoimmune related diseases, disorders, or conditions. The composition can be administered in an amount (e.g., a therapeutically effective amount) sufficient to treat (alleviate symptoms, stop or slow progression of) the condition.

Detailed Description

A commercially available soluble form of TNF receptor (TNFR: Fc) fused to an Fc domain is known as etanercept. Etanercept (trade name)) By interfering with Tumor Necrosis Factor (TNF) as a TNF inhibitor. This dimeric fusion polypeptide, consisting of the extracellular ligand-binding portion of human 75 kilodalton (p75) Tumor Necrosis Factor Receptor (TNFR) linked to the Fc portion of human IgG1, is currently formulated with L-arginine to prevent polypeptide aggregation (see U.S. patent nos. 5,447,851 and 7,648,702, incorporated by reference herein).

Arginine, while tolerated by most people, can cause serious side effects in some people. Severe allergic reactions, known as hypersensitivity, can occur following arginine injection, as well as gastric upset, including nausea, stomach cramps or increased stool frequency. Other potential side effects include low blood pressure and changes in various chemicals and electrolytes in the blood, such as high potassium, high chloride, low sodium, low phosphate, high blood urea nitrogen and high creatinine levels. In theory, arginine may increase the risk of bleeding, raise blood glucose levels, raise potassium levels, and may exacerbate the symptoms of sickle cell disease. Therefore, individuals with liver or kidney disease or those using coagulants should be cautious with arginine.

As discussed in U.S. patent No. 6,748,702, aqueous compositions comprising Fc domain containing polypeptides are believed to require a concentration of about 1mM to about 100mM of L-arginine to prevent polypeptide aggregation. It is also believed that it is necessary to store such aqueous compositions for extended periods of time (e.g., two years or more).

Surprisingly, stable aqueous compositions (e.g., pharmaceutical compositions) that are substantially free of L-arginine (e.g., free of substantial amounts of L-arginine) can be prepared such that they are stable for periods of two years or more. Applicants have found that by increasing the concentration of salt and by decreasing the buffering capacity of the composition, it is still possible to provide a stable polypeptide composition that can be administered subcutaneously to an individual. The term "stable" with respect to long term storage is understood to mean that the active polypeptide of the pharmaceutical composition does not lose more than 20%, more than 15%, more than 10% or more than 5% of its activity with respect to the activity of the composition at the start of storage.

In some embodiments, provided herein are compositions comprising: an isolated polypeptide which is an extracellular ligand-binding portion of human p75 tumor necrosis factor receptor fused to the Fc region of human IgG 1; an aqueous buffer; and a salt in an amount sufficient to prevent aggregation of the polypeptide, thereby stabilizing the composition, wherein the composition comprises L-arginine at a concentration of less than 1 mM. In certain other embodiments, the Fc-containing polypeptide composition is free or substantially free of L-arginine. As used herein, "substantially free" refers to a composition that is free of additional free amino acids such as arginine. It will be appreciated that the polypeptide itself may contain the amino acid arginine in its structure. In some embodiments, the composition does not contain a free arginine amino acid.

As used herein, the phrase "composition" may refer to a formulation comprising a polypeptide prepared in a manner such that it is suitable for injection and/or administration to an individual in need thereof. A "composition" may also be referred to as a "pharmaceutical composition". In certain embodiments, the compositions provided herein are substantially sterile and free of any agent that is unduly toxic or infectious to a recipient. Additionally, as used herein, a solution or aqueous composition may mean a fluid (liquid) formulation comprising one or more chemicals dissolved in a suitable solvent (e.g., water and/or other solvents, such as organic solvents) or a mixture of naturally miscible solvents.

Further, as used herein, the term "about" can mean that there can be 5%, 10%, 15%, or up to and including 20% variation in the concentration of a component of the composition given value. For example, if a composition has about 10mg/ml of an Fc domain-containing polypeptide, the composition can have 8 to 12mg/ml of the polypeptide. In certain embodiments, the composition comprises from about 10mg/ml to about 100mg/ml of the polypeptide. In related embodiments, the composition comprises 50mg/ml or about 50mg/ml of the polypeptide. The composition may comprise more or less polypeptides, as the aspects of the invention are not limited in this respect.

In particular embodiments, the Fc domain-containing polypeptide is a soluble form of a TNF receptor (TNFR: Fc) fused to an Fc domain. Commercially available TNFR Fc is referred to as etanercept (R) ((R))Immunex Corporation), which is a dimeric fusion polypeptide consisting of the extracellular ligand-binding portion of human 75 kilodaltons (p75) Tumor Necrosis Factor Receptor (TNFR) linked to the Fc portion of human IgG 1. The Fc component of etanercept comprises a constant heavy chain 2(CH2) domain, a constant heavy chain 3(CH3) domain, and a hinge region, but does not contain the constant heavy chain 1(CH1) domain of human IgG 1. In some embodiments, the Fc domain may comprise one of the domains described above, while in other embodiments, the Fc domain may comprise all of the domains described above. Etanercept is produced by recombinant DNA technology in a Chinese Hamster Ovary (CHO) mammalian cell expression system. It consists of 934 amino acids and has an apparent molecular weight of about 150 kilodaltons (Physicians' Desk Reference, 2002, Medical Economics Company Inc.).

Other polypeptides contemplated for use in the particular compositions and methods described herein include, but are not limited to, fusion polypeptides comprising at least a portion of an antibody Fc domain. Polypeptides fused to an Fc domain and identical or substantially similar to one of the following polypeptides are suitable for use in the compositions of the invention: flt3 ligand, CD40 ligand, erythropoietin, thrombopoietin, calcitonin, Fas ligand, ligand for NF-. kappa.B Receptor Activator (RANKL), Tumor Necrosis Factor (TNF) -related apoptosis inducing ligand (TRAIL), thymic stromal derived lymphopoietin, granulocyte colony stimulating factor, granulocyte-macrophage colony stimulating factor, mast cell growth factor, stem cell growth factor, epidermal growth factor, RANTES, growth hormone, insulin, insulinotropic hormone, insulin-like growth factor, parathyroid hormone, interferon, nerve growth factor, glucagon, interleukins 1 to 18, colony stimulating factor, lymphotoxin-beta, Tumor Necrosis Factor (TNF), leukemia inhibitory factor, tumor suppressor-M, and various ligands for cell surface molecules ELK and Hek (such as ligands for eph-related kinase or LERKS).

In certain embodiments, the polypeptides include, but are not limited to, recombinant fusion polypeptides comprising the Fc domain of an antibody plus a receptor for any of the above polypeptides or a polypeptide substantially similar to such a receptor. These receptors include, but are not limited to: two forms of TNFR (designated p55 and p75), interleukin-1 receptor (type 1 and type 2), interleukin-4 receptor, interleukin-15 receptor, interleukin-17 receptor, interleukin-18 receptor, granulocyte-macrophage colony stimulating factor receptor, granulocyte colony stimulating factor receptor, receptor for oncostatin-M and leukemia inhibitory factor, NF-. kappa.B Receptor Activator (RANK), receptor for TRAIL (TRAIL receptors 1, 2, 3 and 4) and receptor containing death domains such as Fas or Apoptosis Inducing Receptor (AIR).

In other embodiments, the polypeptide includes, but is not limited to, a differentiation antigen (referred to as a CD polypeptide) or a ligand thereof or a polypeptide substantially similar to either of them fused to the Fc domain of an antibody. Such antigens are described in Leukocyte Typing VI (Proceedings of the VI)^thInternational works and Conference, Kishimoto, Kikutani et al, eds., Kobe, Japan, 1996). Similar CD polypeptides are disclosed in subsequent seminars. Examples of such antigens include CD27, CD30, CD39, CD40, and ligands thereof (CD27 ligand, CD30 ligand, etc.). Many of the CD antigens are members of the TNF receptor family, which also includes 41BB ligand and OX 40. The ligand is typically a member of the TNF family, as are 41BB ligand and OX40 ligand. Thus, members of the TNF and TNFR families can be formulated as described herein.

In certain embodiments, enzymatically active polypeptides or ligands thereof can be used in the compositions and methods described herein. Examples include, but are not limited to, recombinant fusion polypeptides comprising an antibody Fc domain fused to all or a portion of one of the following polypeptides or a ligand thereof or a polypeptide substantially similar to one of these polypeptides: metalloprotease-disintegrin family members, various kinases, glucocerebrosidase, superoxide dismutase, tissue plasminogen activator, factor VIII, factor IX, apolipoprotein E, apolipoprotein A-I, globulin, IL-2 antagonists, alpha-1 antitrypsin, TNF-alpha converting enzyme, ligands for any of the above enzymes, and a variety of other enzymes and ligands therefor.

In some embodiments, the compositions and methods described herein are used to prepare compositions comprising antibodies, human antibodies, humanized antibodies, chimeric antibodies (e.g., antibodies having a human constant antibody immunoglobulin domain linked to one or more murine variable antibody immunoglobulin domains) and/or non-human antibodies or fragments thereof. Specific examples of antibodies suitable for use in the compositions of the invention include, but are not limited to, commercially available antibodies, such as Moluomab-CD 3 (Orthoclone)Ortho Biotech), abciximab (a)Lilly), rituximab (IDEC), Daximab (Roche Laboratories), basiliximab (Novartis), infliximab (b)Centocor), palivizumab (Medmimumone), trastuzumab (Genentech), gemtuzumab ozolomicin (MYLOTARG)^TMWyeth-Ayerst) and alemtuzumab (A)Berlex). Currently, each of the foregoing is available as a lyophilized powder that requires reconstitution prior to administration or as a concentrate that requires dilution. The compositions of the invention no longer require any manipulation, such as rehydration or dilution, prior to administration, but maintain the stability of the active ingredient during long term storage.

In certain embodiments, the compositions described herein are used to store a polypeptide comprising an antibody conjugated to a cytotoxic or luminescent substance. Such materials include, but are not limited to: maytansine derivatives (such as DM1), enterotoxins (such as staphylococcal enterotoxin), iodine isotopes (such as iodine-125), technetium isotopes (such as Tc-99m), cyanine fluorescent dyes (such as Cy5.5.18) and ribosome inactivating polypeptides (such as bouganin, gelonin or saporin-S6).

Examples of antibodies or antibody/cytotoxins or antibody/luminophore conjugates contemplated for use herein include, but are not limited to, those that recognize one or more of the following antigens: CD2, CD3, CD4, CD8, CD11a, CD14, CD18, CD20, CD22, CD23, CD25, CD33, CD40, CD44, CD52, CD80(B7.1), CD86(B7.2), CD147, IL-4, IL-5, IL-8, IL-10, IL-2 receptor, IL-4 receptor, IL-6 receptor, IL-13 receptor, PDGF-beta, VEGF, TGF-beta 2, TGF-beta 1, EGF receptor, VEGF receptor, C5 complement, IgE, tumor antigen CA125, tumor antigen MUC1, PEM antigen, LCG (which is a gene product expressed in association with lung cancer), HER TAG-2, tumor-associated glycoprotein TAG-72, SK-1 antigen, tumor-associated epitopes present at high levels in the serum of individuals with breast and/or pancreatic cancer, tumor-associated with tumor-related epitopes of the colon, pancreatic cancer, pancreatic cells, squamous cells, prostate and/or lung cancer, Cancer-associated epitopes or polypeptides expressed on glioma or neuroblastoma cells, TRAIL receptor 1, 2, 3 and 4, tumor necrosis core, integrin alpha 4 beta 7, integrin VLA-4, B2 integrin, TNF-alpha, adhesion molecule VAP-1, epithelial cell adhesion molecule (EpCAM), intercellular adhesion molecule-3 (ICAM-3), leukocyte integrin adhesion molecule, platelet glycoprotein gp IIb/IIIa, cardiac myosin heavy chain, parathyroid hormone, rNAPC2 (which is an inhibitor of factor VIIa-tissue factor), MHC I, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), Tumor Necrosis Factor (TNF), CTLA-4 (which is a cytotoxic T lymphocyte-associated antigen), Fc-gamma-1 receptor, HLA-DR10 beta, HLA-DR antigen, L-selectin, TNF-gamma-1, TNF-gamma-binding protein, and methods of making such polypeptides, IFN-gamma, respiratory syncytial virus, Human Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV), Streptococcus mutans, and Staphylococcus aureus.

In some embodiments, the compositions described herein are used for anti-idiotypic antibodies or substantially similar polypeptides, including but not limited to anti-idiotypic antibodies directed against: antibodies targeting the tumor antigen gp 72; antibodies against ganglioside GD 3; or an antibody directed against ganglioside GD 2.

In other embodiments, the Fc domain-containing polypeptides for use in the compositions described herein are produced by a living host cell expressing the polypeptide (such as a hybridoma for an antibody) or a host cell genetically engineered to produce the polypeptide for a fusion polypeptide or antibody. Methods for genetically engineering cells to produce polypeptides are well known in the art. See, e.g., Ausubel et al, eds (1990), Current Protocols in Molecular Biology (Wiley, New York). Such methods include introducing into a living host cell a nucleic acid encoding and allowing expression of the polypeptide. These host cells may be, but are not limited to, bacterial cells, fungal cells or animal cells grown in culture. Bacterial host cells include, but are not limited to, E.coli cells. Examples of suitable E.coli strains include, but are not limited to, HB101, DH5 α, GM2929, JM109, KW251, NM538, NM539 and any E.coli strain that is not capable of cleaving foreign DNA. Fungal host cells that may be used include, but are not limited to, Saccharomyces cerevisiae, Pichia pastoris, Aspergillus cells. Several examples of animal cell lines that can be used are CHO, VERO, BHK, HeLa, Cos, MDCK, 293, 3T3 and W138. Novel animal cell lines can be established using methods well known to those skilled in the art (e.g., by transformation, viral infection, and/or screening). Optionally, the polypeptide may be secreted into the culture medium by the host cell.

In certain embodiments, the expressed Fc domain-containing polypeptide is purified by standard methods. When the Fc domain containing polypeptide is produced intracellularly, particulate debris is removed, for example, by centrifugation or ultrafiltration. When the polypeptide is secreted into the culture medium, the supernatant from such an expression system may first be concentrated using a standard polypeptide concentration filter. Protease inhibitors may also be added to inhibit proteolysis, and antibiotics may be included to prevent microbial growth.

In some embodiments, the Fc domain-containing polypeptide is purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, and/or any combination of known or yet to be discovered purification techniques. For example, protein A can be used to purify Fc domain-containing polypeptides based on human gamma 1, gamma 2 or gamma 4 heavy chains (Lindmark et al, 1983, J.Immunol. meth.62: 1-13). Protein G is suggested for all mouse isoforms as well as for human gamma 3(Guss et al, 1986, EMBO J.5: 1567-1575).

Other techniques for polypeptide purification such as fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica, heparin Sepharose^TMChromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation may also be used as desired. Other polypeptide purification techniques/methods may be used.

In certain embodiments, the compositions described herein are prepared by combining a buffer, a salt (e.g., NaCl), and an additional excipient (e.g., sucrose) in addition to the purified polypeptide described above. In some embodiments, the compositions of the invention comprise less than 1mM L-arginine, while in other embodiments, the compositions described herein are free or substantially free of arginine (e.g., L-arginine). It will be understood by those of ordinary skill in the art that the combination of the various components to be included in the composition can be performed in any suitable order, i.e., the buffer can be added first, intermediate, or last, and the tonicity modifier can also be added first, intermediate, or last. One of ordinary skill in the art will also appreciate that some of these chemicals may be incompatible in certain combinations and, therefore, are easily replaced by different chemicals that have similar properties but are compatible in the relevant mixtures.

Aggregation inhibitors reduce the tendency of polypeptides to associate in inappropriate or unwanted ternary or quaternary complexes. Surprisingly, the inventors have found that by increasing the salt concentration and by decreasing the buffering capacity, no further addition of free amino acids (e.g. arginine, lysine, glycine) is required in the composition comprising the Fc-containing polypeptide. The polypeptide in the arginine-free composition remains active (effective) and can be stored for at least 24 months. In certain embodiments, the salt concentration is greater than 100mM, while in other embodiments, the salt concentration is about 140mM or greater. Salts as used herein may include, but are not limited to, sodium chloride (NaCl), potassium chloride (KCl), sodium citrate (Na)₃C₆H_sO₇·2H₂O), magnesium sulfate (MGSO)₄) Calcium chloride (CaCl), sodium hypochlorite (NaClO), sodium nitrate (NaNO)₃) Mercury sulfide (HgS), sodium chromate (Na)₂CrO₄) And magnesium dioxide (MgO)₂). The salts maintain both isotonicity and thermal stability of the composition, e.g., in the absence of arginine (e.g., L-arginine).

Buffering agents maintain the pH within the desired range, and a variety of buffering agents suitable for use in the compositions described herein include, but are not limited to, histidine, potassium phosphate, sodium or potassium citrate, maleic acid, ammonium acetate, tris (hydroxymethyl) aminomethane (tris), various forms of acetate, and diethanolamine. In certain embodiments, the buffering agent is sodium phosphate because of its buffering capacity at or near pH 6.2. In some embodiments, the concentration of the buffer in the composition is about 25mM or less. In some embodiments, the concentration of the buffer is 25 mM. In particular embodiments, the low concentration of buffer is about 10mM or less. In some embodiments, the buffer is at a concentration of 10 mM. Buffers are well known in the art and are manufactured by known methods and are available from commercial suppliers.

When the pH of the composition is set at or near physiological levels, the comfort of the individual is maximized upon administration. In certain embodiments, the pH is about 5.8 to 8.4. In other embodiments, the pH is about 6.2 to 7.4. It is understood that pH can be adjusted as necessary to maximize stability and solubility of the polypeptide in a particular composition, and thus, pH outside of the physiological range, but still tolerable to an individual, is also within the scope of the invention.

In certain embodiments, excipients (also known as chemical additives, co-solutes, or co-solvents) that stabilize the polypeptide in solution (also in dried or frozen form) are added to the composition. Examples include, but are not limited to, sugars/polyols such as sucrose, lactose, glycerol, xylitol, sorbitol, mannitol, maltose, inositol, trehalose, glucose; polymers such as: serum albumin (bovine serum albumin (BSA), human SA or recombinant HA), dextran, PVA, Hydroxypropylmethylcellulose (HPMC), polyethyleneimine, gelatin, polyvinylpyrrolidone (PVP), Hydroxyethylcellulose (HEC); non-aqueous solvents, such as: polyols (e.g., PEG, ethylene glycol, and glycerol), dimethyl sulfoxide (DMSO), and Dimethylformamide (DMF); amino acids such as proline, L-serine, sodium glutamate, alanine, glycine, lysine hydrochloride, sarcosine and gamma-aminobutyric acid; surfactants, such as TWEEN-80^TM(Polysorbate 80), TWEEN-20^TM(polysorbate 20), SDS, polysorbate, polyoxyethylene copolymers; and other excipients, such as: potassium phosphate, sodium acetate, ammonium sulfate, magnesium sulfate, sodium sulfate, trimethylamine N-oxide, betaine, metal ions (e.g., zinc, copper, calcium, manganese, and magnesium), CHAPS, monolaurate, 2-O- β -mannoglycerate, or any combination thereof.

In certain embodiments, the concentration of one or more excipients in the compositions described herein is from about 0.001% to 5% by weight, while in other embodiments, the concentration of one or more excipients is from about 0.1% to 2% by weight. Excipients are well known in the art and are manufactured by known methods and are available from commercial suppliers. In some embodiments, the excipient is sucrose. In other embodiments, sucrose is present in the composition at a concentration of about 1%.

In a particular embodiment, the compositions described herein comprise (or consist of or consist essentially of) from about pH 6.0 to about pH 7.0: about 25 to about 50mg TNFR: Fc (e.g., etanercept), about 10mM to about 50mM sodium phosphate (e.g., monosodium phosphate and/or disodium phosphate), about 0.75% to about 1.25% sucrose, about 50mM to about 150mM NaCl.

In another embodiment, the compositions described herein comprise (or consist of or consist essentially of) at about pH 6.2: about 50mg/ml TNFR: Fc, about 10mM sodium phosphate, about 140mM sodium chloride and about 1% sucrose.

In certain embodiments, provided herein are methods of treating an individual comprising administering to the individual a therapeutically effective amount of a composition described herein, wherein the individual has a disease or disorder that can be beneficially treated by the Fc domain containing polypeptide in the form of a composition. In some embodiments, the Fc domain-containing polypeptide is derived from the same species as the individual to be treated with the composition. In a particular embodiment, the subject is a human in need of treatment. When the Fc domain containing polypeptide of the composition is TNFR: Fc, examples of diseases or conditions that can be treated include, but are not limited to, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, wegener's disease (granuloma), crohn's disease (or inflammatory bowel disease), Chronic Obstructive Pulmonary Disease (COPD), hepatitis c, endometriosis, asthma, cachexia, psoriasis and atopic dermatitis. Additional diseases or disorders that can be treated by TNFR Fc include those described in WO 00/62790, WO 01/62272, and U.S. patent application No. 2001/0021380.

In other aspects, provided herein are polypeptide compositions having improved long term storage such that the active ingredient (e.g., an Fc domain-containing polypeptide) is stable during storage in a liquid (or frozen) state. As used herein, the phrase "long term" storage means that the composition can be stored for three months or more, six months or more, or one year, or two years or more. Long term storage is also understood to mean that the composition is stored as a liquid at 2-8 ℃ or frozen, for example, at a temperature of-20 ℃ or colder. In certain embodiments, the composition may be freeze-thawed multiple times. The term "stable" with respect to long term storage is understood to mean that the active polypeptide of the composition does not lose more than 20% or 15% or even 10% of its activity. In particular embodiments, the active polypeptide of the composition does not lose more than 5% of its activity relative to the activity of the composition at the time of initial storage. The stability of the composition can be assessed based on potency, appearance, concentration, pH, and oxidation, and can be assessed using, for example, Hydrophobic Interaction Chromatography (HIC), capillary electrophoresis-sodium dodecyl sulfate (CE-SDS), high precision (HIAC) liquid particle counter, and/or isoelectric focusing. Other protein stability assays are known in the art and can be used herein.

The appropriate dose or therapeutically effective amount of the Fc domain containing polypeptide of the composition will depend on the condition to be treated, the severity of the condition, previous therapy, and the clinical history and response to the therapeutic agent of the individual. The correct dosage can be adjusted at the discretion of the attending physician so that it can be administered to the individual at one time or over a series of administrations. The compositions can be administered as the sole therapeutic agent or in combination with additional therapies as desired.

In certain embodiments, the effective amount of Fc domain-containing polypeptide per adult is between about 1-500mg/m²Or about 1-200mg/m²Or about 1-40mg/m²Or about 5-25mg/m²Within the range of (1). Alternatively, a fixed dose may be administered, which may range from 2-500 mg/dose, 2-100 mg/dose, or about 10-80 mg/dose. If the dose is to be administered more than once per week, exemplary dosage ranges are the same as or lower than the foregoing dosage ranges, and are preferably administered two or more times per week at each dose range of 25-100 mg/dose. In other embodiments, administration by injection may be acceptableThe receiving dose comprises 80-100 mg/dose, or comprises 80 mg/dose. The dose may be administered once a week, or several weeks apart (e.g., 2 to 8 weeks). In a particular embodiment, 25mg of TNFR: Fc (etanercept) is administered by a single Subcutaneous (SC) injection. Other routes of administration are contemplated.

In many cases, improvement in the condition of an individual will be obtained by up to about 100mg of the composition once to three times a week over a period of at least three weeks, however, longer periods of treatment may be necessary to bring about the desired degree of improvement. For non-treatment of chronic conditions, the regimen may continue indefinitely. For pediatric individuals (age 4-17 years), a suitable regimen involves a 0.4mg/kg to 5mg/kg dose of a polypeptide of the invention administered one or more times per week.

In other embodiments, the compositions described herein are prepared in a bulk formulation and, thus, the components of the composition are adjusted so that they will be higher than desired for administration and appropriately diluted prior to administration.

In certain embodiments, the compositions described herein are administered parenterally, e.g., subcutaneously, intramuscularly, intravenously, intraperitoneally, intracerobrospinally, intraarticularly, intrasynovially, and/or intrathecally. Parenteral administration can be by bolus injection or continuous infusion. Compositions for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. In addition, a variety of recent drug delivery methods have been developed and the compositions of the invention are suitable for administration using these new methods, e.g., injection-ease^TM、GENJECT^TMSuch as GENPEN^TMAnd injection pens such as MEDIJECTOR^TMAnd BIOJECTOR^TMThe needle-free device of (1). The compositions of the present invention may also be suitable for the method of administration to be found. See also Langer, 1990, Science, 249: 1527-1533.

In some embodiments, the compositions described herein are formulated as a depot formulation. Such long acting compositions may be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the composition may be modified by means of suitable polymeric or hydrophobic materials (e.g. as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, e.g. as a sparingly soluble salt.

In other embodiments, the compositions described herein are presented in vials, packets, or dispensing devices that can contain one or more unit dosage forms containing the active ingredient. In some embodiments, the dispensing device comprises a syringe having a single dose of the liquid composition that can be directly injected. The syringe may be accompanied by instructions for administration.

In other aspects, provided herein are kits or containers containing the aqueous compositions of the invention. The concentration of the polypeptide in the aqueous composition may vary within wide ranges. In certain embodiments, it ranges from about 0.05 to about 20,000 milligrams per milliliter (μ g/ml) of the aqueous composition. The kit may also be accompanied by instructions for use.

The compositions are further described below by way of non-limiting examples.

Examples

Example 1: one embodiment of the stabilized polypeptide composition:

table 1: etanercept compositions.

Example 2: stability data of polypeptide compositions at-70 ℃:

table 2; stability data for etanercept at-70 ℃.

Example 3: stability data of polypeptide compositions at 5 ± 3 ℃:

table 3: stability data for etanercept at 5 ± 3 ℃.

Equivalents and ranges

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. The scope of the invention is not intended to be limited to the above "detailed description" but rather is as shown in the appended claims.

In the claims, articles such as "a", "an" and "the" may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Unless indicated to the contrary or otherwise apparent from the context, claims or descriptions including an "or" between one or more members of a group are deemed to be satisfied that one, more than one or all of the group members are present, employed, or otherwise relevant to a given product or process. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., of one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim may be modified to include one or more limitations found in any other claim that is dependent on the same base claim.

When elements are provided in an enumerated form, such as in a Markush group (Markush group), it is to be understood that each subgroup of the elements is also disclosed and that any element can be removed from the group. It will be understood that, in general, when the invention or aspects of the invention are described as including particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist of, or consist essentially of, such elements, features, etc. For simplicity, those embodiments with respect to this aspect are not specifically set forth herein. It should also be noted that the term "comprising" is intended to be open-ended and allows for the inclusion of additional elements or steps.

When ranges are given, the endpoints are also included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can take on any specific value or subrange within the ranges specified in different embodiments of the invention, up to one tenth of the lower limit of the range, unless explicitly specified herein.

The term "about" or "approximately" can mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which range will depend in part on the manner in which the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, as practiced in the art. Alternatively, "about" may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, the term may mean within an order of magnitude of the value, such as within 5-fold or within 2-fold. When particular values are described in the present application and claims, the term "about" should be assumed to be within an acceptable error range for the particular value, unless otherwise specified.

In addition, it should be understood that any particular embodiment of the invention falling within the prior art may be explicitly excluded from any one or more claims. Since such embodiments are considered to be known to those of ordinary skill in the art, they may be excluded even if not explicitly stated to be excluded herein. Any particular embodiment of the method of the present invention may be excluded from any one or more claims for any reason, whether or not relevant to the existence of prior art.

The present application also relates to the following embodiments:

1. a composition, comprising:

an isolated polypeptide which is an extracellular ligand-binding portion of human p75 tumor necrosis factor receptor fused to the Fc region of human IgG 1; and

a salt in an amount sufficient to prevent aggregation of the isolated polypeptide, thereby stabilizing the composition,

wherein the composition comprises L-arginine at a concentration of less than 1 mM.

2. The composition of embodiment 1, wherein said composition comprises L-arginine at a concentration of less than 0.1 mM.

3. The composition of embodiment 2, wherein the composition comprises L-arginine at a concentration of less than 0.01 mM.

4. The composition of embodiment 3, wherein the composition comprises L-arginine at a concentration of less than 0.001 mM.

5. The composition of embodiment 4, wherein the composition is free of L-arginine.

6. The composition according to any one of embodiments 1-5, comprising from about 10mg/ml to about 100mg/ml of the isolated polypeptide.

7. The composition according to any one of embodiments 1-6, wherein the isolated polypeptide is etanercept.

8. The composition of embodiment 1, wherein the composition further comprises an aqueous buffer.

9. The composition of embodiment 8, wherein the aqueous buffer is sodium, histidine, potassium, sodium or potassium citrate, maleic acid, ammonium acetate, tris (hydroxymethyl) aminomethane (tris), acetate, diethanolamine, or a combination thereof.

10. The composition of embodiment 8 or 9, wherein the aqueous buffer is present at a concentration of about 1mM to about 15 mM.

11. The composition of any of embodiments 1-10, wherein the salt is sodium chloride.

12. The composition of any one of embodiments 1-11, wherein the salt is present at a concentration of about 120mM to about 150 mM.

13. The composition according to any one of embodiments 1-12, further comprising an excipient.

14. The composition of any of embodiments 1-13, wherein the excipient is sucrose, lactose, glycerol, xylitol, sorbitol, mannitol, maltose, inositol, trehalose, glucose, Bovine Serum Albumin (BSA), human SA or recombinant HA, dextran, PVA, hydroxypropyl methylcellulose (HPMC), polyethyleneimine, gelatin, polyvinylpyrrolidone (PVP), Hydroxyethylcellulose (HEC), polyethylene glycol, ethylene glycol, glycerol, dimethyl sulfoxide (DMSO), Dimethylformamide (DMF), proline, L-serine, sodium glutamate, alanine, glycine, lysine hydrochloride, sarcosine, gamma-aminobutyric acid, Tween-20, Tween-80, SDS, polysorbate, polyoxyethylene copolymers, potassium phosphate, sodium acetate, ammonium sulfate, magnesium sulfate, sodium sulfate, potassium sulfate, sodium glutamate, glycine, lysine hydrochloride, sarcosine, gamma-aminobutyric acid, Tween-20, Tween-80, SDS, sodium sulfate, sodium phosphate, sodium acetate, sodium sulfate, and the like, Trimethylamine N-oxide, betaine, zinc ion, copper ion, calcium ion, manganese ion, magnesium ion, CHAPS, sucrose monolaurate, 2-O-beta-mannoglycerate, or a combination thereof.

15. The composition according to any one of embodiments 1-14, wherein the excipient is sucrose.

16. The composition of any one of embodiments 1-15, wherein the excipient is sucrose at a concentration of about 0.5% to about 1.5%.

17. The composition according to any of embodiments 1-16, wherein the pH of the composition is from about 5.5 to about 7.8.

18. The composition of embodiment 1, comprising 50mg/ml etanercept, about 10mM sodium phosphate, about 140mM sodium chloride, and about 1% sucrose, wherein the pH of the composition is from about pH 6.0 to about pH 7.0.

19. The composition according to any one of embodiments 1-18, wherein the composition has a commercially viable shelf life of at least 24 months.

20. The composition according to any one of embodiments 1-19, wherein the composition is suitable for subcutaneous administration.

21. The composition of any one of embodiments 1-20, wherein the isolated polypeptide is purified.

22. The composition according to any one of embodiments 1-21, wherein the composition is sterilized.

23. A composition, comprising:

an isolated polypeptide which is an extracellular ligand-binding portion of human p75 tumor necrosis factor receptor fused to the Fc region of human IgG 1;

an aqueous buffer at a concentration of less than 25 mM; and

a salt at a concentration of greater than 100mM,

wherein the composition comprises L-arginine at a concentration of less than 1 mM.

24. The composition of embodiment 23, wherein said composition comprises L-arginine at a concentration of less than 0.1 mM.

25. The composition of embodiment 24, wherein said composition comprises L-arginine at a concentration of less than 0.01 mM.

26. The composition of embodiment 25, wherein said composition comprises L-arginine at a concentration of less than 0.001 mM.

27. The composition of embodiment 26, wherein the composition is free of L-arginine.

28. A composition consisting essentially of:

an isolated polypeptide which is an extracellular ligand-binding portion of human p75 tumor necrosis factor receptor fused to the Fc region of human IgG 1;

an aqueous buffer at a concentration of about 10 mM;

salt at a concentration of about 140 mM; and

sucrose.

29. The composition of embodiment 28, wherein the isolated polypeptide is present at a concentration of 50 mg/mL.

30. The composition of embodiment 28 or 29, wherein the polypeptide is etanercept.

31. A method comprising administering to an individual a composition comprising:

an isolated polypeptide which is an extracellular ligand-binding portion of human p75 tumor necrosis factor receptor fused to the Fc region of human IgG 1;

an aqueous buffer; and

a salt in an amount sufficient to prevent aggregation of the polypeptide, thereby stabilizing the composition,

wherein the composition comprises L-arginine at a concentration of less than 1 mM.

32. The method of embodiment 31, wherein said composition comprises L-arginine at a concentration of less than 0.1 mM.

33. The method of embodiment 32, wherein the composition comprises L-arginine at a concentration of less than 0.01 mM.

34. The method of embodiment 33, wherein said composition comprises L-arginine at a concentration of less than 0.001 mM.

35. The method of embodiment 34, wherein the composition is free of L-arginine.

36. The method according to any one of embodiments 31-35, wherein the composition comprises about 10mg/ml to about 100mg/ml of the isolated polypeptide.

37. The method of any one of embodiments 31-36, wherein the polypeptide is etanercept.

38. The composition of any one of embodiments 31-37, wherein the aqueous buffer is sodium phosphate, histidine, potassium phosphate, sodium or potassium citrate, maleic acid, ammonium acetate, tris (hydroxymethyl) aminomethane (tris), acetate, diethanolamine, or a combination thereof.

39. The method of any one of embodiments 31-38, wherein the aqueous buffer is present at a concentration of about 1mM to about 15 mM.

40. The method of any one of embodiments 31-39, wherein the salt is sodium chloride.

41. The method of any one of embodiments 31-40, wherein the salt is present at a concentration of about 120mM to about 150 mM.

42. The method of any one of embodiments 31-41, wherein the composition further comprises an excipient.

43. The method of embodiment 42, wherein the excipient is sucrose, lactose, glycerol, xylitol, sorbitol, mannitol, maltose, inositol, trehalose, glucose, bovine serum albumin (BSC), human SC or recombinant HC, dextran, PVC, hydroxypropyl methylcellulose (HPMC), polyethyleneimine, gelatin, polyvinylpyrrolidone (PVP), hydroxyethyl cellulose (HEC), polyethylene glycol, ethylene glycol, glycerol, dimethyl sulfoxide (DMSO), Dimethylformamide (DMF), proline, L-serine, sodium glutamate, alanine, glycine, lysine hydrochloride, sarcosine, gamma-aminobutyric acid, Tween-20, Tween-80, SDS, polysorbate, polyoxyethylene copolymer, potassium phosphate, sodium acetate, ammonium sulfate, magnesium sulfate, sodium sulfate, trimethylamine N-oxide, sodium glutamate, Betaine, zinc ion, copper ion, calcium ion, manganese ion, magnesium ion, CHCPS, sucrose monolaurate, 2-O-beta-mannoglycerate, or a combination thereof.

44. The method of embodiment 42 or 43, wherein the excipient is sucrose.

45. The method of embodiment 44, wherein the excipient is sucrose at a concentration of about 0.5% to about 1.5%.

46. The method of any of embodiments 31-45, wherein the composition has a pH of about 5.5 to about 7.8.

47. The method of embodiment 31, wherein the composition comprises 50mg/ml etanercept, about 10mM sodium phosphate, about 140mM sodium chloride, and about 1% sucrose, wherein the pH of the method is from about pH 6.0 to about pH 7.0.

48. The method of any one of embodiments 31-47, wherein the composition has a commercially viable shelf life of at least 24 months.

49. The method of any one of embodiments 31-48, wherein the composition is suitable for subcutaneous administration.

50. The method according to any one of embodiments 31-49, wherein the isolated polypeptide is purified.

51. The method of any one of embodiments 31-50, wherein the composition is sterilized.

52. The method of any one of embodiments 31-51, wherein the individual is diagnosed with a disease or disorder selected from rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Wegener's disease (granuloma), Crohn's disease (or inflammatory bowel disease), Chronic Obstructive Pulmonary Disease (COPD), hepatitis C, endometriosis, asthma, cachexia, psoriasis, or atopic dermatitis.

53. The method of embodiment 52, wherein the composition is administered to the individual in an amount sufficient to treat the disease or disorder.

Claims (10)

1. A composition, comprising:

an isolated polypeptide which is an extracellular ligand-binding portion of human p75 tumor necrosis factor receptor fused to the Fc region of human IgG 1; and

a salt in an amount sufficient to prevent aggregation of the isolated polypeptide, thereby stabilizing the composition,

wherein the composition comprises L-arginine at a concentration of less than 1 mM.

2. The composition of claim 1, wherein the composition comprises L-arginine at a concentration of less than 0.1 mM.

3. The composition of claim 2, wherein the composition comprises L-arginine at a concentration of less than 0.01 mM.

4. The composition of claim 3, wherein the composition comprises L-arginine at a concentration of less than 0.001 mM.

5. The composition of claim 4, wherein the composition is free of L-arginine.

6. The composition of any one of claims 1-5, comprising about 10mg/ml to about 100mg/ml of the isolated polypeptide.

7. The composition of any one of claims 1-6, wherein the isolated polypeptide is etanercept.

8. The composition of claim 1, wherein the composition further comprises an aqueous buffer.

9. The composition of claim 8, wherein the aqueous buffer is sodium, histidine, potassium, sodium or potassium citrate, maleic acid, ammonium acetate, tris (hydroxymethyl) aminomethane (tris), acetate, diethanolamine, or a combination thereof.

10. The composition of claim 8 or 9, wherein the aqueous buffer is present at a concentration of about 1mM to about 15 mM.