Stabilized antibody solutions

Abstract

To provide an antibody protein product that can be used outside the cold chain for a predetermined period of time.SOLUTION: There is provided an aqueous solution comprising an antibody protein and a stabilizing mixture of (i) a chelating agent which is a multi-anion and (ii) a C3 polyol.SELECTED DRAWING: None

Description

Background Art

[0001] (Background of the Invention) When formulated as an aqueous solution, antibody proteins are prone to structural degradation during storage. The processes involved in proteolysis can be physical (e.g., loss of quaternary, tertiary, or secondary structure, aggregation, particle formation) and chemical (i.e., processes involving covalent changes, e.g., deamidation, aspartic acid isomerization, oxidation, hydrolytic clipping, etc.). ) and can be divided into. Degradation products (e.g., soluble aggregates, insoluble aggregates, and chemically modified variants) can each affect the biological activity, toxicity, or immunogenicity of the antibody protein. .

[0002] Therefore, the levels of all degradation products must be maintained within the strict specifications set for each antibody protein product. The rate of the degradation process depends on temperature, and antibody proteins are generally more stable at low temperatures. As a result, commercially available antibody products generally must be stored refrigerated. However, with the increasing trend of subcutaneous products that can be self-administered by patients, there is a strong need for the development of antibody protein products that can be used outside the cold chain for at least a certain period, e.g., 2 weeks, e.g., 4 weeks, e.g., 12 weeks or longer. The ability to store the product outside the cold chain can, in many cases, significantly improve patient convenience during use. Allowing deviation outside the cold chain can also significantly improve the shipping logistics.

[0003] The present invention addresses the problem of antibody protein instability, particularly the problem of antibody protein degradation. ​

[0004] WO2006 / 0096488A2 (Pharmacia & Upjohn Company LLC) describes a composition of a human IgG antibody containing a chelating agent, which is said to exhibit improved chemical and / or physical stability.

[0005] WO2013 / 114122A2 (Arecor Limited) describes an aqueous solution containing an antibody protein at a concentration of at least about 10 mg / mL and an oligomer of ethyleneimine, wherein the number (n) of repeating units of ethyleneimine in the oligomer is in the range of n = 2 to 12.

[0006] WO2010 / 062896A1 (Abbott Laboratories) describes compositions and methods for inhibiting the fragmentation of immunoglobulins containing a λ light chain based on the observation that iron increases the fragmentation of recombinant full - human IgG molecules containing a λ light chain by cleavage in the hinge region in the presence of histidine. SUMMARY OF THE INVENTION

[0007] (SUMMARY OF THE INVENTION) The present invention addresses the problem of the instability of antibody proteins. In one embodiment, the present invention relates to an aqueous solution comprising an antibody protein and (i) a chelating agent that is a polyvalent anion; and (ii) a stabilizing mixture of C3 polyols. In one embodiment, the present invention provides a method for stabilizing an antibody protein in an aqueous solution against storage, the method comprising the step of adding to the solution (i) a chelating agent that is a polyvalent anion; and (ii) a mixture of C3 polyols. DETAILED DESCRIPTION OF THE INVENTION ​​​​​​​​​​

[0008] (Detailed Description of the Invention) The present invention relates to the discovery that an aqueous solution of an antibody protein can be stabilized by a mixture of a chelating agent, which is a polyvalent anion, and a C3 polyol.

[0009] As used herein, the term "aqueous solution" refers to a solution in water, preferably distilled water, deionized water, water for injection, sterile water for injection, or bacteriostatic water for injection. The aqueous solution of the present invention contains a dissolved antibody protein, a chelating agent which is a polyvalent anion, and a C3 polyol, and optionally, one or more additives and / or excipients. The aqueous solution can also contain one or more components, such as additives or excipients, that are partially dissolved or undissolved. The presence of such component(s) results in a multiphase composition, such as a suspension or an emulsion. Preferably, the aqueous solution of the present invention is a homogeneous solution as determined visually or by light scattering.

[0010] As used herein, the term "antibody protein" refers to an antibody, an antibody fragment, an antibody conjugated to an active moiety, a fusion protein containing one or more antibody fragments, such as an immunoglobulin Fc domain, or a derivative of any of the foregoing. Examples of derivatives include conjugated derivatives, such as an antibody or antibody fragment conjugated to another moiety. Such moieties include chemically inert polymers, such as PEG. Preferred antibodies include monoclonal antibodies and polyclonal antibodies, preferably monoclonal antibodies. Monoclonal antibodies can be, for example, from mammals (e.g., mice) or birds, chimeric, such as human / mouse or human / primate chimeric ​​​​​​​​​​​​​​​ It can be a humanized antibody or a fully human antibody. Suitable antibodies include immunoglobulins such as IgG 1 IgG 2 IgG 3 or IgG 4 IgM, IgA such as IgA 1 or IgA 2 IgD, IgE, or IgG including IgY. Suitable antibodies also include single-chain antibodies. Fc Fab, Fab 2 Antibody fragments including ScFv fragments, etc. are also included. Single-domain antibodies including nanobodies are also included.

[0011] In certain embodiments, the antibody is fused or conjugated to a chelating agent that can bind to an active molecule such as a toxin or a radioactive metal ion such as for example 99 Tc 111 Ir 131 I, or 90 Y. In such embodiments, the antibody typically functions as a targeting agent, for example, to direct the active molecule to cells presenting a specific cell surface protein.

[0012] Specific antibodies that can be formulated as described herein include infliximab (chimeric antibody, anti-TNFα), basiliximab (chimeric antibody, anti-IL-2), abciximab (chimeric antibody, anti-GpIIb / IIIa), daclizumab (humanized antibody, anti-IL-2), gemtuzumab (humanized antibody, anti-CD33), alemtuzumab (humanized antibody, anti-CD52), edrecolomab (mouse Ig2a, anti- EpCAM), rituximab (chimeric antibody, anti-CD20), palivizumab (humanized antibody, anti-respiratory syncytial Whole virus), trastuzumab (humanized antibody, anti-HER2 / neu (erbB2) receptor), bevacizumab ( humanized antibody, anti-VEGF), cetuximab (chimeric antibody, anti-EGFR), eculizumab (humanized antibody, anti-complement protein C5), efalizumab (humanized antibody, anti-CD1Ia), ibritumomab (mouse antibody, anti-CD20), muromonab-CD3 (mouse antibody, anti-T cell CD3 receptor), natalizumab (human ized antibody, anti-α4 integrin), nimotuzumab (humanized IgGl, anti-EGF receptor), omalizumab ( humanized antibody, anti-IgE), panitumumab (human antibody, anti-EGFR), ranibizumab (humanized antibody, anti-V EGF), 1-131 tositumomab (humanized antibody, anti-CD20), ofatumumab (human antibody, anti-CD-20), certolizumab (humanized antibody, anti-TNF-α), golimumab (human antibody, anti-TNFα), and denos umab (human antibody, anti-RANK ligand) are included, but not limited thereto. Preferred antibodies include trastuzumab, rituximab, bevacizumab, cetuximab, and ipilimum umab. In one embodiment, the antibody is bevacizumab. In one embodiment it is not an anti-TNF-α antibody.

[0013] Other chimeric antibodies that can be formulated as described herein include babitu ximab (anti-phosphatidylserine), brentuximab (anti-CD30), siltuximab (anti-IL- 6), clenoliximab (anti-CD4), galiximab (anti-CD80), gomiliximab (anti-CD23), keli ximab (anti-CD4), lumiliximab (anti-CD23), priliximab (anti-CD4), teneliximab ( anti-CD40), bapaliximab (anti-VAP1), eclomiximab (anti-GD3), and pagibaximab (anti Examples include staphylococcal lipoteichoic acid).

[0014] Other humanized antibodies that can be formulated as described herein include eprat uzumab (anti-CD22), ofatumumab (anti-CD20), bevacizumab mertansine (anti-CD44), canz umab mertansine (anti-mucin), cetuximab bogatox (anti-TACSTD1), dacetuzumab (anti CD40), elotuzumab (anti-CD319), etaracizumab (anti-α v β 3 -integrin), farlet uzumab (anti-FRα), inotuzumab ozogamicin (anti-CD22), labetuzumab (anti-carcinoembryonic antigen) , rituximab (anti-CD33), miratuximab (anti-CD74), nimotuzumab (anti-EGFR), oportuzma bumab (anti-EpCAM), pertuzumab (anti-HER2), sibrotuzumab (anti-FAP), tecatuzumab tetraxetan (anti-alpha-fetoprotein), tigatuzumab (anti-TRAIL-2), tucotuzumab cell molokin (anti-EpCAM), belzutifan (anti-CD20), acelizumab (anti-CD62L), apolizumab ( anti-HLA-DRB), benralizumab (anti-CD125), cedelizumab (anti-CD4), epratuzumab (anti-CD22) , elulizumab (anti-CD18), fontolizumab (anti-interferon-γ), mepolizumab (anti IL5), ocrelizumab (anti-CD20), pascolizumab (anti-IL4), pecilizumab (anti-complement component 5) , PRO-140 (anti-CCR5), reslizumab (anti-IL5), loncastuximab (anti-interferon-α), robilizumab (anti-CD11, CD18), siprilizumab (anti-CD2), talizumab (anti-IgE), teprilizumab (anti-CD3), tocilizumab (anti-IL6R), vedolizumab (anti-α 4 β 7-Integrin), bisilizumab (anti-CD3), ibalizumab (anti-CD4), tefibazumab (anti-clamping factor A), tadocizumab ( anti-α 11b β 3 -Integrin), bapineuzumab (anti-amyloid-β), solanezumab (anti-amylo id-β), tanezumab (anti-NGF), ulotuxizumab (anti-Escherichia coli (E. coli) Shiga-like toxin II B sub unit), felvizumab (anti-respiratory syncytial virus), motavizumab (anti-respiratory syncytial virus glycoprotein F), and lebrikizumab (anti-IL13) may be mentioned.

[0015] Additional human antibodies that can be formulated as described herein include ato rilumab (anti-Rh factor), fresolimumab (anti-TGFβ-1, -2, and -3), lerdelimumab (anti-TG Fβ-2), metelimumab (anti-TGFβ-1), morolimumab (anti-Rh factor), ipilimumab (anti-CTLA-4) , tremelimumab (anti-CTLA-4), belimumab (anti-CCL11), zanolimumab (anti-CD4), bri kinumab (anti-IL12, -23), canakinumab (anti-IL1β), ustekinumab (anti-IL12, -23), ade castumumab (anti-EpCAM), belimumab (anti-B cell activating factor), cicutumumab anti-IGF-1 receptor), conatumumab (anti-TRAIL-R2), figitumumab (anti-IGF-1 receptor), ilatumumab (anti-CD30), lexatumumab (anti-TRAIL-R2), lucatumumab (anti-CD40), mapatumumab (anti-TRAIL-R4), nes tumumab (anti-EGFR), oralaniumab (anti-PDGF-Rα), purtumumab (anti-vimentin), robatum umab (anti-IGF-1 receptor), botumumab (anti-tumor antigen CTAA16.88), zalutumumab (anti-EGFR), sta Murumab (anti-myostatin), efungumab (anti-fungal HSP90), exibivirumab (anti-hepatitis B surface antigen), foravivumab (anti-rabies glycoprotein), livibizumab (anti-hepatitis B surface antigen) , ravivumab (anti-rabies glycoprotein), legavivumab (anti-cytomegalovirus glycoprotein B), cevivumab (anti-cytomegalovirus), tubivizumab (anti-hepatitis B virus), panobacumab (anti-Pseudomonas aeruginosa serotype IATS 011), raxibacumab (anti- anthrax toxin), ramucirumab (anti-VEGF-R2), and gantenerumab (anti-amyloid-β) are included.

[0016] Fusion proteins containing fragments of immunoglobulin molecules can also be formulated according to the present invention. Suitable fusion proteins include proteins containing one or more immunoglobulin fragments, for example, an active protein domain fused to an Fc domain. Such fusion proteins include dimeric proteins having monomers containing an active protein domain, for example, a soluble receptor or extracellular ligand-binding domain of a receptor, fused to an immunoglobulin Fc domain. The two Fc domains can associate via disulfide bonds to form a dimeric protein. Such fusion proteins include etanercept, abatacept, and belatacept.

[0017] Conjugated derivatives containing an antibody (or one or more antibody fragments) and a chemically inert polymer, for example, PEG, can also be formulated according to the present invention. Such derivatives include certolizumab pegol.

[0018] The antibody protein can be isolated from a natural source or is a recombinant protein and can be.

[0019] In certain embodiments, the antibody protein is substantially pure, i.e., the composition contains a single antibody protein and no substantial amount of any additional protein. In a preferred embodiment, the antibody protein comprises at least 99% of the total protein content of the composition, preferably at least 99.5%, more preferably at least about 99.9%. In a preferred embodiment, the antibody protein is sufficiently pure to be used as in a pharmaceutical composition and is pure.

[0020] The antibody protein is preferably a therapeutic antibody protein. Such an antibody protein has the desired therapeutic or prophylactic activity and is adapted for the treatment, inhibition, or prevention of a disease or medical disorder.

[0021] In one embodiment, the antibody protein is a monoclonal antibody, e.g., trastuzumab, rituximab, bevacizumab, cetuximab, or ipilimumab. In another embodiment the antibody protein is a fusion protein comprising an active protein domain fused to one or more immunoglobulin Fc fragments, e.g., etanercept, abatacept, or belatacept. In a further embodiment, the antibody is a derivative of the antibody protein and is a conjugated derivative comprising one or more antibodies or antibody fragments and a chemically inert polymer, e.g., certolizumab pegol.

[0022] ​​​The antibody protein is preferably present at a concentration of about 1 mg / mL to about 300 mg / mL, for example, about 10 mg / mL to about 300 mg / mL, about 1 mg / mL to about 200 mg / mL, or about 10 mg / mL to about 200 mg / mL. L, about 1 mg / mL to about 200 mg / mL, or about 10 mg / mL to about 200 mg / mL.

[0023] The aqueous solution of the present invention contains a chelating agent, which is a polyvalent anion, as a stabilizer. A polyvalent anion means a species having at least two anionic centers per molecule at a specific pH of the solution. A chelating agent means a drug that can form a complex with metal ions, such as calcium, magnesium, iron, and / or or zinc ions. Preferably, the chelating agent can form a complex with zinc ions. Usually, the polyvalent anion has at least two anionic centers per molecule, where the pH of the solution is about pH 4.0 to about pH 8.0. In one embodiment, the chelating agent, which is a polyvalent anion, is ethylenediaminetetraacetic acid (EDTA) . The EDTA anion is preferably introduced into the aqueous solution in the form of a salt of ethylenediaminetetraacetic acid, such as the disodium salt or the tetrasodium salt. Alternatively, it can be introduced in the form of ethylenediaminetetraacetic acid and then the pH can be adjusted to the required level . Further examples of the chelating agent, which is a polyvalent anion, include other chelating ions having four ionic centers, such as ethylene glycol-bis(β-aminoethyl ether)-N,N,N ',N'-tetraacetate (EGTA) and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA), as well as citrate, pyrophosphate, and alginate. The chelating agent, which is a polyvalent anion, is used in a suitable salt form (for example, the sodium salt ), such as ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetate (EGTA) and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA), and citrate, pyrophosphate, and alginate are also included. The chelating agent, which is a polyvalent anion, is used in a suitable salt form (for example, the sodium salt ',N'-tetraacetate (EGTA) and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA), and citrate, pyrophosphate, and alginate are also included. The chelating agent, which is a polyvalent anion, is used in a suitable salt form (for example, the sodium salt ), such as ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetate (EGTA) and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA), and citrate, pyrophosphate, and alginate are also included. The chelating agent, which is a polyvalent anion, is used in a suitable salt form (for example, the sodium salt ), such as ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetate (EGTA) and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA), and citrate, pyrophosphate, and alginate are also included. The chelating agent, which is a polyvalent anion, is used in a suitable salt form (for example, the sodium salt It may also be used as a polyvalent anion-forming acid form in a solution. Chelating agent mixtures may be used. The chelating agent, which is a polyvalent anion, has a stabilizing effect and is usually present at a concentration of about 0.1 mM to about 50 mM, for example, about 0.1 mM to about 20 mM, for example, about 0.1 mM to about 10 mM. Preferably, the chelating agent is not citrate.

[0024] In one embodiment, the chelating agent, which is a polyvalent anion, does not contain a cationic center. In one embodiment, the chelating agent, which is a polyvalent anion, contains only anionic centers.

[0025] In one embodiment, the chelating agent, which is a polyvalent anion, contains 4 anionic centers per molecule. In one embodiment, the chelating agent, which is a polyvalent anion, is of a tetracoordinate type.

[0026] In one embodiment, the chelating agent, which is a polyvalent anion, has a metal-binding stability constant for zinc ion binding of > 5.5 at 25 °C, for example, a log K for zinc binding of > 6, > 6.5, > 7, > 7.5, > 8, > 8.5, > 9, > 9.5, > 10, > 10.5, > 11, > 11.5, > 12, > 1 2.5, > 13, > 13.5, > 14, or > 14.5 at 25 °C. In one embodiment, the chelating agent, which is a polyvalent anion, has a log K for zinc ion binding of > 5.5 to 15 at 25 °C, for example, a log K for zinc binding of > 6 to 15, > 6.5 to 15, > 7 to 15, > 7.5 to 15, > 8 to 15, > 8.5 to 15, > 9 to 15, > 9.5 to 15, > 10 to 15, > 10.5 to 15, > 11 to 15, > 11.5 to 15, > 12 to 15, > 12.5 to 15, > 13 to 15, > 13.5 to 15, > 14 to 15, or > 14.5 to 15 at 25 °C. American National Standards 15, > 9 to 15, > 9.5 to 15, > 10 to 15, > 10.5 to 15, > 11 to 15, > 11.5 to 15, > 12 to 15, > 12.5 to 15, > 13 to 15, > 13.5 to 15, > 14 to 15, or > 14.5 to 15 at 25 °C. American National Standards The metal-binding stability constants listed in the reference database 46 of the National Institute of Standards and Technology (severely selected stability constants of metal complexes) can be used. This database usually lists the logK constants determined at 25 °C, for example, phosphate (logK = 4.93), EDTA (logK = 14.6), EGTA (logK = 12.6), BAPTA (logK = 10.26), pyrophosphate (logK = 8.71), and alginate (logK = 6.91). Preferably, the chelating agent that is a polyvalent anion has a logK for zinc ion binding of >5.5 to 15 at 25 °C.

[0027] The aqueous solution of the present invention also contains a C3 polyol as a stabilizer preferably selected from 1,2-propanediol (also known as propane-1,2-diol or propylene glycol) and glycerol (1,2,3-propanetriol, glycerin, or also known as glycerol). In one embodiment, the C3 polyol is 1,2-propanediol. In another embodiment, the C3 polyol is glycerol. In a further embodiment, the C3 polyol is a mixture of 1,2-propanediol and glycerol. The C3 polyol is preferably present at a concentration of about 100 mM to about 500 mM, for example, about 150 mM to about 400 mM, or about 150 mM to about 300 mM. When multiple C3 polyols are present in the aqueous solution, the concentration refers to the total concentration of the C3 polyols.

[0028] Generally, the pH of the aqueous solution of the present invention is from about pH 4.0 to about pH 8.0, for example, from about pH 5.0 to about pH 7.0 or is from about pH 5.0 to about pH 6.5.

[0029] In one embodiment, the aqueous solution of the invention further comprises a buffer to stabilize the pH of the formulation, which can also be selected to enhance antibody protein stability. Suitable buffers are selected from the group consisting of histidine, succinate, maleate, acetate, phosphate, and TRIS. In certain embodiments, the buffer is a phosphate buffer. In one embodiment, the buffer is selected to have a pK

[0030] close to the pH of the composition; for example, a histidine is preferably utilized as a buffer when the pH of the composition is in the range of 5.0 to 7.0. As another example, phosphate is preferably utilized as a buffer when the pH of the composition is in the range of 6.1 to 8.1. Alternatively, in another embodiment, the solution of the invention is a formulation comprising a protein and one or more additives, further stabilized as disclosed in WO2008 / 084 237A2, which is substantially free of compounds having ionizable groups having a pK within one unit of the pH of the formulation at the intended storage temperature range of the system, e.g., 25°C, of a conventional buffer, i.e., a buffer of the composition. a In this embodiment, the pH of the formulation is set to the value at which the formulation has the maximum measurable stability with respect to pH; one or more additives (substituted buffers) can exchange protons with the insulin compound and have a pK value at least one unit greater or less than the pH of the formulation at the intended storage temperature range of the formulation. The additive is from 1 to 5 pH units of the pH of the aqueous formulation at the intended storage temperature range of the composition (e.g., 25°C), a The additive is from 1 to 5 pH units of the pH of the aqueous formulation at the intended storage temperature range of the composition (e.g., 25°C), Preferably, it has a pK of 1 to 3 pH units, most preferably 1.5 to 2.5 pH units a and can have an ionizable group. Such additives can usually be used at a concentration of 0.5 to 10 mM, for example, 2 to 5 mM .

[0031] Usually, the buffer is present at a concentration of about 0.5 mM to about 50 mM, for example, about 1 mM to about 20 mM, for example, about 2 mM to about 5 mM .

[0032] The aqueous solution of the present invention can optionally contain a surfactant. In one embodiment, the surfactant is a non-ionic surfactant, for example, an alkyl glycoside, for example, dodecyl maltoside; a polysorbate surfactant, for example, polysorbate 80 or polysorbate 20; for example, an alkyl ether of polyethylene glycol selected from polyethylene glycol (2) dodecyl ether, polyethylene glycol (2) oleyl ether, and polyethylene glycol (2) hexadecyl ether; a block copolymer of polyethylene glycol and polypropylene glycol, for example, poloxamer 188, poloxamer 407, poloxamer 171, or poloxamer 185; or an alkyl phenyl ether of polyethylene glycol, for example, 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol . Preferably, the non-ionic surfactant is present at a concentration of about 10 μg / mL to about 2000 μg / mL, for example, about 50 μg / mL to about 1000 μg / mL, for example, about 100 μg / mL to about 500 μg / mL . .

[0033] .

[0033] The aqueous solution of the present invention includes hypotonic, isotonic, and hypertonic aqueous solutions and has a wide range of osmolalities can reach. Preferably, the aqueous solution of the present invention is substantially isotonic. In one embodiment the aqueous solution of the present invention is isotonic. Preferably, the osmolality of the aqueous solution is selected so as to minimize, for example, pain during injection depending on the administration route. A preferred aqueous solution has an osmolality in the range of about 200 mOsm / L to about 500 mOsm / L. Preferably, the osmolality is in the range of about 250 to about 350 mOsm / L. More preferably, the osmolality is about 300 mOsm / L.

[0034] The osmotic pressure of the aqueous solution can be adjusted with an osmotic pressure regulator. The osmotic pressure regulator may be charged or uncharged.

[0035] Examples of charged osmotic pressure regulators include salts such as sodium, potassium, magnesium or calcium ions combined with chloride, sulfuric acid, carbonic acid, sulfurous acid, nitric acid, lactic acid, succinic acid acetic acid or maleic acid ions (especially sodium chloride or sodium sulfate, especially sodium chloride). Amino acids such as glycine, histidine, or arginine can also be used for this purpose. In one embodiment, the charged osmotic pressure regulator is selected from the group consisting of sodium chloride, sodium sulfate, sodium acetate, sodium lactate, glycine, histidine and arginine. Such charged osmotic pressure regulators are usually present at a concentration of about 25 mM to about 500 mM, for example, about 50 mM to about 250 mM, for example, about 150 mM.

[0036] Examples of uncharged osmotic pressure regulators include sugars, sugar alcohols, and other polyols such as ​​Sucrose, trehalose, mannitol, raffinose, lactose, dextrose , sorbitol, or lactitol, or polyethylene glycol, for example, PEG300 or PEG400. In one embodiment, the non-charged osmotic regulator is sucrose , trehalose, mannitol, sorbitol, PEG300, or PEG400. The C3 polyol, which is a necessary component of the aqueous solution of the present invention, can function as a non-charged osmotic regulator . However, the reference to the aqueous solution of the present invention that "further" contains a non-charged osmotic regulator is intended to refer to an additional further component that will be added to the solution . Therefore, the aqueous solution can further contain a non-charged osmotic regulator that is other than the C3 polyol, and in particular, other than 1,2-propanediol and glycerol. Such a non-charged osmotic regulator is usually present at a concentration of about 50 mM to about 1000 mM, for example, about 100 mM to about 500 mM, for example , about 300 mM .

[0037] The aqueous solution of the present invention can optionally contain a preservative preferably selected from phenol, m-cresol, chlorocresol, benzyl alcohol , propyl paraben, methyl paraben, benzalkonium chloride, and benzethonium chloride . When present, the preservative is at a concentration of about 0.01 mM to about 100 mM. The preservative selected from phenol, m-cresol, chlorocresol , benzyl alcohol, propyl paraben, and methyl paraben can be present, for example , at a concentration of about 10 mM to about 100 mM, for example, about 20 mM to about 80 mM, for example, about 25 mM to about 50 mM . The preservative selected from benzalkonium chloride and benzethonium chloride The agent can be present at a concentration of, for example, about 0.01 mM to about 1 mM, for example, about 0.05 mM to about 0.5 mM, for example, about 0.05 mM to about 0. 2 mM.

[0038] The inventors have discovered that the stability of antibody proteins in aqueous solutions is improved by the addition of (i) a chelating agent that is a polyvalent anion; and (ii) a mixture of C3 polyols. The addition of a chelating agent that is a polyvalent anion, such as EDTA, has been observed to enhance the stability of antibody proteins in aqueous solutions. Surprisingly, the stabilizing effect of EDTA is further increased by the addition of C3 polyols. Without wishing to be bound by theory, the stabilizing effect of a chelating agent that is a polyvalent anion is thought to be due to a combination of (i) charge interaction with positively charged patches on the protein surface and (ii) removal of trace metals that can catalyze the degradation process. Without wishing to be bound by theory, the further stabilizing effect of C3 polyols is thought to be due to the optimal hydrophobicity and hydrogen bond interactions of small polyols on the protein surface, which make the three-dimensional structure between protein molecules more compact, modify the interfacial tension, and then reduce the exposure of reactive sites and the possibility of irreversible aggregation events. -ting agent; and (ii) a mixture of C3 polyols. The addition of a chelating agent that is a polyvalent anion, such as EDTA, has been observed to enhance the stability of antibody proteins in aqueous solutions. Surprisingly, the stabilizing effect of EDTA is further increased by the addition of C3 polyols. Without wishing to be bound by theory, the stabilizing effect of a chelating agent that is a polyvalent anion is thought to be due to a combination of (i) charge interaction with positively charged patches on the protein surface and (ii) removal of trace metals that can catalyze the degradation process. Without wishing to be bound by theory, the further stabilizing effect of C3 polyols is thought to be due to the optimal hydrophobicity and hydrogen bond interactions of small polyols on the protein surface, which make the three-dimensional structure between protein molecules more compact, modify the interfacial tension, and then reduce the exposure of reactive sites and the possibility of irreversible aggregation events. The addition of a chelating agent that is a polyvalent anion, such as EDTA, has been observed to enhance the stability of antibody proteins in aqueous solutions. Surprisingly, the stabilizing effect of EDTA is further increased by the addition of C3 polyols. Without wishing to be bound by theory, the stabilizing effect of a chelating agent that is a polyvalent anion is thought to be due to a combination of (i) charge interaction with positively charged patches on the protein surface and (ii) removal of trace metals that can catalyze the degradation process. Without wishing to be bound by theory, the further stabilizing effect of C3 polyols is thought to be due to the optimal hydrophobicity and hydrogen bond interactions of small polyols on the protein surface, which make the three-dimensional structure between protein molecules more compact, modify the interfacial tension, and then reduce the exposure of reactive sites and the possibility of irreversible aggregation events. Without wishing to be bound by theory, the stabilizing effect of a chelating agent that is a polyvalent anion is thought to be due to a combination of (i) charge interaction with positively charged patches on the protein surface and (ii) removal of trace metals that can catalyze the degradation process. Without wishing to be bound by theory, the stabilizing effect of a chelating agent that is a polyvalent anion is thought to be due to a combination of (i) charge interaction with positively charged patches on the protein surface and (ii) removal of trace metals that can catalyze the degradation process. Without wishing to be bound by theory, the stabilizing effect of a chelating agent that is a polyvalent anion is thought to be due to a combination of (i) charge interaction with positively charged patches on the protein surface and (ii) removal of trace metals that can catalyze the degradation process. Without wishing to be bound by theory, the stabilizing effect of a chelating agent that is a polyvalent anion is thought to be due to a combination of (i) charge interaction with positively charged patches on the protein surface and (ii) removal of trace metals that can catalyze the degradation process. Without wishing to be bound by theory, the further stabilizing effect of C3 polyols is thought to be due to the optimal hydrophobicity and hydrogen bond interactions of small polyols on the protein surface, which make the three-dimensional structure between protein molecules more compact, modify the interfacial tension, and then reduce the exposure of reactive sites and the possibility of irreversible aggregation events. Without wishing to be bound by theory, the further stabilizing effect of C3 polyols is thought to be due to the optimal hydrophobicity and hydrogen bond interactions of small polyols on the protein surface, which make the three-dimensional structure between protein molecules more compact, modify the interfacial tension, and then reduce the exposure of reactive sites and the possibility of irreversible aggregation events. Without wishing to be bound by theory, the further stabilizing effect of C3 polyols is thought to be due to the optimal hydrophobicity and hydrogen bond interactions of small polyols on the protein surface, which make the three-dimensional structure between protein molecules more compact, modify the interfacial tension, and then reduce the exposure of reactive sites and the possibility of irreversible aggregation events. be due to the optimal hydrophobicity and hydrogen bond interactions of small polyols on the protein surface, which make the three-dimensional structure between protein molecules more compact, modify the interfacial tension, and then reduce the exposure of reactive sites and the possibility of irreversible aggregation events.

[0039] In one embodiment, the ratio (mM / mM) of the chelating agent that is a polyvalent anion to C3 polyol is about 1:5 to about 1:500, for example, about 1:20 to about 1:200. In one embodiment, the ratio (mM / mM) of the chelating agent that is a polyvalent anion to C3 polyol is about 1:5 to about 1:500, for example, about 1:20 to about 1:200.

[0040] In one embodiment, the ratio (wt / wt) of the antibody protein to the chelating agent that is a polyvalent anion is from about 1:1 to about 500:1, for example, from about 10:1 to about 200:1. In another embodiment, the antibody tan The ratio (wt / wt) of the protein to the chelating agent, which is a polyvalent anion, is from about 10:1 to about 1000:1, for example, about 5 0:1 to about 200:1.

[0041] In one embodiment, the ratio (wt / wt) of the antibody protein to the C3 polyol is from about 1:5 to about 200:1 , for example, from about 1:1 to about 50:1. In another embodiment, the ratio (wt / wt) of the antibody protein to the C3 polyol is from about 1:2 to about 200:1, for example, from about 2:1 to about 50:1.

[0042] The addition of a mixture of a chelating agent, which is a polyvalent anion, and a C3 polyol to an aqueous solution of the antibody protein is considered to enhance the stability of the antibody protein, as shown in Example 1, for example. Therefore, the mixture of the chelating agent, which is a polyvalent anion, and the C3 polyol is referred to as a stabilizing mixture.

[0043] The "stability" or "stabilizing mixture" of the antibody protein generally refers to a reduction in the degradation of the antibody protein during storage. In one embodiment, "stability" / "stabilization" refers to physical stability, for example , loss of quaternary, tertiary, or secondary structure, aggregation, or particle formation. In another embodiment , "stability" / "stabilization" refers to chemical stability, for example, processes involving covalent changes, such as deamidation, aspartic acid isomerization, oxidation, or hydrolytic clipping.

[0044] The addition of a mixture of a chelating agent, which is a polyvalent anion, and a C3 polyol to an aqueous solution containing the antibody protein enhances the stability of the antibody protein and, in particular, compared to the same solution lacking the chelating agent and the C3 polyol after storage for the same length of time under the same conditions. is considered to be able to reduce the rate of antibody protein aggregation.

[0045] Accordingly, the present invention provides a method for stabilizing an antibody protein in an aqueous solution against storage comprising adding to the solution (i) a chelating agent which is a polyvalent anion; and (ii) a mixture of C3 polyols. Also provided is the use of (i) a chelating agent which is a polyvalent anion; and (ii) a mixture of C3 polyols for stabilizing an antibody protein in an aqueous solution against storage. All of the embodiments described above herein in connection with the aqueous solution of the present invention apply equally to the methods and uses of the present invention.

[0046] The method of the present invention refers to "the step of adding to the solution (i) a chelating agent which is a polyvalent anion; and (ii) a mixture of C3 polyols". It should be understood that the chelating agent which is a polyvalent anion and the C3 polyols can be added to the solution simultaneously, or sequentially and in any order (i.e., the "step" can actually include a plurality of steps).

[0047] Also provided is a method for inhibiting the formation of high molecular weight species of an antibody protein in an aqueous solution during storage, comprising adding to the solution a mixture of a chelating agent which is a polyvalent anion and C3 polyols.

[0048] Also provided is a method for inhibiting the formation of visible particles of an antibody protein in an aqueous solution during storage, comprising adding to the solution a mixture of a chelating agent which is a polyvalent anion and C3 polyols.

[0049] ​​​​​​​​​​Also provided is a method for inhibiting the formation of related species of antibody proteins in an aqueous solution during storage, the method comprising adding to the solution a mixture of a chelating agent which is a polyvalent anion and a C3 polyol.

[0050] Also provided is a method for inhibiting the deamidation of antibody proteins in an aqueous solution during storage, the method comprising adding to the solution a mixture of a chelating agent which is a polyvalent anion and a C3 polyol.

[0051] Also provided is a method for inhibiting the formation of low molecular weight degradation products of antibody proteins in an aqueous solution during storage, the method comprising adding to the solution a mixture of a chelating agent which is a polyvalent anion and a C3 polyol.

[0052] Also provided is the use of a mixture of a chelating agent which is a polyvalent anion and a C3 polyol for inhibiting the formation of high molecular weight species of antibody proteins in an aqueous solution during storage.

[0053] Also provided is the use of a mixture of a chelating agent which is a polyvalent anion and a C3 polyol for inhibiting the formation of visible particles of antibody proteins in an aqueous solution during storage.

[0054] Also provided is the use of a mixture of a chelating agent which is a polyvalent anion and a C3 polyol for inhibiting the formation of related species of antibody proteins in an aqueous solution during storage.

[0055] Also provided is the use of a mixture of a chelating agent which is a polyvalent anion and a C3 polyol for inhibiting the deamidation of antibody proteins in an aqueous solution during storage.

[0056] ​​​​​​​ Also provided is the use of a mixture of a chelating agent, which is a polyvalent anion, and a C3 polyol to inhibit the formation of low molecular weight degradation products in an aqueous solution of an antibody protein during storage.

[0057] As used herein, the term "high molecular weight species" refers to any component of an antibody protein content having an apparent molecular weight of at least about twice that of the parent active antibody protein. That is, the high molecular weight species are multimeric aggregates of the parent antibody protein. The multimeric aggregates can contain parent antibody protein molecules with a considerably altered three-dimensional structure, or they can be an assembly of iterative or native-like three-dimensional structure parent protein units. The determination of high molecular weight species can be carried out using methods known in the art, including size exclusion chromatography, electrophoresis, analytical ultracentrifugation / sedimentation velocity, light scattering, dynamic light scattering, static light scattering, and field flow fractionation.

[0058] As used herein, the term "low molecular weight degradation product" refers to any component of an antibody protein content having an apparent molecular weight less than that of the parent active antibody protein. That is, the low molecular weight degradation products are fragments of the parent antibody protein. The determination of low molecular weight degradation products can be carried out using methods known in the art, including size exclusion chromatography, electrophoresis, analytical ultracentrifugation / sedimentation velocity, light scattering, dynamic light scattering, static light scattering, and field flow fractionation.

[0059] As used herein, the term "related species" refers to those resulting from chemical modification of the parent antibody protein. Refers to any component of the antibody protein content formed, e.g., deamidated species or oxidized species is. Related species are preferably detected by cation exchange chromatography, reverse phase chromatography, or capillary electrophoresis.

[0060] Preferably, the aqueous solution of the present invention is sufficiently stable such that it remains substantially free of visible particles after storage at 30 °C for at least 1, 2, or 3 months. Visible particles are preferably detected using 2.9.20. European Pharmacopoeia Monograph (Particulate Contamination: Visible Particles).

[0061] Preferably, the aqueous solution of the present invention is sufficiently stable such that the concentration of related species remains low upon long-term storage.

[0062] In one embodiment, the aqueous solution of the present invention, after storage at 30 °C for 1, 2, or 3 months, (by weight of the total antibody protein) retains at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99% of the parent antibody protein. The percentage of antibody protein (by weight of the total antibody protein) can be determined by size exclusion chromatography, cation exchange chromatography, reverse phase chromatography, or capillary electrophoresis.

[0063] In one embodiment, the presence of a mixture of a chelating agent that is a polyvalent anion and a C3 polyol results in an increase in high molecular weight antibody protein species (by weight of the total antibody protein) after storage at 40 °C for 1 month. ​​​​​Restrict it to 5% or less by weight, preferably 3% or less, more preferably 2% or less. In one embodiment In the presence of a mixture of a chelating agent which is a polyvalent anion and a C3 polyol, storage at 2-8 °C After storage for up to 2 years, the increase in high molecular weight antibody protein species (by weight of total antibody protein) Is restricted to 5% or less, preferably 3% or less, more preferably 2% or less. The determination of high molecular weight species Is as a weight percentage of total antibody protein in the aqueous solution.

[0064] In one embodiment, the presence of a mixture of a chelating agent which is a polyvalent anion and a C3 polyol After storage under the same conditions and for the same length of time, compared to an aqueous solution lacking the chelating agent which is a polyvalent anion and C3 poly All but otherwise identical, restricts the increase in high molecular weight antibody protein species by at least 10%, preferably at least 25%, more preferably at least 50%.

[0065] In one embodiment, the presence of a mixture of a chelating agent which is a polyvalent anion and a C3 polyol Maintains the aqueous solution of the antibody protein in a state free of visible aggregates, while under the same conditions After storage for the same length of time, in an aqueous solution lacking the chelating agent which is a polyvalent anion and the C3 polyol mixture but otherwise identical, the formation of visible aggregates is observed To occur. Quantification of visible aggregates can be performed by measurement of turbidity or other types of light scattering .

[0066] Preferably, the aqueous solution of the present invention contains 5% or less of high molecular weight species (by weight of total protein) after storage at 40 °C for at least 1, 2, or 3 months. In one embodiment, the amount of high molecular weight species After storage at 40 °C for at least 1, 2, or 3 months, it increases by 5% or less (by weight of the total antibody protein), preferably 3% or less. The quantification of the high molecular weight species is as a weight percentage of the total antibody protein in the aqueous solution. Preferably, the aqueous solution of the present invention lacks a mixture of a chelating agent that is a polyvalent anion and C3 polyol after storage under the same conditions and length of time, but is at least 10% lower, preferably at least 25% lower, more preferably at least 50% lower than an otherwise identical aqueous solution in terms of the increase in high molecular weight species during storage. In one embodiment, the aqueous solution of the present invention is a pharmaceutical composition suitable for administration of a therapeutic antibody protein to a subject in need thereof. Such a composition can be used in a method of administering the therapeutic protein to the subject.

[0067] Preferably, the aqueous solution of the present invention lacks a mixture of a chelating agent that is a polyvalent anion and C3 polyol after storage under the same conditions and length of time, but is at least 10% lower, preferably at least 25% lower, more preferably at least 50% lower than an otherwise identical aqueous solution in terms of the increase in high molecular weight species during storage. Preferably, the aqueous solution of the present invention lacks a mixture of a chelating agent that is a polyvalent anion and C3 polyol after storage under the same conditions and length of time, but is at least 10% lower, preferably at least 25% lower, more preferably at least 50% lower than an otherwise identical aqueous solution in terms of the increase in high molecular weight species during storage. Preferably, the aqueous solution of the present invention lacks a mixture of a chelating agent that is a polyvalent anion and C3 polyol after storage under the same conditions and length of time, but is at least 10% lower, preferably at least 25% lower, more preferably at least 50% lower than an otherwise identical aqueous solution in terms of the increase in high molecular weight species during storage. Preferably, the aqueous solution of the present invention lacks a mixture of a chelating agent that is a polyvalent anion and C3 polyol after storage under the same conditions and length of time, but is at least 10% lower, preferably at least 25% lower, more preferably at least 50% lower than an otherwise identical aqueous solution in terms of the increase in high molecular weight species during storage.

[0068] In one embodiment, the aqueous solution of the present invention is a pharmaceutical composition suitable for administration of a therapeutic antibody protein to a subject in need thereof. Such a composition can be used in a method of administering the therapeutic protein to the subject. In one embodiment, the aqueous solution of the present invention is a pharmaceutical composition suitable for administration of a therapeutic antibody protein to a subject in need thereof. Such a composition can be used in a method of administering the therapeutic protein to the subject. In one embodiment, the aqueous solution of the present invention is a pharmaceutical composition suitable for administration of a therapeutic antibody protein to a subject in need thereof. Such a composition can be used in a method of administering the therapeutic protein to the subject.

[0069] In another embodiment, the present invention provides a method of administering a therapeutic antibody protein to a subject in need thereof. The method includes the step of administering an aqueous solution containing the antibody protein, a chelating agent that is a polyvalent anion, and C3 polyol. Preferably, the composition is administered by intravenous, subcutaneous, or intramuscular injection or infusion. More preferably, the composition is administered by subcutaneous injection. In another embodiment, the present invention provides a method of administering a therapeutic antibody protein to a subject in need thereof. The method includes the step of administering an aqueous solution containing the antibody protein, a chelating agent that is a polyvalent anion, and C3 polyol. Preferably, the composition is administered by intravenous, subcutaneous, or intramuscular injection or infusion. More preferably, the composition is administered by subcutaneous injection. In another embodiment, the present invention provides a method of administering a therapeutic antibody protein to a subject in need thereof. The method includes the step of administering an aqueous solution containing the antibody protein, a chelating agent that is a polyvalent anion, and C3 polyol. Preferably, the composition is administered by intravenous, subcutaneous, or intramuscular injection or infusion. More preferably, the composition is administered by subcutaneous injection. In another embodiment, the present invention provides a method of administering a therapeutic antibody protein to a subject in need thereof. The method includes the step of administering an aqueous solution containing the antibody protein, a chelating agent that is a polyvalent anion, and C3 polyol. Preferably, the composition is administered by intravenous, subcutaneous, or intramuscular injection or infusion. More preferably, the composition is administered by subcutaneous injection. In another embodiment, the present invention provides a method of administering a therapeutic antibody protein to a subject in need thereof. The method includes the step of administering an aqueous solution containing the antibody protein, a chelating agent that is a polyvalent anion, and C3 polyol. Preferably, the composition is administered by intravenous, subcutaneous, or intramuscular injection or infusion. More preferably, the composition is administered by subcutaneous injection.

[0070] In another embodiment, the present invention provides a packaged pharmaceutical composition suitable for administration to a subject in need thereof. The pharmaceutical composition includes an aqueous solution containing the antibody protein, a chelating agent that is a polyvalent anion, and C3 polyol. The pharmaceutical composition preferably contains the solution In another embodiment, the present invention provides a packaged pharmaceutical composition suitable for administration to a subject in need thereof. The pharmaceutical composition includes an aqueous solution containing the antibody protein, a chelating agent that is a polyvalent anion, and C3 polyol. The pharmaceutical composition preferably contains the solution In another embodiment, the present invention provides a packaged pharmaceutical composition suitable for administration to a subject in need thereof. The pharmaceutical composition includes an aqueous solution containing the antibody protein, a chelating agent that is a polyvalent anion, and C3 polyol. The pharmaceutical composition preferably contains the solution It is packaged in a vial suitable for introducing a needle for extraction. In one embodiment, the medicament composition is packaged in a glass vial with a rubber stopper. The packaged pharmaceutical composition is provided as a kit further comprising instructions for use and optionally a syringe suitable for intramuscular or subcutaneous administration can be. Alternatively, the packaged pharmaceutical composition can be provided in the form of a disposable prefilled syringe suitable for intramuscular or subcutaneous administration. A prefilled autoinjector device would also be suitable for intramuscular or subcutaneous administration.

[0071] As used herein, the term "pharmaceutically acceptable" refers to components of a pharmaceutical composition that are without undue adverse effects, such as toxicity, irritation, and allergic response, and that are suitable for their intended use and mode of administration to the body of a human or animal, such as a mammal, with a reasonable risk / benefit ratio. (Abbreviations)

Table 1

Examples

[0072] (Example) (Materials) Disodium EDTA salt (Mw 372 Da), 1,2-propanediol (Mw 76 Da), glycerol (Mw 9 2 Da), mannitol (Mw 182 Da), NaCl (Mw 58 Da), trehalose (Mw 342 Da) were obtained from Sigma Aldrich chem.

[0073] (Method for evaluating the stability of antibody proteins) (a) Visual evaluation Visible particles were preferably counted according to 2.9.20. European Pharmacopoeia (Particulate contamination: Visible particles) Detect using ((ntamination: Visible Particles)). The required equipment consists of the following A viewing station including: · A matte black panel of appropriate size held in a vertical position · An anti-reflective white panel of appropriate size held in a vertical position next to the black panel · An adjustable lamp holder equipped with a suitable white light source with a hood and a suitable diffuser (each A viewing illuminator containing two 13W fluorescent tubes each 525mm long is preferred). The illumination intensity at the viewing point is maintained between 2000 lux and 3750 lux.

[0074] Remove any adhesive labels from the container, wash the outside, and dry it. Gently rotate or invert the container while ensuring no air bubbles enter, and observe in front of the white panel for about 5 seconds. Repeat this procedure in front of the black panel. Record the presence of any particles. Rank the visual scores as follows:

[0075] Visual score 1: A clear solution containing few particles Visual score 2: ~5 very small particles Visual score 3: ~10 to 20 very small particles Visual score 4: 20 - 50 particles including large particles Visual score 5: >50 particles including large particles

[0076] Particles in samples with visual scores 4 and 5 are clearly detectable by casual visual assessment under normal light, while samples with visual scores 1 - 3 usually appear as clear solutions in the same assessment. Samples with visual scores 1 - 3 are considered "qualified"; samples with visual scores 4 - 5 are considered "unqualified".

[0077] ​​​​ (b) Size Exclusion Chromatography (SEC) The amount of high molecular weight species is measured using a 300×7.8 mm S3000 (or equivalent) size exclusion column equipped with a guard column. The mobile phase is potassium phosphate pH 6.5, the flow rate is 0.4 ml / min and the injection volume is 1 μl, detected at 210 nm and 280 nm. The results are expressed as % high molecular species (HMWS), i.e., the sum of all peak areas corresponding to aggregated proteins relative to the sum of all protein-related peaks on the chromatogram is represented as. Regarding the absolute value of % HMWS, for example, slight variations between time points may be observed due to repeated use of the size exclusion column . However, within a given time point, samples are tested using columns under the same conditions and the values obtained within that time point are an excellent indicator of the relative stability of the proteins in the tested aqueous solution .

[0078] (b) Cation Exchange Chromatography (CEX) The amount of related species is measured using a Protein-Pak Hi Res SP column. Mobile phase A is 20 mM sodium phosphate (pH 6.5); mobile phase B is 20 mM sodium phosphate + 0.5 M NaCl (pH 6.0) . The following gradient elution is used: 0 min - 100% A, 4 min - 80% A, 10 min - 55% A, 12 min - 0% A . The flow rate is 1.0 ml / min; the injection volume is 3 μl, and UV detection is performed at 214 nm. The results are expressed as % main peak (i.e., native protein), % acidic species, and % basic species. % related species = % acidic species + % basic species.

[0079] (Example 1) The effects of EDTA and C3 polyols on the stability of abatacept (125 mg / ml) were investigated. This effect was tested in a background solution containing sodium phosphate (5 mM) and polysorbate 80 (0.5 mg / ml). All formulations tested were adjusted to pH 6.5. The additional excipients in the tested formulations are shown in Table 1. Table 1: Additional components in the tested abatacept formulations. All formulations contained abatacept (125 mg / ml), sodium phosphate (5 mM), and polysorbate 80 (0.5 mg / ml) and were adjusted to pH 6.5.

Table 2

[0080] The stability of Formulations 1 - 12 (Table 1) was tested at 25 °C and 40 °C by visual evaluation and size exclusion chromatography (SEC). The results are shown in Tables 2 and 3. In the absence of EDTA, the stability of abatacept was shown to be slightly better in the presence of uncharged osmotic regulators (trehalose or 1,2 - propanediol) than in the presence of charged osmotic regulator (NaCl). The addition of EDTA appeared to improve the stability of abatacept both with respect to visual evaluation and with respect to the formation of HMWS. The degree of improvement was greater in compositions containing uncharged species. Surprisingly, the degree of improvement was greater in compositions containing C3 polyol (1,2 - propanediol) than in compositions containing the larger polyol (trehalose). This indicates a synergistic effect between EDTA and 1,2 - propanediol. The highest level of EDTA tested (50 mM) resulted in the best stability with respect to high molecular weight species but a slightly worse visual score. It also seemed to be so. This may be due to the fact that more soluble aggregates (i.e., HMWS) are converted into insoluble aggregates in the presence of a high concentration of ED TA. Table 2: Visual scores of abatacept formulations 1 - 12 after storage at 25 °C and 40 °C. Visual score 1: Clear solution containing almost no particles; Visual score 2: ~5 very small particles; Visual score 3: ~10 to 20 very small particles; Visual score 4: 20 - 50 particles containing large particles ; Visual score 5: >50 particles containing large particles [Table 3] Table 3: Stability of abatacept (125 mg / ml) in formulations 1 - 12 evaluated by SEC. After storage at 25 °C and 4 0 °C, the formation of HMWS was evaluated. [Table 4]

[0081] (Example 2) The effects of EDTA and polyols on the stability of bevacizumab (25 mg / ml) were investigated at 40 °C. This effect was tested in a buffer solution containing sodium phosphate (5 mM) and polysorbate 20 (0.4 mg / ml). All formulations tested were adjusted to pH 6.2. Further excipients in the tested formulations are shown in Table 4. The stability was also compared with the composition of the currently marketed bevacizumab product (Avas tin (registered trademark)). Table 4: Further components in the tested abatacept formulations. All formulations contained bevacizumab (25 mg / ml) and polysorbate 20 (0.5 mg / ml) and were adjusted to pH 6.2. [Table 5]

[0082] The stability of Formulations 1-9 (Table 4) was tested by visual evaluation at 40°C. The results are shown in Table 5. Formulation 1 (i.e., the composition of Avastin®) resulted in a visual score of 5 after storage at 40°C for 10 weeks Similarly, a visual score of 5 was achieved with a composition containing either 5 mM sodium phosphate and a charged osmotic regulator (NaCl) or an uncharged osmotic regulator (mannitol, trehalose, or glycerol). A better visual score was observed in the presence of EDTA (10 or 50 mM). However, the use of EDTA in the presence of NaCl still resulted in a worse visual score than the use of EDTA in the presence of a C3 polyol (glycerol), indicating a synergistic effect between EDTA and the C3 polyol. Table 5: Visual scores of bevacizumab Formulations 1-9 after storage at 40°C. Visual score 1: a clear solution containing few particles; visual score 2: ~5 very small particles; visual score 3: ~10 to 20 very small particles; visual score 4: 20-50 particles containing large particles; visual score 5: >50 particles containing large particles. [Table 6]

[0083] Throughout this specification and the following claims, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" and variations such as thereof are to be construed as including the stated integer, step, group of integers or group of steps It should be understood that it does not mean the exclusion of any other integer, step, group of integers, or group of steps. will be understood.

[0084] Although the present invention has been shown and described with respect to its preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as encompassed by the appended claims. It should also be understood that the embodiments described herein are not mutually exclusive and that features from various embodiments may be combined in whole or in part in accordance with the present invention. It will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as encompassed by the appended claims. It should also be understood that the embodiments described herein are not mutually exclusive and that features from various embodiments may be combined in whole or in part in accordance with the present invention. It should also be understood that the embodiments described herein are not mutually exclusive and that features from various embodiments may be combined in whole or in part in accordance with the present invention. It should also be understood that the embodiments described herein are not mutually exclusive and that features from various embodiments may be combined in whole or in part in accordance with the present invention.

[0085] All publications, patents, patent applications, Internet sites, and accession numbers / database sequences (including both polynucleotide and polypeptide sequences) cited are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, Internet site, and accession number / database sequence were specifically and individually indicated to be incorporated by reference in this manner. / database sequences (including both polynucleotide and polypeptide sequences) are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, Internet site, and accession number / database sequence were specifically and individually indicated to be incorporated by reference in this manner. All publications, patents, patent applications, Internet sites, and accession numbers / database sequences (including both polynucleotide and polypeptide sequences) cited are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, Internet site, and accession number / database sequence were specifically and individually indicated to be incorporated by reference in this manner. All publications, patents, patent applications, Internet sites, and accession numbers / database sequences (including both polynucleotide and polypeptide sequences) cited are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, Internet site, and accession number / database sequence were specifically and individually indicated to be incorporated by reference in this manner. All publications, patents, patent applications, Internet sites, and accession numbers / database sequences (including both polynucleotide and polypeptide sequences) cited are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, Internet site, and accession number / database sequence were specifically and individually indicated to be incorporated by reference in this manner. are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, Internet site, and accession number / database sequence were specifically and individually indicated to be incorporated by reference in this manner.

Claims

1. Stabilization of an antibody protein and (i) a chelating agent that is a polyanion; and (ii) a C3 polyol. and a mixture thereof.

2. 1. A method for stabilizing an antibody protein in an aqueous solution against storage, comprising: (ii) adding a mixture of a chelating agent which is a polyanion; and (iii) a C3 polyol. The method.

3. (i) a polyanion for stabilizing antibody proteins in aqueous solutions against storage; and (ii) the use of a mixture of C3 polyols.

4. 2. The aqueous solution of claim 1, wherein the chelating agent is a chelating ion having four ionic centers.

4. The solution, the method of claim 2, or the use of claim 3.

5. The water-soluble polymer according to any one of claims 1 to 4, wherein the polyanion chelating agent is EDTA. Solution, method, or use.

6. The polyanion chelating agent is about 0.1 mM to about 50 mM, for example, about 0.1 mM to about 20 mM, The aqueous solution of any one of claims 1 to 5, for example, present at a concentration of about 0.1 mM to about 10 mM. , method, or use.

7. The water-soluble polyol according to any one of claims 1 to 6, wherein the C3 polyol is 1,2-propanediol. Solution, method, or use.

8. 7. The aqueous solution of claim 1, wherein the C3 polyol is glycerol. Method or use.

9. Claims 1 to 6, wherein the C3 polyol is a mixture of 1,2-propanediol and glycerol.

2. The aqueous solution, method or use according to any one of claims 1 to 11.

10. The C3 polyol is about 100 mM to about 500 mM, for example, about 150 mM to about 400 mM, or about 150 mM to about 10. The aqueous solution, method or use of any one of claims 1 to 9, wherein the aqueous solution, method or use is present in a concentration of about 300 mM. 。

11. The antibody protein according to any one of claims 1 to 10, wherein the antibody protein is a therapeutic antibody protein. Aqueous solutions, methods, or uses of.

12. The antibody protein may be an antibody, an antibody fragment, an antibody conjugated to an active moiety, or one or more A fusion protein comprising the above antibody fragment, or a derivative of any of the foregoing.

12. The aqueous solution, method or use according to any one of 1 to 11.

13. 13. The method of claim 12, wherein the antibody protein is a monoclonal antibody. is used.

14. The monoclonal antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody.

14. The aqueous solution, method or use of claim 13.

15. The monoclonal antibody is selected from the group consisting of trastuzumab, rituximab, bevacizumab, and cetaxel.

14. The aqueous solution, method or use of claim 13, wherein the anti-inflammatory agent is selected from the group consisting of mab and ipilimumab.

16. 16. The aqueous solution or method of claim 15, wherein the monoclonal antibody is bevacizumab. use.

17. The antibody protein comprises an active protein domain fused to one or more immunoglobulin Fc fragments.

13. The aqueous solution, method or use of claim 12, wherein the antibody is a fusion protein comprising an amino acid sequence similar to that of claim 12.

18. The antibody protein is etanercept, abatacept, or belatacept.

13. The aqueous solution, method or use according to claim 12.

19. The derivatives include one or more antibodies or antibody fragments and a chemically inert polymer.

13. The aqueous solution, method or use of claim 12, which is a condensed derivative.

20. 20. The method of claim 19, wherein the conjugated derivative is certolizumab pegol. Solution, method, or use.

21. The antibody protein is at about 1 mg / mL to about 300 mg / mL, for example, at about 10 mg / mL to about 300 mg / mL, 21. The method of claim 1, wherein the composition is present in a concentration of from about 10 mg / mL to about 200 mg / mL, or from about 10 mg / mL to about 200 mg / mL.

2. The aqueous solution, method or use according to any one of claims 1 to 11.

22. The pH of the solution is from about pH 4.0 to about pH 8.0, for example, from about pH 5.0 to about pH 7.0 or from about pH 5.0 to about 22. The aqueous solution, method or use of any one of claims 1 to 21, having a pH of 6.

5.

23. 23. The aqueous solution, method or use of any one of claims 1 to 22, further comprising a buffering agent.

24. The buffering agent is selected from the group consisting of histidine, succinate, maleate, acetate, phosphate, and TRIS.

24. The aqueous solution, method or use of claim 23, selected from the group consisting of:

25. The buffering agent has a concentration of about 0.5 mM to about 50 mM, for example, about 1 mM to about 20 mM, for example, about 2 mM to about 5 mM.

25. The aqueous solution, method or use of claim 23 or claim 24, wherein the aqueous solution, method or use is present at 100% by mass.

26. The aqueous solution according to any one of claims 1 to 25, further comprising a non-ionic surfactant. law or use.

27. The nonionic surfactant is an alkyl glycoside, for example, dodecyl maltoside.

27. The aqueous solution, method or use of claim 26.

28. The non-ionic surfactant is a polysorbate surfactant, for example, polysorbate 80. or polysorbate 20.

29. The nonionic surfactant is an alkyl ether of polyethylene glycol. Item 27. The aqueous solution, method or use according to item 26.

30. The alkyl ether of polyethylene glycol is polyethylene glycol (2) dodecyl ether. Polyethylene glycol (2) oleyl ether, polyethylene glycol (2) oleyl ether, and polyethylene glycol 30. The aqueous solution, method or use of claim 29, wherein the aryl ether is selected from the group consisting of aryl (2) hexadecyl ether and aryl (2) hexadecyl ether. For.

31. The nonionic surfactant is a mixture of polyethylene glycol and polypropylene glycol. Block copolymers, such as poloxamer 188, poloxamer 407, poloxamer 171, or poloxamer 185.

32. The nonionic surfactant is an alkyl phenyl ether of polyethylene glycol, For example, 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol.

27. The aqueous solution, method or use of claim 26.

33. The nonionic surfactant is from about 10 μg / mL to about 2000 μg / mL, for example, from about 50 μg / mL to about 10 33. Any of claims 26 to 32, wherein the α-aminobutyric acid is present in a concentration of about 100 μg / mL, for example about 100 μg / mL to about 500 μg / mL.

2. The aqueous solution, method or use according to claim 1.

34. Uncharged osmotic modifiers, such as sucrose, trehalose, mannitol, sorbitol 34. The aqueous solution according to any one of claims 1 to 33, further comprising PEG, PEG300, or PEG400. Method or use.

35. The non-charged osmotic agent is at about 50 mM to about 1000 mM, for example, at about 100 mM to about 500 mM, for example 35. The aqueous solution, method or use of claim 34, wherein the aqueous solution, method or use is present in a concentration of about 300 mM.

36. For example, sodium chloride, sodium sulfate, sodium acetate, sodium lactate, glycerol, and a charged osmolality adjusting agent selected from the group consisting of arginine, histidine, and arginine.

36. The aqueous solution, method or use of any one of claims 1 to 35, comprising:

37. The charged osmotic modifier is at about 25 mM to about 500 mM, for example, at about 50 mM to about 250 mM, for example, at about 37. The aqueous solution, method or use of claim 36, which is present in a concentration of 150 mM.

38. 38. The aqueous solution of claim 1, wherein the aqueous solution is isotonic. use.

39. 39. The aqueous solution, method or use of any one of claims 1 to 38, further comprising a preservative.

40. The preservative is phenol, m-cresol, chlorocresol, benzyl alcohol, Propylparaben, methylparaben, benzalkonium chloride, and benzethonium chloride 40. The aqueous solution, method or use of claim 39, selected from the group consisting of:

41. 41. The aqueous solution of claim 39 or claim 40, wherein the preservative is present at a concentration of about 0.01 mM to about 100 mM. Solutions, methods, or uses.

42. The method of stabilizing the antibody protein prevents the formation of high molecular weight species of the antibody protein upon storage. The method according to any one of claims 2 or 4 to 41, which is a method for inhibiting

43. The method of stabilizing the antibody protein prevents the formation of related species of the antibody protein upon storage. The method according to any one of claims 2 or 4 to 41, which is a method for inhibiting

44. The method for stabilizing an antibody protein inhibits deamidation of the antibody protein during storage. The method according to any one of claims 2 or 4 to 41,

45. The method for stabilizing the antibody protein comprises the step of: preventing low molecular weight degradation products in the aqueous solution during storage; The method of any one of claims 2 or 4 to 41, which is a method for inhibiting formation.

46. The method for stabilizing the antibody protein includes preventing the antibody protein from becoming visible in a composition during storage.

42. The method of claim 2 or any one of claims 4 to 41, which is a method for inhibiting particle formation.

47. Any of claims 3 to 41 for inhibiting the formation of high molecular weight species of said antibody protein upon storage.

2. Use according to any one of the preceding claims.

48. Any of claims 3 to 41 for inhibiting the formation of related species of said antibody protein during storage. or the use as described in any one of claims 1 to 4.

49. Any of claims 3 to 41 for inhibiting deamidation of the antibody protein during storage.

10. The use according to claim 1.

50. Claims 3 to 4 for inhibiting the formation of low molecular weight degradation products of said antibody protein during storage.

2. The use according to any one of claims 1 to 11.

51. To inhibit the formation of visible particles in compositions of said antibody protein upon storage, 3. The use according to any one of claims 3 to 41.

52. The solution is administered by subcutaneous or intramuscular injection or by intravenous injection or infusion.

42. The aqueous solution of claim 1 or any one of claims 4 to 41, for administration by.

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