formulation

Abstract

FORMULATION The present invention relates to the field of pharmaceutical formulations. More particularly, it relates to a liquid formulation comprising an anti-TG2 antibody and a method for producing such a formulation. The liquid formulation according to the present invention is stable when stored at a temperature of about 2-25° C. for an appropriate period of time.

Description

[Technical field]

[0001] The present invention relates to the field of pharmaceutical formulations. More particularly, it relates to liquid formulations comprising anti-TG2 antibodies and methods for producing such formulations. The liquid formulations according to the present invention are stable when stored at a temperature of about 2-25° C. for an appropriate period of time. [Background technology]

[0002] Tissue transglutaminase (TG2) is an enzyme that forms crosslinks between proteins via epsilon (gamma-glutamyl) lysine crosslinks. High expression of TG2 leads to abnormal protein crosslinks and is associated with several pathologies, such as various types of tissue scarring, formation of neurofibrillary tangles in some brain diseases, and resistance to chemotherapy in some cancers. Various TG2 inhibitors, such as small molecules, silencing RNAs, or antibodies (e.g., US Pat. No. 5,399,411, US Pat. No. 5,499,421 or US Pat. No. 5,499,431) have been disclosed for the potential treatment of TG2-mediated disorders. Antibodies against TG2 have been described in the literature, but no stable formulations have been proposed so far. When preparing a pharmaceutical composition containing a bioactive protein such as an antibody, the composition must be formulated in such a way that the protein is stable for an appropriate period of time. A decrease in protein activity / stability can result from chemical or physical instability of the protein, especially due to denaturation, aggregation, oxidation. Therefore, the resulting product may not be pharma- ceutical acceptable. The use of excipients is known to increase the stability of a given protein, but the stabilizing effect of these excipients depends largely on the nature of the excipient and the bioactive protein itself. There remains a need for liquid formulations containing an anti-TG2 antibody as an active ingredient, which formulations are stable for a suitable period of time and are suitable for use in injections, such as intravenous or subcutaneous injections, that would be useful for administration in the treatment of diseases or disorders mediated by TG2. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2006 / 100679 [Patent Document 2] International Publication No. 2012 / 146901 [Patent Document 3] International Publication No. 2013 / 175229 Summary of the Invention [Problem to be solved by the invention]

[0004] The object of the present invention is to provide a novel formulation comprising an anti-TG2 antibody. More specifically, the formulation is a stable liquid formulation comprising an anti-TG2 antibody. The present invention also provides a method for preparing the liquid formulation of the present invention. The liquid formulations described herein are useful for administration in the treatment of diseases or disorders mediated by TG2. [Means for solving the problem]

[0005] In a first aspect, the present invention provides a stable liquid formulation comprising or consisting of an anti-TG2 antibody, a buffer that maintains a pH of 5.0 or about 5.0-7.0 or about 7.0, a stabilizer (such as an amino acid or a salt), and optionally a polysorbate surfactant. In a preferred embodiment, the buffer is a histidine or citrate buffer, and the stabilizer is either an amino acid, preferably glycine, or a salt, preferably NaCl. In a further preferred embodiment, the buffer maintains a pH of 5.5 or about 5.5-6.5 or about 6.5. In a further preferred embodiment, the anti-TG2 antibody is in an amount of 10 or about 10 mg / mL to 200 or about 200 mg / mL. Preferably, the anti-TG2 antibody comprises a light chain variable region defined by SEQ ID NO:1 and a heavy chain variable region defined by SEQ ID NO:2.

[0006] In a second aspect, the present invention provides a method for producing a stable liquid formulation, comprising forming a mixture of an anti-TG2 antibody with a buffer, a stabilizer (such as an amino acid or a salt), and optionally a polysorbate surfactant. In a preferred embodiment, the buffer is a histidine or citrate buffer and the stabilizer is either an amino acid, preferably glycine, or a salt, preferably NaCl. In a preferred embodiment, the buffer maintains a pH of 5.0 or about 5.0-7.0 or about 7.0, more particularly 5.5 or about 5.5-6.5 or 6.5. Preferably, the anti-TG2 antibody comprises a light chain variable region as defined in SEQ ID NO:1 and a heavy chain variable region as defined in SEQ ID NO:2.

[0007] In a third aspect, there is provided an article of pharmaceutical or veterinary manufacture comprising a container containing a stable liquid formulation according to the invention.

[0008] In a fourth aspect, the present invention provides a stable liquid formulation according to the invention for use in therapy.

[0009] In a fifth aspect, the present invention provides a method of treating a disease or disorder by administering a stable liquid formulation according to the present invention.

[0010] Definition: - the term "about" means approximately or nearly, and in the context of numerical values ​​described herein, preferably means ±10% around the recited or claimed numerical value. - When a range of values ​​is stated or claimed, the range is intended to include the stated values.

[0011] - As used herein, the term "anti-TG2 antibody" intends an antibody molecule that binds to tissue transglutaminase (TG2) protein, an enzyme that forms crosslinks between proteins via epsilon (gamma-glutamyl) lysine bridges. Examples of such antibodies are described in WO 2013 / 175229. Without being limited thereto, an anti-TG2 antibody that can be used according to the present invention comprises, for example, a light chain variable region as defined in SEQ ID NO:1 and a heavy chain variable region as defined in SEQ ID NO:2.

[0012] - The term "antibody" as used herein includes, but is not limited to, monoclonal antibodies, polyclonal antibodies and recombinant antibodies produced by recombinant techniques as known in the art. "Antibody" includes antibodies of any species, in particular mammalian antibodies, such as human antibodies of any isotype, such as IgG1, IgG2a, IgG2b, IgG3, IgG4, IgE, IgD and antibodies produced as dimers of these basic structures, including pentamers such as IgGA1, IgGA2 or IgM, as well as modified variants thereof; non-human primate antibodies, such as antibodies from chimpanzees, baboons, rhesus monkeys or cynomolgus monkeys; rodent antibodies, such as antibodies from mice or rats; rabbit or goat or horse antibodies; camelid antibodies (e.g. antibodies from camels or llamas such as Nanobodies®) and derivatives thereof; antibodies of avian species, such as chicken antibodies; or antibodies of fish species, such as shark antibodies. The term "antibody" also refers to a "chimeric" antibody in which a first portion of at least one heavy and / or light chain antibody sequence is from a first species and a second portion of said heavy and / or light chain antibody sequence is from a second species. Chimeric antibodies of interest herein include "primatized" antibodies that contain variable domain antigen-binding sequences from a non-human primate (e.g., an Old World monkey such as a baboon, rhesus monkey, or cynomolgus monkey) and human constant region sequences. A "humanized" antibody is a chimeric antibody that contains sequences from a non-human antibody. In most cases, a humanized antibody is a human antibody (recipient antibody) in which residues from the recipient's hypervariable regions are replaced by residues from the hypervariable regions [or complementarity determining regions (CDRs)] of a non-human species (donor antibody) such as mouse, rat, rabbit, chicken, non-human primate, etc., to provide the desired specificity, affinity, and activity. In most cases, residues of the human (recipient) antibody outside the CDRs, i.e., in the framework regions (FR), are further replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody.These modifications are made to further refine the properties of the antibody. Humanization reduces the immunogenicity of non-human antibodies in humans, facilitating the application of antibodies in the treatment of human diseases. Humanized antibodies and techniques for their production are well known in the art. The term "antibody" also refers to human antibodies, which can be generated as an alternative to humanization. For example, it is possible to generate transgenic animals (e.g. mice) that are capable of producing a full repertoire of human antibodies upon immunization in the absence of endogenous mouse antibody production. Other methods for obtaining human antibodies / antibody fragments in vitro are based on display technologies, such as phage display and ribosome display technology, using recombinant DNA libraries that are at least partially artificially generated or generated from a donor immunoglobulin variable (V) domain gene repertoire. Phage and ribosome display technologies for generating human antibodies are well known in the art. Human antibodies can also be generated from isolated human B cells ex vivo immunized with an antigen of interest, which can then be fused to generate hybridomas and screened for optimal human antibodies. The term "antibody" refers to both glycosylated and aglycosylated antibodies. Furthermore, as used herein, the term "antibody" refers not only to full-length antibodies, but also to antibody fragments, more particularly antigen-binding fragments thereof. Antibody fragments contain at least one heavy or light chain immunoglobulin domain and bind to one or more antigens, as known in the art. Examples of antibody fragments according to the present invention include Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, Fab-Fv-Fv, scFv and Bis-scFv fragments. The fragments may also be single domain antibodies (dAbs) such as diabodies, tribodies, triabodies, tetrabodies, minibodies, sdAbs, VL, VH, VHH or camelid antibodies (e.g. from camel or llama such as Nanobodies®) and VNAR fragments.An antigen-binding fragment according to the invention may also comprise a Fab linked to one or two scFvs or dsscFvs, each scFv or dsscFv binding the same or different targets (e.g. one scFv or dsscFv that binds a therapeutic target and one scFv or dsscFv that increases half-life, e.g. by binding albumin). Examples of such antibody fragments include FabdsscFv (also called BYbe®) or Fab-(dsscFv)2 (also called TrYbe®, see e.g. WO 2015 / 197772). The antibody molecules as defined above, including antigen-binding fragments thereof, are known in the art.

[0013] - The term "stability" as used herein refers to the physical, chemical and conformational stability (including maintenance of biological potency) of an anti-TG2 antibody formulation according to the present invention. Instability of said antibody formulation may be caused by chemical degradation or aggregation of the antibody to form higher order polymers, deglycosylation, modified glycosylation, oxidation or other structural modification that reduces at least one biological activity of the antibody.

[0014] - The term "stable formulation" refers to a formulation in which the protein of interest (here, an anti-TG2 antibody) essentially retains its physical, chemical and biological properties upon storage. To measure the stability of an antibody in a formulation, various analytical methods are within the knowledge of the skilled artisan (see some examples in the Examples section). Stability is typically assessed at a selected temperature (e.g., -70°C, 2-8°C, 25°C, 35°C or higher) and for a selected period of time (e.g., 3 months, 6 months, 12 months or more). Since an antibody, once formulated, is typically stored in a refrigerator (typically 2-8°C) or at room temperature (typically 15-25°C) before being administered to a patient, it is important that the formulated antibody is stable over time at a temperature range of at least 2-25°C, e.g., 2-8°C and 25°C, as indicated herein. To conclude about the stability over a given period of time, various values ​​can be used, for example but not limited to: 1) less than 90% of the antibody is in monomeric form, 2) the change in the monomeric form of the antibody is less than 10% (compared to the initial data), 3) the amount of high molecular weight species (HMW or HMWS; also referred to herein as aggregates) is less than 5%, or 4) the pH fluctuates less than ±0.2 units (compared to the initial data).

[0015] - The term "stabilizing agent", "stabilizer" or "isotonicity agent" as used herein is a compound that is physiologically acceptable and confers suitable stability / tonicity to the formulation. It significantly prevents the net flow of water through cell membranes in contact with the formulation. Compounds such as glycerin are often used for such purposes. Other suitable stabilizers include, but are not limited to, amino acids or proteins (e.g., glycine or albumin), salts (e.g., sodium chloride), and sugars (e.g., dextrose, mannitol, sucrose and lactose).

[0016] - The term "buffer" as used herein refers to a solution of a compound known to be safe in a pharmaceutical or veterinary formulation and effective to maintain or control the pH of the formulation in the pH range desired for the formulation. Acceptable buffers for controlling pH at moderately acidic to moderately basic pH include, but are not limited to, phosphate, acetate, citrate, arginine, histidine and TRIS (2-amino-2-hydroxymethyl-1,3,-propanediol, the term including pharma- ceutically acceptable salts thereof) buffers.

[0017] - The term "surfactant" as used herein refers to a soluble compound that can be used specifically to increase the water solubility of hydrophobic, oily substances or to increase the miscibility of two substances with different hydrophobicity. For this reason, these polymers are often used in industrial applications, cosmetics and pharmaceuticals. They are also used as model systems for drug delivery applications, especially to alter the absorption of drugs and their delivery to target tissues. Well-known surfactants include polysorbates (polyoxyethylene derivatives; Tweens) and poloxamers (i.e. copolymers based on ethylene oxide and propylene oxide, also known as Pluronics®).

[0018] - The term "vial" or "container" as used herein refers broadly to a reservoir suitable for holding an anti-TG2 antibody formulation in liquid form. Examples of vials that can be used in the present invention include ampoules, tubes, bottles, syringes (such as pre-filled syringes), cartridges, and other reservoirs suitable for delivering an anti-TG2 antibody formulation to a patient via injection, preferably intravenous or subcutaneous injection.

[0019] - The term "solvent" as used herein refers to an aqueous or non-aqueous liquid solvent. The choice of solvent depends inter alia on the solubility of the drug compound in the solvent and the mode of administration. Aqueous solvents may consist of water alone or water and one or more miscible solvents, and may also contain dissolved solutes such as sugars, buffers, salts, and other excipients. The more commonly used non-aqueous solvents are short-chain organic alcohols such as methanol, ethanol, propanol, short-chain ketones such as acetone, and polyhydric alcohols such as glycerol. According to the present invention, the preferred solvents are aqueous solvents such as water or saline.

[0020] - The term "treating" or "treatment" of a disease state includes: (i) inhibiting the disease state, i.e., preventing the onset of the disease state or its clinical symptoms, or (ii) palliating the disease state, i.e., causing the temporary or permanent regression of the disease state or its clinical symptoms.

[0021] - The terms "preventing" or "prevention" of a disease state include keeping clinical symptoms of the disease state from developing in a subject who may be exposed to or susceptible to the disease state, but who has not yet experienced or exhibited symptoms of the disease state. In all embodiments of the present invention, the "pharmaceutical composition" may also be called, without any distinction, a "stable pharmaceutical composition".

[0022] Detailed description of the invention: The present invention is based on the combination of a stabilizer selected from the group consisting of glycine or NaCl with a buffer such as histidine or citrate buffer that maintains a pH of 5.0-7.0, which allows the preparation of a pharmaceutical composition suitable for human use of an anti-TG2 antibody without affecting the processability of the pharmaceutical composition and the long-term stability of the antibody. The present inventors have found that the pharmaceutical composition according to the present invention is stable over time, particularly when stored at about 2-25°C, as shown in the Examples section at 2-8°C and 25°C.

[0023] The main object of the present invention is a stable liquid formulation comprising or consisting of an anti-TG2 antibody, a buffer that maintains a pH of about 5.0 to about 7.0, and a stabilizer. In a preferred embodiment, the buffer is a histidine buffer or a citrate buffer, and the stabilizer is selected from the group consisting of glycine or NaCl. Optionally, the formulation may further comprise a surfactant, such as a polysorbate surfactant.

[0024] The invention further provides a method for producing any of the stable liquid formulations of anti-TG2 antibody described herein, comprising combining the anti-TG2 antibody with a buffer, a stabilizer, and optionally a surfactant, such as a polysorbate surfactant. Said step is typically carried out by buffer exchange according to conventional procedures. As an example, to prepare a suitable stable formulation, a predetermined amount of anti-TG2 antibody is buffer exchanged with 1) a citrate or histidine buffer that maintains a pH of 5.0-7.0 or about 5.0-7.0, and 2) a stabilizer (preferably selected from the group consisting of glycine or NaCl). If the formulation contains a surfactant, it is preferably added after the buffer exchange step. After buffer exchange, the formulation is filtered (final filtration). Depending on the target concentration of the antibody, the formulation can be concentrated between the steps of buffer exchange and final filtration. Each of these compounds (i.e., anti-TG2 antibody, buffer, stabilizer, and optionally, surfactant) can be used according to the concentrations, pH, and / or ratios described herein. The resulting mixture is dispensed into vials. Variations on this process will be apparent to those skilled in the art.

[0025] The invention also provides a pharmaceutical or veterinary article of manufacture comprising a container containing any of the stable liquid formulations described herein, the formulation comprising or consisting of an anti-TG2 antibody, a buffer, a stabilizer, and optionally a surfactant. Each of these components (i.e., the anti-TG2 antibody, the buffer, the stabilizer, and optionally a surfactant) may be used according to the concentrations, pH, and / or ratios described herein.

[0026] Also described is packaging that provides instructions for use. Preferably, the anti-TG2 antibody used according to the present invention comprises in its entirety (see also Table A): 1) a light chain variable domain having the sequence defined in SEQ ID NO:1 and a heavy chain variable domain having the sequence defined in SEQ ID NO:2; 2) a light chain variable domain having at least 80% identity or similarity, preferably 90% identity or similarity, to the sequence defined in SEQ ID NO:1, and a heavy chain variable domain having at least 80% identity or similarity, preferably 90% identity or similarity, to the sequence defined in SEQ ID NO:2; 3) a light chain having the sequence defined in SEQ ID NO:3 and a heavy chain having the sequence defined in SEQ ID NO:4; or 4) A light chain having at least 80% identity or similarity, preferably 90% identity or similarity, to the sequence defined in SEQ ID NO:3, and a heavy chain having at least 80% identity or similarity, preferably 90% identity or similarity, to the sequence defined in SEQ ID NO:4.

[0027] [Table A]

[0028] In the context of the present invention as a whole, the amount of anti-TG2 antibody in the formulation is preferably 10 or about 10 to 200 or about 200 mg / mL, preferably 30 or about 30 mg / mL to 180 or about 180 mg / mL, or preferably 50 or about 50 mg / mL to 150 or about 150 mg / mL, such as 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 or 150 mg / mL. Alternatively, the anti-TG2 antibody is present in the protein formulation in an amount expressed as % by weight (% w / v) per 100 mL. In such cases, the anti-TG2 antibody contained in the formulation of the present invention as a whole is present in an amount of about 1 to 20 or about 20% w / v, preferably about 3 to 18 or about 18% w / v, or preferably about 5 to 15 or about 15% w / v, for example, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, or 15.0% w / v.

[0029] The anti-TG2 antibody may, for example, comprise (but is not limited to) a light chain variable region defined in SEQ ID NO:1 and a heavy chain variable region defined in SEQ ID NO:2.

[0030] The preferred buffer solution according to the present invention is a histidine (preferably L-histidine) or citrate buffer solution as a whole, and is maintained at a pH of about 5.0 to about 7.0, preferably about 5.2 to about 6.0, for example, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, and 6.0 in the case of histidine buffer solution, preferably about 6.2 to about 7.0, for example, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 in the case of citrate buffer solution. More preferably, the pH is 5.5 or about 5.5 in the case of histidine buffer solution, and 6.5 or about 6.5 in the case of citrate buffer solution. In all embodiments of the invention, unless otherwise indicated, pH values ​​are measured at room temperature and are preferably within ±0.1 or ±0.2 of the target pH unit (e.g., 5.5±0.1 or 5.5±0.2 for histidine buffers, and 6.5 or about 6.5±0.1 or 6.5 or about 6.5±0.2 for citrate buffers).

[0031] In the context of the present invention as a whole, the buffer concentration is preferably 10 or about 10 to 100 or about 100 mM. In a preferred embodiment, the buffer concentration is 20 or about 20 to 80 or about 80, more preferably about 40 to about 60 mM, for example 40, 45, 50, 55 or 60 mM. Preferably, the buffer concentration is 50 or about 50 mM.

[0032] In the context of the present invention as a whole, the stabilizer is selected from the group consisting of glycine (preferably L-glycine) or NaCl. When the stabilizer is glycine, its concentration is preferably 150 or about 150 mM to 350 or about 350 mM, preferably 200 or about 200 to 300 or about 300 mM, more preferably 220 or about 220 to 280 or about 280 mM, for example 220, 230, 240, 250, 260, 270 or 280 mM. When the stabilizer is NaCl, the concentration is preferably 100 or about 100 mM to 200 or about 200 mM, more preferably 125 or about 125 to 175 or about 175 mM, for example, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170 and 175 mM.

[0033] In the context of the overall invention, a surfactant may optionally be present. If present, it is preferably a polysorbate surfactant, such as polysorbate 20 (PS20, also known as Tween® 20) or polysorbate 80 (PS80, also known as Tween® 80). Preferably, said surfactant is present in the formulation in an amount of 0.01 or about 0.01 to 5 or about 5 mg / mL, more preferably 0.01 or about 0.01 to 1 or about 1 mg / mL, more particularly 0.1 or about 0.1 to 0.6 or about 0.6 mg / mL, such as 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55 or 0.6 mg / mL. Alternatively, said polysorbate surfactant is preferably present in the protein formulation in an amount expressed as % by weight (% w / v) per 100 mL. In such cases, the polysorbate surfactant contained in the formulation of the present invention as a whole is 0.001 to 0.5% w / v, preferably 0.01 to 0.1% w / v, more preferably 0.01 to 0.06% w / v, for example, 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.045, 0.05, 0.055 or 0.06% w / v.

[0034] In a preferred embodiment, the stable liquid formulation of the present invention comprises or consists of, as a whole, 50 or about 50 to 200 or about 200 mg / mL of an anti-TG2 antibody, about 10 to about 100 mM histidine at a pH of about 5.5 (or about 10 to about 100 mM citrate buffer at a pH of about 6.5), about 150 to about 350 mM glycine or about 100 to about 200 mM NaCl, and, optionally, about 0.001 mg / mL to about 0.5 mg / mL of a surfactant (such as a polysorbate surfactant). Alternatively, the stable liquid formulation comprises or consists of about 5.0 to about 20% w / v anti-TG2 antibody, about 10 to about 100 mM histidine at pH about 5.5 (or about 10 to about 100 mM citrate buffer at pH about 6.5), about 150 to about 350 mM glycine or about 100 to about 200 mM NaCl, and optionally, 0.001 to 0.5% w / v surfactant (polysorbate surfactant, etc.). As a specific example (but not limited to these), a stable liquid formulation is provided that comprises or consists of about 100 mg / mL anti-TG2 antibody, about 50 mM histidine buffer maintaining a pH of about 5.5, about 250 mM glycine, and optionally, about 0.02 to 0.06% w / v PS80. As a further specific example (but not limited to), a stable liquid formulation is provided that comprises or consists of about 100 mg / mL of an anti-TG2 antibody, about 50 mM histidine buffer maintaining a pH of 5.5 or about 5.5, about 150 mM NaCl, and optionally about 0.02-0.06% w / v PS80. As a further example (but not limited to), a stable liquid formulation is provided that comprises or consists of about 100 mg / mL of an anti-TG2 antibody, about 50 mM citrate buffer maintaining a pH of about 5.5, about 250 mM glycine, and optionally about 0.02-0.06% w / v PS80. The anti-TG2 antibody may, for example, comprise a light chain variable region defined by SEQ ID NO:1 and a heavy chain variable region defined by SEQ ID NO:2.

[0035] Preferably, the formulations of the invention retain at least 80% of the biological activity of the anti-TG2 antibody at the time of formulation and / or packaging for a period of at least 12 months, preferably at least 24 months, more preferably at least 36 months, and even more preferably at least 48 months (prior to first use). Anti-TG2 antibody activity can be measured according to routine methods such as Elisa or cell-based assays.

[0036] Additional excipients for use within pharmaceutical compositions of the present invention include, but are not limited to, viscosity enhancing agents, bulking agents, solubilizing agents, or combinations thereof.

[0037] The present invention also provides a container comprising a pharmaceutical composition according to the present invention. In particular, the container may be, without limitation, a vial, ampoule, tube, bottle or syringe (such as a pre-filled syringe) containing the pharmaceutical composition.

[0038] The container may be part of a kit-of-parts comprising one or more containers containing a pharmaceutical composition of the invention, a delivery device such as a syringe, prefilled syringe, auto-injector, needle-free device, implant or patch, or other parental administration device, and instructions for use.

[0039] The liquid formulation of the present invention can be stored for at least about 12 months to about 48 months. Under preferred storage conditions, prior to first use, the formulation is stored away from bright light (preferably in the dark) at a temperature of about 2-25°C, e.g., room temperature (25 or about 25°C) or 2-8°C (see examples below). The formulation minimizes loss of the active principle, i.e., anti-TG2 antibody. It has also been found that the formulation is less susceptible to acidification and degradation, such as formation of protein aggregates.

[0040] The present invention provides stable liquid formulations of anti-TG2 antibodies for use in therapy. For example, the stable liquid formulations of anti-TG2 antibodies described herein are suitable for pharmaceutical or veterinary use. The present invention also provides methods of treating a disease or disorder by administering the stable liquid formulations of anti-TG2 antibodies.

[0041] The stable liquid formulation containing the anti-TG2 antibody of the present invention can be administered to improve or treat a disease or disorder mediated by TG2. Such diseases or disorders mediated by TG2 include, for example, celiac disease, wound healing disorders, scar formation, keloids and hypertrophic scars, ocular scar formation, inflammatory bowel disease, macular degeneration, Graebe's ophthalmopathy, drug-induced ergotism, psoriasis, fibrotic diseases or fibrosis-related diseases, atherosclerosis, restenosis, inflammatory diseases, autoimmune diseases, neurodegenerative / neurological diseases (e.g., Huntington's disease, Alzheimer's disease, Parkinson's disease, polyglutamine deficiency syndrome, etc.). , spinobulbar muscular atrophy, dentatorubral-pallidoluysian atrophy, spinocerebellar ataxia 1, 2, 3, 6, 7 and 12, rubro-pallidal atrophy, spinocerebellar palsy) and / or cancer (e.g. glioblastoma such as glioblastoma of Li-Fraumeni syndrome and sporadic glioblastoma, malignant melanoma, pancreatic ductal adenocarcinoma, myeloid leukemia, acute myeloid leukemia, myelodysplastic syndrome, myeloproliferative syndrome, gynecological cancer, Kaposi's sarcoma, leprosy, collagen colitis).

[0042] The pharmaceutical compositions of the present invention can be administered in a therapeutically effective amount. As used herein, the term "therapeutically effective amount" refers to the amount of therapeutic agent (i.e., antibody) required to treat, ameliorate, or prevent a disease or disorder mediated by TG2, or to exhibit a detectable therapeutic, pharmacological, or prophylactic effect. For any antibody, the therapeutically effective amount can be initially estimated either in cell culture assays or in animal models (usually rodents, rabbits, dogs, pigs, primates). The animal models can also be used to determine appropriate concentration ranges and routes of administration. Such information can then be used to determine useful doses and routes of administration for administration in humans.

[0043] In treating the above diseases and / or disorders, the appropriate dosage will vary depending, for example, on the particular antibody employed, the subject being treated, the mode of administration, and the nature and severity of the condition being treated. In certain embodiments, the pharmaceutical composition of the present invention is administered by intravenous or subcutaneous route. When administered intravenously, it can be administered by bolus injection or continuous infusion. The pharmaceutical composition according to any embodiment of the present invention can also be administered by intramuscular injection. Depending on the mode of administration, the formulations described herein can be diluted with a solvent (such as NaCl) prior to use. The pharmaceutical composition can be injected using an injection device such as a syringe, an auto-injector, a needleless device, an implant, and a patch. The liquid pharmaceutical formulation of the present invention is suitably administered to a patient at one time or over a series of treatments and can be administered to a patient at any time following diagnosis; it can be administered as the sole treatment or in combination with other drugs or treatments useful in treating conditions such as those described herein above.

[0044] The anti-TG2 antibody may be the only active ingredient in the liquid pharmaceutical formulation. Alternatively, the antibody may be administered in combination with one or more other therapeutically active ingredients, e.g., simultaneously, sequentially, or separately. As used herein, an active ingredient refers to an ingredient that has a pharmacological effect, such as a therapeutic effect, at an appropriate dose. In some embodiments, the antibody in the pharmaceutical composition may be accompanied by other active ingredients, including other antibodies or non-antibody components, administered by the same or different administration route, to treat other inflammatory or autoimmune diseases. In one embodiment, the subject is administered simultaneously or sequentially (before and / or after) other antibody components, such as anti-TNF antibodies, or non-antibody components, such as small drug molecules.

[0045] The following examples are provided to further illustrate the preparation of the formulations and compositions of the present invention. The scope of the present invention should not be construed as consisting solely of the following examples. EXAMPLES

[0046] material: Anti-TG2 antibody: The anti-TG2 monoclonal antibody (mAb) used in the following examples comprises a light chain variable region defined in SEQ ID NO:1 and a heavy chain variable region defined in SEQ ID NO:2, and is designated mAb1 in the following examples.

[0047] method: Protein concentration: The protein concentration was calculated using ultraviolet-visible spectroscopy according to the following formula: Concentration (mg / mL)=[(A280) / a×b] where A280 = absorbance at 280 nm (AU), a = mass extinction coefficient (1.34 mL mg -1 cm -1 ), b=path length (1 cm).

[0048] Thermal stability: Proteins can unfold over a specific temperature range. This temperature, also called melting temperature (Tm), is an intrinsic parameter that describes the thermal stability of a protein. Tm is the peak temperature of protein unfolding. The Tm of mAb1 was determined using capillary differential scanning calorimetry (DSC). Using an autosampler, wells were filled with approximately 300 μL each of buffer and mAb1 sample. Reference wells were filled with buffer only. DSC scans were analyzed using MicroCal Origin® software. After subtracting the respective buffer scans, protein scans were normalized to protein concentration. Tm values ​​were determined using the "Pick Peak" tool in MicroCal Origin® software.

[0049] Diffusion behavior: Dynamic Light Scattering (DLS) was used for that purpose, according to standard methods. Among the various parameters evaluated: main species peak diameter and width: each peak represents a distinct and separable species or population of particles. Peak diameter represents the hydrodynamic diameter (expressed in nm) of the separated chemical species. Peak width is a measure of the polydispersity of the population of particles within the peak. "Peak 1" is considered as the main species peak and generally corresponds to the non-aggregated form of the protein.

[0050] Aggregation and fragmentation: Size exclusion chromatography (SEC), e.g., size exclusion high performance liquid chromatography (SE-HPLC), was used according to standard methods. The percentage values ​​of the peak areas of the main species and species eluting before and after the main species, designated high molecular weight (HMW) and low molecular weight (LMW), respectively, were evaluated.

[0051] Purity under reducing or non-reducing conditions: Purity (IgG monomer) under reducing or non-reducing conditions was assessed using capillary gel electrophoresis (CGE) or on-chip based electrophoresis (Bioanalyzer) according to standard methods (separation of proteins based on hydrodynamic size differences under denaturing conditions). The different mAb1 species were detected by a 220 nm photodiode array (PDA) detector.

[0052] Acidic and basic species: The presence of acidic and basic species was assessed using isoelectric focusing capillary electrophoresis (iCE) according to standard iCE techniques (which separate proteins based on differences in charge). Typically, peaks eluting before the main peak are labeled as acidic species and those eluting after the main peak are labeled as basic species. Osmolality: Osmolality was assessed according to standard methods prior to analysis of mAb1 samples using an Osmette XL 5007 osmometer calibrated with standard solutions of deionized water (0 mOsm / kg), 100 mOsm / kg, 500 mOsm / kg and 1500 mOsm / kg. pH: The pH was assessed according to standard methods using a pH meter equipped with a temperature compensated electrode.

[0053] viscosity: Viscosity measurements were performed using a Rheosense MicroVisc® according to standard methods. Four separate measurements were performed and the average dynamic viscosity results are reported.

[0054] Example 1 - Preliminary Screening 1.1. Buffers and pH Screening Five buffers, each with two to three different pH values, were evaluated for their effect on the thermal and physical stability of mAb1. mAb1 samples were buffer exchanged into the buffers shown in Table 1 according to standard methods. The target protein concentration for this preliminary screen was 2 mg / mL. Prepared samples were analyzed for pH, protein concentration, thermal stability, and conformational stability. The pH of each sample was measured after the buffer exchange step. The pH of each sample was within 0.2 pH units of the target pH value (data not shown). The mAb1 concentration of each sample was measured using UV spectroscopy (see Table 1). Overall, recoveries ranged from 52% to 127%. Given the variability of these results, no clear trends were observed.

[0055] [Table 1]

[0056] The mAb1 samples were then analyzed by DLS for diffusion behavior and DSC for aggregated species (data not shown). The Tm of the formulation samples was relatively similar, ranging from 75.3°C to 77.6°C, but the onset temperatures were more informative. Formulations with pH < 5.5 showed onset temperatures ≤ 53°C. Furthermore, all formulations with pH < 5.5 showed three thermal transitions, whereas the other formulations showed only two. In view of the data, a buffer system with pH ≥ 5.5 appears more optimal. Based on these results, phosphate buffer was eliminated from further evaluation due to its lack of advantages over other buffer systems and potential freeze-thaw issues. Furthermore, pH 5.5 was selected for excipient screening based on the DSC data.

[0057] 1.2. Excipient Screening Based on the buffer and pH screen results, four buffers at pH 5.5 were evaluated in combination with various excipients (see Table 2) to determine their ability to confer stability to mAb1. mAb1 samples were buffer exchanged into the buffers shown in Table 6 according to standard methods. The target mAb1 concentration was 2 mg / mL. The pH and recovery of each mAb1 sample were measured, and the thermal and conformational stability of mAb1 in various buffers was measured (using DSC and DLS, data not shown).

[0058] [Table 2]

[0059] The pH of each sample was measured after the buffer exchange step. With the exception of three formulations (acetate buffers with either glycine, sodium chloride (NaCl), or sorbitol), each pH was within 0.2 units of the target pH of 5.5 (data not shown). The pH of acetate buffers with glycine, NaCl, and sorbitol was 5.8, 5.8, and 5.9, respectively.

[0060] The mAb1 concentration of each sample was measured using UV spectroscopy (see Table 2). Three samples (acetic acid / arginine, histidine / arginine, and histidine / sorbitol) had poor recoveries of ≦60%. The remaining formulations had reasonable recoveries of ≧84%. The variability between these formulations did not suggest a clear trend. The mAb1 samples were then analyzed by DLS (data not shown). All samples containing sucrose showed impurity peaks characteristic of sucrose that hindered observation. Overall, no clear trends were observed. It is noteworthy that the succinic acid / sorbitol sample showed aggregated species that were not observed in the other formulations containing sorbitol. Additionally, the succinic acid / NaCl sample had the highest monomeric breadth compared to all other samples. The DSC results are shown in Table 3. Whatever the buffer system, the formulations with glycine showed higher onset and Tm final temperatures than the formulations with arginine. Formulations containing NaCl exhibited higher onset temperatures than arginine for all formulations except histidine. Onset temperatures for sorbitol and sucrose were comparable, within 1°C of each other.

[0061] [Table 3]

[0062] In summary, the DSC data indicated that glycine and NaCl were preferred over arginine; therefore, arginine was excluded from further evaluation. Both DLS and DSC data indicated that sucrose and sorbitol were equivalent; however, the characteristic sucrose impurity peak made protein aggregation difficult to detect; therefore, sorbitol was preferred and sucrose was not selected for further evaluation. Finally, the succinic acid / sorbitol sample contained aggregated species that were not observed in the other sorbitol samples, therefore succinic acid was not selected for further evaluation.

[0063] 1.3. Solubility test: The solubility of mAb1 was evaluated at various concentrations in the buffers shown in Table 4.

[0064] [Table 4]

[0065] mAb1 samples were buffer exchanged into the appropriate buffer following standard methods. Three mAb1 concentrations were targeted for this study based on volume reduction: 100 mg / mL, 150 mg / mL, and 200 mg / mL. The concentration, recovery, and visual observations for each sample are shown in Table 5.

[0066] Recovery rates were calculated based on the concentrations measured in the most recent evaluation. In this solubility evaluation range, the designation "clear" by visual observation is considered equivalent to "clear, colourless". All formulations achieved the target concentrations of 100 mg / mL and 150 mg / mL with recoveries ranging from 54% to 79% and 59% to 98%, respectively. All samples were confirmed to be colourless and clear (CC). All samples were further concentrated to the target of 200 mg / mL. The acetate / glycine and acetate / sorbitol samples reached 178 mg / mL and 162 mg / mL, respectively. All other samples achieved concentrations >180 mg / mL with recoveries >80%. At these higher concentrations, all samples were observed to be clear and gel-like (CG), except for the histidine / NaCl sample, which was clear and viscous (CV). Overall, recoveries showed no clear trends across all concentrations.

[0067] [Table 5] CC = clear, colorless, CV = clear, viscous, CG = clear, gelatinous, Avg Conc = average concentration, % rec = percent recovery, Obs = visual observation.

[0068] Example 2 - DOE (Design of Experiments) Study: Based on the preliminary studies performed according to Example 1, a DOE was prepared evaluating buffer type (focusing on histidine and citrate buffers, which showed more promise), buffer strength, pH, and excipients (see Table 6).

[0069] [Table 6-1] [Table 6-2]

[0070] Protein samples were buffer exchanged into buffer / excipient combinations (see Table 6) according to standard methods. Where necessary, after buffer exchange, PS80 surfactant was spiked into the appropriate samples at the concentrations indicated. Vials of each formulation were placed at 5°C or 37°C and incubated for 4 and 7 weeks. Osmolality: Osmolality at T0 was within the expected range (340–380 mOsm / kg) in all samples (data not shown).

[0071] viscosity: The viscosity was within the acceptable range (2.5–4.0 at T0) for all samples (data not shown). pH: The pH values ​​of all samples were within 0.2 pH units of the buffer pH, except for sample #21, which had a pH of 6.2 at TO, 4 weeks at 5°C, 4 weeks at 37°C, and 7 weeks at 5°C (data not shown).

[0072] Visual Observation: The appearance of each sample was evaluated at T0, 4 weeks (5°C, 37°C), and 7 weeks (5°C, 37°C) (data not shown). All samples were observed to be colorless, clear liquids with no visible particulates at T0. All samples stored at 5°C for 4 weeks were observed to be colorless, clear liquids with no visible particulates, except for samples 1, 2, and 4, which were slightly yellowish, clear liquids with no visible particulates. All samples stored at 37°C for 4 weeks were observed to be colorless, clear liquids with no visible particulates, except for samples 12, 22, 28, 30, 32, 35, and 36, which were colorless, clear liquids (no visible particulates). All samples stored at 5°C for 7 weeks were observed to be colorless, clear liquids with no visible particulates. All samples stored at 37°C for 7 weeks were observed to be clear liquids (no visible particulates) with a slight yellowish tint.

[0073] DSC: The DSC results at time T0 are shown in Table 7. The thermogram of sample 8-23 showed a broader peak and a lower signal than expected.

[0074] [Table 7]

[0075] SEC: The SEC results at 4 weeks (5° C., 37° C.) and 7 weeks (5° C., 37° C.) are shown in Table 8.

[0076] [Table 8-1] [Table 8-2] #=Sample#.

[0077] cIEF: The results of cIEF (capillary isoelectric focusing) at 4 and 7 weeks (5° C., 37° C.) are shown in Table 9.

[0078] [Table 9-1] [Table 9-2]

[0079] The primary objective of the pre-formulation development studies shown in Examples 1 and 2 was to identify formulation components that would provide optimal chemical and physical stability of the anti-TG2 antibody. A summary of the available data indicated that a citrate or histidine buffer in the pH range of 5.5-6.5 in combination with 250 mM glycine or 150 mM NaCl was optimal. The SEC and iCE results from the DoE studies were reanalyzed using JMP software (data not shown). Comparisons were made between each data set and the preferred formulations recommended by JMP (for the SEC data set, percent monomer, percent HMWS, and percent LMWS at both 5°C and 37°C; for the iCE data set, percent main peak, percent acidic species, and percent basic species at both 5°C and 37°C). Overall, the statistical analysis revealed the following: - Both citrate and histidine have very good buffering properties, with histidine being slightly preferred. - The pH of citrate as a buffer solution is preferably 6.5, and the pH of histidine is preferably 5.0 to 5.5. - A buffer strength of 50 mM is most preferred; - NaCl and glycine also gave good results. - The benefit of adding surfactants was not clear.

[0080] Example 3 - Long-term (12 month) stability study of selected formulations: Based on the above examples, the following four formulations were selected for long-term testing (all containing 100 mg / ml of anti-TG2 antibody mAb1): - F1: 50 mM histidine, 250 mM glycine, pH 5.5, - F2: 50 mM citrate, 250 mM glycine, 0.06% w / v PS80, pH 5.6, - F3: 50 mM histidine, 150 mM NaCl, pH 5.0, - F4: 50 mM histidine, 250 mM glycine, 0.05% w / v PS80, pH 5.5. Four storage conditions were tested: -60°C (data not shown), 2-8°C, 25°C, and 40°C.

[0081] exterior: No significant changes in appearance (particles, precipitate, color, etc.) were observed over time during testing. The only noticeable change was a slow, slight yellow color change of the solution along the 40°C and 25°C stability lines. Samples thawed from storage at ≦-60°C may appear very slightly cloudy due to suspended microbubbles.

[0082] Protein concentration and pH (data not shown): No significant variation in concentration was observed during the study. Similarly, a stable pH was observed between formulations during the study.

[0083] Viscosity and osmolality: The viscosity and osmolality were both within acceptable ranges, 2.3-3.5 cP and 335-385 mOsm / kg water, respectively (data not shown).

[0084] SEC Results: No significant changes were observed in HMWS% for samples stored at ≦60°C. As shown in Tables 10-15, the increase in HMWS% over time was linear up to 3 months when samples were stored at 2-8°C, 25°C / 60% RH, and 40°C / 75% RH. After this 3 month period, the HMWS% tended to stabilize. Quarterly HMWS% rates (slope x 3) were calculated based on the slope (rate / month) of the results measured during the first 3 months for samples stored at 2-8°C, 25°C / 60% RH, and 40°C / 75% RH (data not shown). In summary, at 2-8°C and 25°C / 60% RH, the histidine buffer formulation was the most stable over time. No significant differences were observed between the histidine / glycine or histidine / NaCl formulations. The addition of PS80 to the histidine / glycine formulation did not add any obvious value to the stability of the molecule. At 40°C / 75% RH, the citrate buffer formulation was the most stable.

[0085] [Table 10]

[0086] [Table 11]

[0087] [Table 12]

[0088] iCE Results: No significant changes in major chemical species were observed when samples were stored at ≤60°C and 2-8°C. At 25°C / 60% RH and 40°C / 75% RH, the histidine buffer formulation was the most stable, and the addition of PS80 did not add any added value to the stability of the formulation.

[0089] [Table 13]

[0090] [Table 14]

[0091] [Table 15]

[0092] Example 4 - Additional long-term (12-48 months) stability studies: Based on the results of Example 3, the four formulations tested were very stable, however, long-term storage (up to 4 years) of F1 was tested at 2-8° C. Stability was further evaluated based on osmolality, charge variants, aggregates, and fragmentation.

[0093] [Table 16]

[0094] The data show that the selected formulation (F1) was stable over time for more than 36 months (up to 48 months) at temperatures between 2 and 8°C.

[0095] Overall conclusion: Surprisingly, anti-TG2 antibodies have been shown to be stabilized in the presence of glycine or NaCl. The most stable formulations containing 10% anti-TG2 antibodies were: 1) F1: 50 mM histidine, 250 mM glycine, pH 5.5, and 2) F2: 50 mM citrate, 250 mM glycine, 0.06% w / v PS80, pH 6.5 or 5.6. Formulations containing 150 mM NaCl instead of 250 mM glycine were also very promising. Formulation F1 was tested over time. In particular, it was shown to be very stable over time for 36-48 months at a temperature range of 2-8°C. Given the promising results of this formulation at 25°C for 12 months, it is expected that this stability will extend to longer periods. F2 to F4 would also be good options.

[0096] References: 1) International Publication No. 2006100679 2) International Publication No. 2012146901 3) International Publication No. 2013175229

Claims

1. A stable liquid formulation comprising an anti-TG2 antibody, a buffer that maintains the pH at about 5.0 to 7.0, and a stabilizer selected from the group consisting of glycine or NaCl.

2. 2. The stable liquid formulation of claim 1, wherein the buffer is a histidine or citrate buffer.

3. 3. The stable liquid formulation of claim 2, wherein the histidine buffer maintains a pH at or about 5.5±0.2, or the citrate buffer maintains a pH at or about 6.5±0.

2.

4. 2. The stable liquid formulation of claim 1, wherein the concentration of the buffer is at or about 10 to 100 mM, preferably 20 to 80 mM, more preferably 40 to 60 mM.

5. 2. The stable liquid formulation of claim 1, wherein the stabilizer is glycine in an amount of approximately 150 mM to 350 mM, preferably 200 to 300 mM, more preferably 220 to 280 mM.

6. 2. The stable liquid formulation of claim 1, wherein the stabilizing agent is NaCl in an amount of approximately 100 mM to 200 mM, preferably 125 to 175 mM.

7. 10. The stable liquid formulation of claim 1, further optionally comprising a polysorbate surfactant.

8. 8. The stable liquid formulation of claim 7, wherein the concentration of the polysorbate surfactant is at or about 0.01 to 0.5 mg / mL.

9. 2. The stable liquid formulation of claim 1, wherein the concentration of the anti-TG2 antibody is from about 10 mg / mL to about 200 mg / mL, preferably from about 30 mg / mL to about 180 mg / mL, or preferably from about 50 mg / mL to about 150 mg / mL.

10. 10. The stable liquid formulation of claim 1, wherein the formulation comprises about 100 mg / mL of anti-TG2 antibody, about 50 mM histidine buffer maintaining a pH at or about 5.5, about 250 mM glycine or about 150 mM NaCl, and optionally about 0.02-0.06 mg / mL of polysorbate.

11. 10. The stable liquid formulation of claim 1, wherein the formulation comprises about 100 mg / mL of an anti-TG2 antibody, about 50 mM citrate buffer maintaining a pH at or about 6.5, about 250 mM glycine or about 150 mM NaCl, and optionally about 0.02-0.06 mg / mL of a polysorbate.

12. 2. The stable liquid formulation of claim 1, wherein the anti-TG2 antibody comprises: 1) a light chain variable domain having the sequence defined in SEQ ID NO:1 and a heavy chain variable domain having the sequence defined in SEQ ID NO:2; 2) a light chain variable domain having at least 80% identity or similarity, preferably at least 90% identity or similarity, to the sequence defined in SEQ ID NO: 1 and a heavy chain variable domain having at least 80% identity or similarity, preferably at least 90% identity or similarity, to the sequence defined in SEQ ID NO: 2; 3) a light chain having the sequence defined in SEQ ID NO: 3 and a heavy chain having the sequence defined in SEQ ID NO: 4, or 4) A light chain having at least 80% identity or similarity, preferably at least 90% identity or similarity, to the sequence defined in SEQ ID NO:3 and a heavy chain having at least 80% identity or similarity, preferably at least 90% identity or similarity, to the sequence defined in SEQ ID NO:

4.

13. 13. A method for producing the stable liquid formulation of any one of claims 1 to 12, comprising forming a mixture of an anti-TG2 antibody with 1) a histidine or citrate buffer, 2) glycine or NaCl, and, optionally, 3) a polysorbate surfactant.

14. An article of manufacture comprising a container containing the stable liquid formulation of any one of claims 1 to 12.

15. A stable liquid formulation according to any one of claims 1 to 12 for use in therapy.

16. A method for treating a disease or disorder by administering the stable liquid formulation of any one of claims 1 to 12.