compositions

Abstract

FIELD: pharmaceuticals; medicine.SUBSTANCE: stable liquid composition comprising an antibody against tissue transglutaminase (TG2) for binding to a tissue transglutaminase (TG2) protein, a histidine or citrate buffer at a concentration of 40 to 60 mM or approximately 40 to 60 mM, maintaining a pH between approximately 5.0 and 7.0, and a stabilizer selected from the group consisting of glycine in an amount of approximately 220 to 280 mM or NaCl in an amount of approximately 125 to 175 mM, where the antibody against TG2 comprises a light chain having a sequence defined in SEQ ID NO: 3, and a heavy chain having the sequence defined in SEQ ID NO: 4, and wherein the concentration of the anti-TG2 antibody is from about 50 to about 150 mg / ml, as well as to a method for preparing a stable liquid composition, to an article containing a container containing a stable liquid composition, and to a method for treating a TG2-mediated disease or disorder.EFFECT: stability of the antibody composition.13 cl, 17 tbl, 4 ex

Description

[0001] Field of invention

[0002] The invention relates to the field of pharmaceutical compositions. More specifically, the invention relates to liquid compositions containing antibodies against TG2 and to methods for producing such compositions. Liquid compositions according to the invention are stable when stored at a temperature of from about 2 to 25°C for a suitable period of time.

[0003] Prior Art

[0004] Tissue transglutaminase (TG2) is an enzyme that forms cross-links between proteins via epsilon (gamma-glutamyl) lysine bridge bonds. Increased expression of TG2 leads to abnormal protein cross-linking, which is associated with several pathologies, including various types of tissue scars, the formation of neurofibrillary tangles in some brain disorders, and chemotherapy resistance in some cancers. Various TG2 inhibitors, such as small molecules, RNA silencing, or antibodies (e.g., WO 2006100679, WO 2012146901, or WO 2013175229), have been disclosed for the potential treatment of TG2-mediated disorders.

[0005] Although antibodies directed against TG2 have been described in the literature, stable formulations have not been proposed so far.

[0006] When preparing a pharmaceutical composition containing a biologically active protein, such as an antibody, said composition must be prepared in such a way that the protein is stable for a suitable period of time. Loss of protein activity / stability may result from chemical or physical instability of the protein, particularly due to denaturation, aggregation, or oxidation. Therefore, the resulting products may be pharmaceutically unacceptable. Although the use of excipient(s) is known to enhance the stability of a given protein, the stabilizing effects of these excipients are highly dependent on the nature of the excipients and the biologically active protein itself.

[0007] There remains a need for liquid formulations containing an anti-TG2 antibody as an active ingredient, wherein said formulations are stable for a suitable period of time and suitable for use by injection, such as intravenous or subcutaneous injection. Said formulations may be useful for administration in the treatment of disorders or diseases mediated by TG2.

[0008] Brief summary of the invention

[0009] The objective of the present invention is to provide novel compositions containing an anti-TG2 antibody. More specifically, said compositions are stable liquid compositions containing an anti-TG2 antibody. The invention also provides methods for preparing liquid compositions according to the present invention. The liquid compositions described herein may be useful for administration in the treatment of disorders or diseases mediated by TG2.

[0010] The first aspect of the invention provides stable liquid compositions comprising or consisting of an anti-TG2 antibody, a buffer that maintains a pH of or from about 5.0 to 7.0, a stabilizer (such as an amino acid or salt), and optionally a polysorbate surfactant. In a preferred embodiment, the buffer is a histidine or citrate buffer, and the stabilizer is an amino acid, preferably glycine, or a salt, preferably NaCl. In another preferred embodiment, the buffer maintains a pH of 5.5 to 6.5 or about 5.5 to 6.5. In yet another preferred embodiment, the anti-TG2 antibody is present in an amount of 10 mg / ml or about 10 mg / ml to 200 mg / ml or up to about 200 mg / ml. 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.

[0011] In the second aspect of the invention, there is provided a method for preparing a stable liquid composition of an anti-TG2 antibody, comprising the steps of: forming a mixture of an anti-TG2 antibody together with a buffer, a stabilizer (such as an amino acid or salt) and, optionally, a surfactant polysorbate. In a preferred embodiment, the buffer is a histidine or citrate buffer, and the stabilizer is an amino acid, preferably glycine, or a salt, preferably NaCl. In a preferred embodiment, the buffer maintains a pH of from 5.0 to 7.0 or from about 5.0 to 7.0, and more specifically from 5.5 or from about 5.5 to 6.5 or up to about 6.5. Preferably, the anti-TG2 antibody comprises the light chain variable region defined in SEQ ID NO: 1 and the heavy chain variable region defined in SEQ ID NO: 2.

[0012] In a third aspect, there is provided an article for pharmaceutical or veterinary use comprising a container containing a stable liquid composition according to the invention.

[0013] In a fourth aspect of the invention, there is provided a stable liquid composition according to the invention for use in therapy.

[0014] In a fifth aspect of the invention, there is provided a method of treating a disease or disorder by administering a stable liquid composition according to the invention.

[0015] Definitions

[0016] The term "approximately" means approximately or about, and in the context of a numerical value set forth herein, preferably means + / - 10% around the numerical value set forth or stated.

[0017] When a range of quantities is stated or claimed, the range is presumed to include the quantities stated.

[0018] The term "anti-TG2 antibody" as used herein is an antibody molecule that binds tissue transglutaminase (TG2), an enzyme that forms cross-links between proteins through epsilon (gamma-glutamyl) lysine bridge bonds. Examples of such antibodies are described in WO2013175229. Without any limitation, an anti-TG2 antibody that can be used according to the present invention comprises, for example, the light chain variable region defined in SEQ ID NO: 1 and the heavy chain variable region defined in SEQ ID NO: 2.

[0019] The term "antibody" as used herein includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, and recombinant antibodies that are produced using recombinant technologies as known in the art. "Antibody" includes antibodies of any species, particularly mammalian species, such as human antibodies of any isotype, including IgG1, IgG2a, IgG2b, IgG3, IgG4, IgE, and IgD, and antibodies that are produced as dimers of this basic structure, including IgGA1, IgGA2, or pentamers such as IgM and modified variants thereof; antibodies of non-human primates, such as chimpanzee, baboon, rhesus macaque, or cynomolgus macaque; rodent antibodies, such as mouse or rat; rabbit; goat or horse; camelid antibodies (e.g. from camels or llamas, such as Nanobodies™) and their derivatives; avian species antibodies, such as chicken antibodies, or fish species antibodies, such as shark antibodies.The term "antibody" also refers to "chimeric" antibodies in which a first fragment of at least a first heavy and / or light chain sequence of the antibody is derived from a first species, and a second fragment of the heavy and / or light chain sequence of the antibody is derived from a second species. Chimeric antibodies of interest in this invention include "primatized" antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g., an Old World monkey such as a baboon, rhesus macaque, or cynomolgus monkey) and human constant region sequences. "Humanized" antibodies are chimeric antibodies that comprise a sequence derived from non-human antibodies.Humanized antibodies are essentially human antibodies (the recipient antibody) in which the hypervariable region residues of the recipient are replaced by the hypervariable region (or complementarity determining region, CDR) residues of a non-human species (the donor antibody), such as mouse, rat, rabbit, chicken, or non-human primate, that exhibit the desired specificity, affinity, and activity. In most cases, residues of the human (recipient) antibody outside the CDR, i.e., in the framework region (FR), are additionally replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not found in the recipient antibody or the donor antibody. These modifications are performed to further improve the antibody's properties.Humanization reduces the immunogenicity of non-human antibodies in humans, thereby facilitating the use of antibodies to treat human diseases. Humanized antibodies and several different technologies for their production are well known in the art. The term "antibody" also refers to human antibodies that can be produced as an alternative to humanization. For example, transgenic animals (e.g., mice) can be created that, after immunization, produce a full repertoire of human antibodies in the absence of endogenous murine antibody production. Other methods for producing human antibodies / antibody fragments in vitro rely on display technologies such as phage display or ribosome display technology, which utilize recombinant DNA libraries that are at least partially produced artificially or from the variable (V) domain gene repertoires of donor immunoglobulins.Phage and ribosome display technologies for generating human antibodies are well known in the art. Human antibodies can also be produced from isolated human B cells that are immunized ex vivo with the antigen of interest and then fused to produce hybridomas, which can then be selected for the optimal human antibody. 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, particularly their antigen-binding fragments. An antibody fragment comprises at least one immunoglobulin heavy or light chain domain known in the art and binds to one or more antigens.Examples of antibody fragments according to the invention include a Fab fragment, a modified Fab, a Fab', a modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, Fab-Fv-Fv, scFv and Bis-scFv. Said fragment can also be a diabody, tribody, triabody, tetrabody, minibody, single-domain antibody (dAb), such as sdAb, VL, VH, VHH or a camelid antibody (e.g., camelid or llama, such as Nanobody™) and a VNAR fragment. The antigen-binding fragment according to the invention may also comprise a Fab linked to one or two scFvs or dsscFvs, wherein each scFv or dsscFv binds the same or a different target (e.g., one scFv or dsscFv binding a therapeutic target and one scFv or dsscFv that increases half-life by binding, for example, albumin). Examples of fragments of such antibodies are FabdsscFv (also referred to as BYbe®) or Fab-(dsscFv)2 (also referred to as TrYbe®, see, for example, WO2015 / 197772).The antibody molecules defined above, including their antigen-binding fragments, are known in the art.

[0020] The term "stability" as used herein refers to the physical, chemical, and conformational stability of the anti-TG2 antibody composition according to the present invention (including the maintenance of biological activity). Instability of said antibody composition may be caused by chemical degradation or aggregation of the antibody to form higher-order polymers, deglycosylation, glycosylation modification, oxidation, or any other structural modification that reduces at least one biological activity of the antibody.

[0021] The term "stable formulation" refers to a formulation in which the protein of interest (here, an anti-TG2 antibody) substantially retains its physical, chemical, and biological properties during storage. Various analytical methods are well known to those skilled in the art for measuring the stability of an antibody in a formulation (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) for a selected period of time (e.g., 3 months, 6 months, 12 months, or longer). Since the antibody, after preparation, is typically stored in a refrigerator (typically 2-8°C) or at room temperature (typically 15-25°C) prior to administration to a patient, it is important that said prepared antibody is stable over time at least at a temperature in the range of 2 to 25°C, as shown here, for example, at 2-8°C and 25°C.Various values ​​may be used to infer approximate stability over a given period of time, such as (and without limitation): 1) no less than 90% monomeric form of the antibody, 2) no more than 10% change in monomeric form of the antibody (compared to the starting data), 3) no more than 5% high molecular weight species (HMW or HMWS; also referred to herein as aggregates), or 4) no more than + / - 0.2 pH units change (compared to the starting data).

[0022] As used herein, the term "stabilizing agent," "stabilizer," or "isotonizing agent" is a compound that is physiologically tolerable and imparts suitable stability / tonicity to the composition. This, in particular, prevents water from penetrating through cell membranes in contact with the drug. Compounds such as glycerol are commonly used for such purposes. Other suitable stabilizing agents 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).

[0023] The term "buffer" as used herein refers to solutions of compounds known to be safe in a pharmaceutical or veterinary formulation and which have the effect of maintaining or controlling the pH of the formulation within the desired pH range. Acceptable buffers for controlling pH from a moderately acidic pH to a 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 includes any pharmacologically acceptable salt thereof).

[0024] The term "surfactant" as used herein refers to a soluble compound that can be used primarily to increase the water solubility of hydrophobic, oily substances or otherwise enhance the miscibility of two substances with differing hydrophobicities. For this reason, these polymers are commonly used in industrial applications, cosmetics, and pharmaceuticals. They are also used as model systems for drug delivery applications, primarily to modify drug absorption or delivery to target tissues. Well-known surfactants include polysorbates (polyoxyethylene derivatives; Tween) and poloxamers (i.e., copolymers based on ethylene oxide and propylene oxide, also known as Pluronics®).

[0025] The term "vial" or "container" as used herein broadly refers to a reservoir suitable for storing an anti-TG2 antibody composition in liquid form. Examples of vials that can be used in the present invention include ampoules, test tubes, bottles, syringes (such as pre-filled syringes), cartridges, or other such reservoirs suitable for delivering the anti-TG2 antibody composition to a patient by injection, preferably by intravenous or subcutaneous injection.

[0026] The term "solvent" as used herein refers to a liquid solvent, both aqueous and non-aqueous. The choice of solvent depends, in particular, on the solubility of the drug compound in said solvent and the route of administration. An aqueous solvent may consist solely of water or may consist of water together with one or more miscible solvents and may contain solutes such as sugars, buffers, salts, or other excipients. The most commonly used non-aqueous solvents are short-chain organic alcohols such as methanol, ethanol, and propanol, short-chain ketones such as acetone, and polyalcohols such as glycerol. According to the present invention, a preferred solvent is an aqueous solvent such as water or a saline solvent.

[0027] The term "process of treating" or "treating" a disease state includes: (1) suppressing the disease state, i.e., stopping the development of the disease state or its clinical symptoms, or (2) alleviating the disease state, i.e., causing temporary or permanent regression of the disease state or its clinical symptoms.

[0028] The term "preventing" or "preventing" a disease state includes preventing the development of clinical symptoms of a disease state in a subject who may be susceptible or predisposed to the disease state but who is not yet experiencing or demonstrating symptoms of the disease state.

[0029] In all embodiments of the invention, the "pharmaceutical composition" may also be referred to as a "stable pharmaceutical composition" without any distinction.

[0030] Detailed description of the invention

[0031] The invention is based on a combination of a stabilizer selected from the group consisting of glycine or NaCl and a buffer solution, such as a histidine or citrate buffer, maintaining a pH between 5.0 and 7.0, for the preparation of a suitable pharmaceutical composition for human use of an anti-TG2 antibody without affecting the processing ability of the pharmaceutical composition and the long-term stability of the antibody. The inventors have found that the pharmaceutical compositions according to the invention are stable over time, in particular when stored at approximately 2-25°C, as presented in the examples section at 2-8°C and 25°C.

[0032] The main object of the present invention is a stable liquid composition comprising or consisting of an anti-TG2 antibody, a buffer maintaining a pH between approximately 5.0 and approximately 7.0, and a stabilizer. In a preferred embodiment, the buffer is a histidine buffer or citrate buffer, and the stabilizer is selected from the group consisting of glycine or NaCl. Optionally, the composition may additionally contain a surfactant, such as a polysorbate surfactant.

[0033] The invention further provides a method for preparing any of the stable liquid anti-TG2 antibody compositions described herein, wherein the method comprises the steps of combining the anti-TG2 antibody with a buffer, a stabilizer, and optionally a surfactant, such as a polysorbate surfactant. This step is typically carried out by buffer exchange in accordance with conventional methods. As an example, to prepare a suitable stable composition, a given amount of the anti-TG2 antibody is buffer exchanged with 1) a citrate or histidine buffer that maintains a pH of 5.0 to 7.0 or about 5.0 to 7.0, 2) a stabilizer (preferably selected from the group consisting of glycine or NaCl). If the composition contains a surfactant, it is preferably added after the buffer exchange step. After the buffer exchange, the composition is filtered (final filtration).Depending on the target antibody concentration, the composition can be concentrated between the buffer exchange step and final filtration. Each of these compounds (i.e., the anti-TG2 antibody, buffer, stabilizer, and optionally a surfactant) can be used in accordance with the concentrations, pH, and / or ratios described herein. The resulting mixture is then dispensed into vials. Variations of this method will be apparent to those skilled in the art.

[0034] The invention also provides an article for pharmaceutical or veterinary use, comprising a container containing any of the stable liquid compositions described herein, wherein said composition comprises or consists of an anti-TG2 antibody, a buffer, a stabilizer, and optionally a surfactant. Each of these compounds (i.e., the anti-TG2 antibody, the buffer, the stabilizer, and optionally the surfactant) can be used depending on the concentrations, pH, and / or ratios described herein.

[0035] Also described is the packaging material providing instructions for use.

[0036] Preferably, the anti-TG2 antibody used according to the invention generally comprises (see also Table A):

[0037] 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;

[0038] 2) a light chain variable domain having at least 80% identity or similarity, preferably 90% identity or similarity with 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 with the sequence defined in SEQ ID NO: 2;

[0039] 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

[0040] 4) a light chain having at least 80% identity or similarity, preferably 90% identity or similarity with the sequence defined in SEQ ID NO: 3, and a heavy chain having at least 80% identity or similarity, preferably 90% identity or similarity with the sequence defined in SEQ ID NO: 4.

[0041] Table A Amino acid sequences of anti-TG2 antibody

[0042]

[0043] In the context of the invention as a whole, the amount of the anti-TG2 antibody in the compositions is preferably from 10 mg / ml or from about 10 mg / ml to 200 mg / ml or to about 200 mg / ml, preferably from 30 mg / ml or from about 30 mg / ml to 180 mg / ml or to about 180 mg / ml, or preferably from or from about 50 mg / ml to or to 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 compositions in an amount expressed as weight per 100 ml (% w / v). In this case, the anti-TG2 antibody contained in the compositions according to the present invention may generally be present in an amount of from about 1 to 20% w / v or up to about 20% w / v, preferably in an amount of from about 3 to 18% w / v or up to about 18% w / v.or preferably in an amount of from about 5 to or up to about 15% w / v, such as 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. The anti-TG2 antibody may, for example (but not limited to), comprise 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.

[0044] Preferred buffers according to the present invention are generally histidine (preferably L-histidine) or citrate buffers and maintain a pH in the range between about 5.0 and about 7.0, preferably between about 5.2 and about 6.0, such as 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 and 6.0 for a histidine buffer, and preferably between about 6.2 and about 7.0, such as 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0 for a citrate buffer. Even more preferably, the pH is 5.5 or about 5.5 for a histidine buffer and 6.5 or about 6.5 for a citrate buffer.In all embodiments of the present invention, unless otherwise stated, the pH value is measured at room temperature and is preferably ±0.1 or ±0.2 relative to the target pH value (e.g., 5.5±0.1 or 5.5±0.2 for a histidine buffer and is or is approximately 6.5±0.1 or 6.5±0.2 for a citrate buffer).

[0045] In the context of the invention as a whole, the buffer concentration is preferably or is about 10 to 100 mM. In a preferred embodiment, the buffer concentration is from 20 or is from about 20 to 80 or up to about 80 or even preferably from about 40 to about 60 mM, such as 40, 45, 50, 55 or 60 mM. Preferably, the buffer concentration is or is about 50 mM.

[0046] In the context of the invention as a whole, the stabilizer is selected from the group consisting of glycine (preferably L-glycine) or NaCl. The stabilizer should be glycine, its concentration is preferably from 150 mM or is from about 150 mM to 350 mM or up to about 350 mM, preferably is from 200 or is from about 200 to 300 or up to about 300 mM or even preferably is from 220 or is from about 220 to 280 or up to about 280 mM, such as 220, 230, 240, 250, 260, 270 or 280 mM. If the stabilizer is NaCl, then its concentration is preferably or is from about 100 mM to or about 200 mM, preferably is or is from about 125 to or about 175 mM, such as 125, 130, 135, 140, 145, 150, 155, 160, 165, 170 and 175 mM.

[0047] In the context of the invention, a surfactant may generally be present. When present, the surfactant 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, the surfactant is present in the composition in an amount of from 0.01 or about 0.01 to 5 or to about 5 mg / mL, more preferably from 0.01 or about 0.01 to 1 or to about 1 mg / mL, more particularly from 0.1 or about 0.1 to 0.6 or to 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, the polysorbate surfactant is preferably present in the protein composition in an amount expressed as wt. % per 100 ml (% w / v).). In such a case, the polysorbate surfactant contained in the composition according to the present invention may generally be present in an amount of from 0.001 to 0.5% w / v, preferably from 0.01 to 0.1% w / v, or even preferably from 0.01 to 0.06% w / v, such as 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055 or 0.06% w / v.

[0048] In a preferred embodiment, the stable liquid compositions according to the present invention generally comprise or consist of an anti-TG2 antibody at a concentration of or comprising from about 50 to 200 mg / mL, from about 10 to about 100 mM histidine buffer at a pH of about 5.5 (or from about 10 to about 100 mM citrate buffer at a pH of about 6.5), from about 150 to about 350 mM glycine buffer or from about 100 to about 200 mM NaCl, and optionally a surfactant (such as a polysorbate surfactant) from about 0.001 mg / mL to about 0.5 mg / mL. Alternatively, the stable liquid compositions comprise or consist of an anti-TG2 antibody from about 5.0 to about 20% w / v., from about 10 to about 100 mM histidine buffer at a pH of about 5.5 (or from about 10 to about 100 mM citrate buffer at a pH of about 6.5), from about 150 to about 350 mM glycine buffer, or from about 100 to about 200 mM NaCl, and optionally from 0.001 to 0.5% w / v of a surfactant (such as a polysorbate surfactant).

[0049] As a specific example (but not limitation), provided herein is a stable liquid composition comprising or consisting of an anti-TG2 antibody at a concentration of about 100 mg / mL, about 50 mM histidine buffer maintaining a pH at about 5.5, about 250 mM glycine, and optionally PS80 in an amount of about 0.02-0.06% w / v. As another specific example (but not limitation), provided herein is a stable liquid composition comprising or consisting of an anti-TG2 antibody at a concentration of about 100 mg / mL, about 50 mM histidine buffer maintaining a pH at or about 5.5, about 150 mM NaCl, and optionally PS80 at about 0.02-0.06% w / v.In yet another non-limiting example, a stable liquid composition is provided that comprises or consists of an anti-TG2 antibody at a concentration of about 100 mg / mL, about 50 mM citrate buffer maintaining a pH of about 5.5, about 250 mM glycine, and optionally PS80 in an amount of about 0.02-0.06% w / v. The anti-TG2 antibody may, for example, comprise the light chain variable region defined in SEQ ID NO: 1 and the heavy chain variable region defined in SEQ ID NO: 2.

[0050] Preferably, the compositions according to the invention retain at least 80% of the biological activity of the anti-TG2 antibody at the time of preparation and / or packaging of the composition for a period of at least 12 months, preferably at least 24 months, or even preferably at least 36 months, or even more preferably at least 48 months (before the first use). The activity of the anti-TG2 antibody can be measured according to standard methods, such as enzyme-linked immunosorbent assay (ELISA) or cell-based assays.

[0051] Additional excipients for use in the pharmaceutical compositions according to the invention include, but are not limited to, viscosity increasing agents, fillers, solubilizers, or combinations thereof.

[0052] The present invention also provides a container containing the pharmaceutical composition according to the invention. In particular, the container may be, without any limitation, a vial, ampoule, test tube, flask, or syringe (such as a pre-filled syringe) containing the pharmaceutical composition.

[0053] The container may be part of a kit of parts comprising one or more containers containing pharmaceutical compositions according to the invention and delivery devices such as a syringe, pre-filled syringe, autoinjector, needle-free device, implant or patch, or other parenteral administration devices and directions for use.

[0054] Liquid compositions according to the invention can be stored for at least about 12 months to about 48 months. Under preferred storage conditions, before first use, the compositions are protected from bright light (preferably in the dark), at a temperature of from about 2 to 25°C, for example, at room temperature (at or at about 25°C) or at 2-8°C (see the following examples). Said compositions minimize the loss of the active ingredient, i.e., the anti-TG2 antibody. Said compositions have also been found to be less prone to oxidation or degradation, such as the formation of protein aggregates.

[0055] The present invention provides stable liquid formulations of an anti-TG2 antibody for use in therapy. For example, the stable liquid formulations of the anti-TG2 antibody described herein are suitable for pharmaceutical or veterinary use. The present invention also provides a method for treating a disease or disorder by administering the stable liquid formulations of the anti-TG2 antibody.

[0056] A stable liquid composition containing an anti-TG2 antibody according to the present invention can be administered to improve or treat TG2-mediated disorders or diseases. For example, such TG2-mediated disorders or diseases may be selected from the group consisting of celiac disease, abnormal wound healing, scarring, keloids and hypertrophic scars, ocular scars, inflammatory bowel disease, macular degeneration, Graves' orbitopathy, drug-induced ergotism, psoriasis, fibrotic diseases or fibrotic-related diseases, atherosclerosis, restenosis, inflammatory diseases, autoimmune diseases, neurodegenerative / neurological diseases (e.g., Huntington's disease, Alzheimer's disease, Parkinson's disease, polyglutamine disease, spinobulbar muscular atrophy, dentato-rubro-pallido-Lewis atrophy, spinocerebellar ataxias 1, 2, 3, 6, 7, and 12, rubropallidal atrophy,spinal cerebellar palsy) and / or cancer (e.g. glioblastomas such as glioblastoma in Li-Fraumeni syndrome and sporadic glioblastoma, malignant melanomas, pancreatic ductal adenocarcinomas, myeloid leukemia, acute myelogenous leukemia, myelodysplasia syndrome, myeloproliferative syndrome, gynecologic cancer, Kaposi's sarcoma, Hansen's disease, collagenous colitis).

[0057] The pharmaceutical composition according to the invention can be administered in a therapeutically effective amount. As used herein, the term "therapeutically effective amount" refers to the amount of a therapeutic agent (i.e., an antibody) required to treat, ameliorate, or prevent a disorder or disease mediated by TG2, or to demonstrate 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, typically in rodents, rabbits, dogs, pigs, or primates. An animal model can also be used to determine a suitable concentration range and route of administration. Such information can then be used to determine useful doses and routes of administration in humans.

[0058] For the treatment of the above diseases and / or disorders, the appropriate dosage will vary depending on, for example, the particular antibody used, the subject being treated, the route of administration, and the nature and severity of the condition being treated. In a specific embodiment, the pharmaceutical composition according to the invention is administered via the intravenous or subcutaneous route. When administered via intravenous injection, it can be administered as a bolus injection or as a continuous infusion. The pharmaceutical composition according to any of the embodiments of the invention can also be administered by intramuscular injection. Depending on the route of administration, the compositions described herein can be dissolved in a solvent (such as NaCl) before use. The pharmaceutical composition can be injected using a syringe, an injection device such as an autoinjector, a needle-free device, an implant, or a patch.

[0059] The liquid pharmaceutical composition according to the invention is suitably administered to the patient in a single administration or as a series of treatments, and it can be administered to the patient at any time after diagnosis; it can be administered as a single treatment or in combination with other drugs or treatment methods useful in treating the conditions described above.

[0060] The anti-TG2 antibody may be the sole active ingredient in a liquid pharmaceutical composition. Alternatively, the antibody may be administered in combination, for example, simultaneously, sequentially, or separately, with one or more other therapeutically active ingredients. As used herein, an active ingredient refers to an ingredient that, at an appropriate dose, has a pharmacological effect, such as a therapeutic effect. In some embodiments, the antibody in the pharmaceutical composition may be accompanied by other active ingredients, including other antibodies or ingredients different from the antibodies administered via the same or a different route of administration, for the treatment of other inflammatory or autoimmune diseases.In one embodiment, the subject is administered simultaneously or sequentially (before and / or after) other ingredients of the antibody, such as anti-TNF (tumor necrosis factor (TNF)) antibodies or ingredients other than antibodies, such as small molecule drugs.

[0061] The following examples are provided to further illustrate the preparation of pharmaceutical compositions and formulations according to the invention. The scope of the invention should not be construed as consisting simply of the following examples.

[0062] Examples

[0063] Material

[0064] Anti-TG2 antibody: an anti-TG2 monoclonal antibody (mAb) used in the following examples, comprising the light chain variable region defined in SEQ ID NO: 1 and the heavy chain variable region defined in SEQ ID NO: 2. In the following examples, it is referred to as mAb1.

[0065] Methods

[0066] Protein concentration: The protein concentration was determined using the UV-visible spectroscopy method using the following equation: Concentration (mg / mL) = [(A280) / a × b],

[0067] where A280 = absorbance at 280 nm (AU); a = mass extinction coefficient (1.34 ml mg -1 cm -1 ); b = Path length (1 cm).

[0068] Thermal Stability: Proteins can undergo unfolding within a specific temperature range. This temperature, also known as the melting temperature (Tm), is an intrinsic parameter describing the thermal stability of proteins. Tm represents the maximum temperature during unfolding. Capillary differential scanning calorimetry (DSC) was used to determine Tm for mAb1. An autosampler was used to fill wells with approximately 300 µL of buffer and mAb1 sample, respectively. Reference wells were filled with buffer only. DSC scans were analyzed using MicroCal Origin® software. After subtracting the corresponding buffer scan, protein scans were normalized for protein concentration. Tm values ​​were determined using the "Pick Peak" tool in MicroCal Origin® software.

[0069] Diffusion behavior: Dynamic light scattering (DLS) was used for this task according to standard methods. Among the various parameters evaluated were the peak diameter and peak width of the major species: each peak represents individual and separable species or multiple species. The peak diameter represents the hydrodynamic diameter (expressed in mm) of the separated species. The peak width represents a measure of the polydispersity of the multiple species in the peak. "Peak 1" is considered the peak of the major species and typically corresponds to non-aggregated forms of the protein.

[0070] Aggregation and fragmentation: Size exclusion chromatography (SEC), such as size exclusion high-performance liquid chromatography (SE-HPLC), was used according to standard methods. The peak area percentages of the major species as well as the species eluting before and after the major peak, designated as high molecular weight (HMW) and low molecular weight (LMW) species, respectively, were estimated.

[0071] Purity under reducing or non-reducing conditions: Purity under reducing or non-reducing conditions (IgG monomer) was assessed using capillary gel electrophoresis (CGE) or chip-based electrophoresis (Bioanalyzer) according to standard methods (separation of proteins based on their hydrodynamic size differences under denaturing conditions). Different mAb1 species were detected using a photodiode array detector (PDA) at 220 nm.

[0072] Acidic and Basic Species: The presence of acidic and basic species was assessed using isoelectric capillary electrophoresis (iCE) according to standard iCE (charge-based separation of proteins) methods. Generally, peaks eluting before the main peak are designated as acidic species, and peaks eluting after the main peak are designated as basic species.

[0073] Osmolality: Osmolality was assessed according to standard methods using an Osmette XL 5007 osmometer calibrated with deionized water (zero mOsm / kg), 100 mOsm / kg, 500 mOsm / kg and 1500 mOsm / kg standard solutions prior to analysis of mAb1 samples.

[0074] pH: pH was estimated according to standard methods using a pH meter equipped with a temperature compensation electrode.

[0075] Viscosity: Viscosity measurements were performed according to standard methods using a Rheosense Micro Vise®. Viscosity measurements were performed in quadruplicate, and the average dynamic viscosity is presented.

[0076] Example 1 - Preliminary selection

[0077] 1.1 Buffers and pH selection

[0078] Five different buffer types, each at two or three different pH values, were evaluated for their effect on the thermal and physical stability of mAb1. mAb1 samples were buffer exchanged according to standard methods with the buffers listed in Table 1. The target protein concentration for this prescreening was 2 mg / mL. The prepared samples were analyzed for pH, protein concentration, and thermal and conformational stability. The pH of each sample was determined after the buffer exchange process. Each sample's pH was within 0.2 pH units of the target pH (data not shown). The mAb1 concentration for each sample was determined using UV spectroscopy (see Table 1). Overall, recovery ranged from 52% to 127%. Due to the variability of these results, no clear trend was observed.

[0080]

[0081]

[0082] The mAb1 samples were then analyzed using DLS for diffusion behavior and DSC for aggregated species (data not shown). Although the Tt of the prepared samples were relatively similar, ranging from 75.3°C to 77.6°C, the initial temperatures were more informative. Formulations with a pH < 5.5 displayed initial temperatures ≤ 53°C. Furthermore, it was found that all formulations with a pH < 5.5 exhibited three thermal transitions, compared with only two transitions for the other formulations. In light of these data, buffer systems with a pH > 5.5 appear to be more optimal.

[0083] Based on these results, phosphate buffer was excluded from further evaluation due to its lack of advantage over other buffer systems and potential freeze / thaw issues. Additionally, based on DSC data, pH 5.5 was chosen for excipient selection.

[0084] 1.2. Selection of excipient

[0085] Based on the buffer and pH screening results, four buffers at pH 5.5 were evaluated in combination with various excipients (see Table 2) to determine their ability to impart stability to mAb1. mAb1 samples were buffer exchanged with the buffers listed in Table 6 according to standard methods. The target mAb1 concentration was 2 mg / mL. The pH and percent recovery of each mAb1 sample, as well as the thermal and conformational stability of mAb1 in different buffers (using DSC and DLS; data not shown) were determined.

[0086]

[0087] The pH of each sample was determined after the buffer exchange process. With the exception of three compositions (acetate buffer with any of 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 for the acetate buffer with glycine, NaCl, and sorbitol was 5.8, 5.8, and 5.9, respectively.

[0089] The mAb1 concentration for each sample was determined using UV spectroscopy (see Table 2). Three samples (acetate / arginine, histidine / arginine, and histidine / sorbitol) demonstrated poor recoveries of ≤60%. The remaining compositions demonstrated adequate recoveries of ≥84%. The differences between these compositions did not show a clear trend.

[0090] The mAb1 samples were then analyzed by DLS (data not shown). All sucrose-containing samples exhibited a characteristic sucrose impurity peak, preventing any observations. Overall, the results did not reveal a clear trend. It was noted that the succinate / sorbitol sample exhibited aggregated species that were not detected in other sorbitol-containing formulations. Furthermore, the succinate / NaCl sample also exhibited the highest monomer width compared to all other samples.

[0091] The DSC results are shown in Table 3. Regardless of the buffer system, the compositions with glycine showed higher initial and temperature compared to arginine. Compositions containing NaCl demonstrated a higher initial temperature than arginine in all compositions except the histidine one. Sorbitol and sucrose demonstrated comparable initial temperatures within 1°C of each other.

[0092]

[0093] Thus, DSC data demonstrated that glycine and NaCl were preferred over arginine. Therefore, arginine was excluded from further evaluation. Both DLS and DSC data demonstrated that sucrose and sorbitol were comparable. However, the characteristic sucrose impurity peak makes protein aggregation detection problematic. Sorbitol itself was preferred, and sucrose was not selected for further evaluation. Finally, since the succinate / sorbitol sample contained aggregated species not detected in any other sorbitol samples, succinate was not selected for further evaluation.

[0094] 1.3. Solubility Study

[0095] The solubility of mAb1 was assessed at different concentrations in the buffers listed in Table 4.

[0096]

[0097] The mAb1 samples were buffer exchanged with suitable buffers according to standard methods. In this study, three mAb1 concentrations were targeted based on volume reduction: 100 mg / mL, 150 mg / mL, and 200 mg / mL. The concentration, percentage recovery, and visual detection results for each sample are presented in Table 5.

[0098] The percentage recovery was calculated based on the determined concentration as a result of the previous assessment. For the purposes of this dissolution study, the visual detection designation "clear" is considered equivalent to "clear, colorless." All formulations achieved target concentrations of 100 mg / mL and 150 mg / mL, with recovery percentages ranging from 54% to 79% and 59% to 98%, respectively. All samples were found to be clear and colorless (CC). All samples were further concentrated to the target concentration of 200 mg / mL. The acetate / glycine and acetate / sorbitol samples achieved 178 mg / mL and 162 mg / mL, respectively. All other samples achieved concentrations >180 mg / mL with recovery percentages >80%. At these high concentrations, all samples were found to be clear and gelatinous (CG), with the exception of the histidine / NaCl sample, which was clear and viscous (CV). Overall, the recoveries did not show a clear trend across all concentrations.

[0099]

[0100] Example 2 - Study of the SPE (ROE) (experimental design)

[0101] Based on the preliminary study conducted according to Example 1, DOE was performed to evaluate the buffer type (focusing on histidine and citrate buffers, which were more promising), buffer concentration, pH, and excipient (see Table 6).

[0102]

[0103]

[0104] Protein samples were buffer exchanged with buffer / excipient combinations (see Table 6) according to standard methods. Where appropriate, the surfactant PS80 was added to the respective samples at the indicated concentration after buffer exchange. Vials containing each composition were incubated at either 5°C or 37°C for 4-week and 7-week periods.

[0105] Osmolality: Osmolality for T0 was within the expected range for all samples (between 340 and 380 mOsm / kg) (data not shown).

[0106] Viscosity: Viscosity was within the acceptable range for all samples (between 2.5 and 4.0 for T0) (data not shown).

[0107] pH: The pH values ​​for all samples were within 0.2 pH units of the pH buffer except for sample #21, which was pH 6.2 at T0, 5°C 4 weeks, 37°C 4 weeks, and 5°C 7 weeks (data not shown).

[0108] Visual Detection: The appearance of each sample was assessed at T0, 4 weeks (5°C, 37°C) and 7 weeks (5°C, 37°C) (data not shown). All samples appeared as colorless transparent liquids with no visible particles at T0. All samples stored at 5°C for 4 weeks were found to be colorless transparent liquids with no visible particles, except for samples 1, 2 and 4, which were slightly yellow transparent liquids with no visible particles. All samples stored at 37°C for 4 weeks were found to be slightly yellow transparent liquids with no visible particles, except for samples 12, 22, 28, 30, 32, 35 and 36, which were colorless transparent liquids (no visible particles). All samples stored at 5°C for 7 weeks were found to be a colourless, transparent liquid with no visible particles.All samples stored at 37°C for 7 weeks were found to be a slightly yellow, clear liquid (no visible particles).

[0109] DSC: The DSC results for time T0 are shown in Table 7. The thermograms for samples 8-23 showed broader peaks and a signal lower than expected.

[0110]

[0111]

[0112]

[0113]

[0114] The primary objective of the preliminary formulation development studies presented in Examples 1 and 2 was to identify formulation components that provide optimal chemical and physical stability for the anti-TG2 antibody. Collectively, the available data indicate that optimal formulations may include citrate or histidine buffers with pH ranges from 5.5 to 6.5 in combination with 250 mM glycine or 150 mM NaCl.

[0115] The SEC and iCE results obtained during the DoE study were re-analyzed using JMP software (data not shown). The JMP-proposed preferred compositions from each data set (SEC data set at 5°C and 37°C for % monomer, % HMWS, and % LMWS, and iCE data set at 5°C and 37°C for % base peak, % acidic species, and % basic species) were compared. Overall, it is evident from this statistical analysis that:

[0116] - Both citrate and histidine had very good buffering properties, with a slight advantage for histidine,

[0117] - pH 6.5 was preferred for citrate as a buffering agent and pH 5.0-5.5 for histidine,

[0118] - 50 mM buffer concentration was the most desirable,

[0119] - Both NaCl and glycine provided good results,

[0120] - The benefit of adding surfactant is not obvious.

[0121] Example 3 - Long-term (12 months) stability study on selected compositions

[0122] Based on the above examples, four formulations were selected for long-term studies (all containing 100 mg / mL anti-TG2 mAb1 antibody):

[0123] - F1: 50 mM histidine, 250 mM glycine, pH 5.5;

[0124] - F2: 50mM citrate, 250mM glycine, 0.06% w / v PS80, pH 5.6;

[0125] - F3: 50 mM histidine, 150 mM NaCl, pH5.0;

[0126] - 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.

[0127] Appearance: No significant changes in appearance (particles, sediment, color, etc.) were observed over time. The only minor observation was discoloration of the liquid composition, which slowly turned slightly yellow depending on stability at 40°C and 25°C. A sample thawed at storage conditions ≤-60°C may appear slightly cloudy due to suspended microbubbles.

[0128] Protein concentration and pH (data not shown): No significant variation in concentration was observed during the study. Similarly, a stable pH was observed across the formulations during the study.

[0129] Viscosity and osmolality: Both viscosity and osmolality were within the acceptable ranges, respectively, between 2.3 and 3.5 cP and between 335 and 385 mOsm / kg water (data not shown).

[0130] SEC Results: No significant change in HMWS% could be detected for the sample stored at ≤-60°C. As shown in Tables 10-15, the increase in HMWS% over time was linear up to 3 months when the samples were stored at 2-8°C, 25°C / 60%RH, and 40°C / 75%RH. After these 3 months, HMWS% showed a tendency to be stable. The rate of HMWS% per quarter (slope x 3) was calculated based on the slope (rate / month) of the results measured during the first three months for samples stored at 2-8°C, 25°C / 60%RH, 40°C / 75%RH (data not shown). In conclusion, at 2-8°C and 25°C / 60% RH, the histidine-buffered formulations were the most stable over time. No significant difference was observed between the histidine / glycine or histidine / NaCl formulations. The addition of PS80 to the histidine / glycine formulation did not demonstrate any apparent additional benefit in terms of molecule stability.At 40°C / 75% RH, the citrate buffer formulation was the most stable.

[0131]

[0132]

[0133] ICE results: No significant changes in the major species were detected when storing samples at temperatures ≤ -60°C and 2-8°C. At 25°C / 60% RH and 40°C / 75% RH, the histidine-buffered formulations were the most stable, and the addition of PS80 did not demonstrate any additional benefit in terms of formulation stability.

[0134]

[0135] Case 4 - Additional Long-Term (12 to 48 Months) Stability Study

[0136] Based on the results of Example 3, although the four tested formulations were very stable, F1 was tested for long-term storage (up to 4 years) at 2-8°C. Stability was further assessed based on osmolality, charge variants, aggregates, and fragmentation.

[0137]

[0138] The data demonstrate that the selected composition (F1) was stable for more than 36 months (and up to 48 months) at temperatures ranging from 2 to 8°C.

[0139] General conclusions

[0140] It was unexpectedly demonstrated that anti-TG2 antibodies can 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 also showed great promise. Formulation F1 was studied over the long term. In particular, it was demonstrated to be very stable over time for up to 36-48 months at temperatures ranging from 2 to 8°C. Given the promising results over 12 months at 25°C for this composition, it is expected that this stability is maintained for longer periods. F2-F4 may also represent good alternatives.

[0141] Links:

[0142] 1) WO 2006100679.

[0143] 2) WO 2012146901.

[0144] 3) WO 2013175229.

[0145] --->

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Claims

1. A stable liquid composition comprising an antibody against tissue transglutaminase (TG2) for binding to a tissue transglutaminase (TG2) protein, a histidine or citrate buffer at a concentration of 40 to 60 mM or about 40 to 60 mM, maintaining a pH between about 5.0 and 7.0, and a stabilizer selected from the group consisting of glycine in an amount of about 220 to 280 mM or NaCl in an amount of about 125 to 175 mM, wherein the anti-TG2 antibody comprises 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, and wherein the concentration of the anti-TG2 antibody is from about 50 to about 150 mg / ml.

2. The stable liquid composition of claim 1, wherein the histidine buffer maintains a pH of 5.3 to 5.7 or about 5.3 to 5.7, or wherein the citrate buffer maintains a pH of 6.3 to 6.7 or about 6.3 to 6.

7.

3. A stable liquid composition according to any one of claims 1, 2, additionally containing a surfactant polysorbate.

4. The stable liquid composition of claim 3, wherein the concentration of the polysorbate surfactant is or is approximately 0.01 to 5 mg / mL.

5. A stable liquid composition according to claim 1, comprising about 100 mg / ml of an antibody against TG2, about 50 mM of a histidine buffer that maintains a pH of 5.5 or about 5.5, about 250 mM of glycine, or about 150 mM of NaCl.

6. A stable liquid composition according to claim 1, comprising about 100 mg / ml of an antibody against TG2, about 50 mM citrate buffer that maintains a pH of 6.5 or about 6.5, about 250 mM glycine, or about 150 mM NaCl.

7. The stable liquid composition of claim 5 or 6, further comprising approximately 0.02-0.06% w / v polysorbate.

8. A stable liquid composition according to claim 1, comprising approximately 100 mg / ml of an antibody against TG2, 50 mM of a histidine buffer that maintains a pH of 5.5, and 250 mM of glycine.

9. A method for preparing a stable liquid composition according to any one of claims 1-8, comprising the steps of forming a mixture of an antibody against TG2 together with 1) a histidine or citrate buffer, 2) glycine or NaCl.

10. The method for preparing a stable liquid composition according to claim 9, further comprising the step of adding a polysorbate surfactant.

11. An article of manufacture comprising a container containing a stable liquid composition according to any one of claims 1 to 8, wherein the composition comprises an antibody against tissue transglutaminase (TG2) for binding to a tissue transglutaminase (TG2) protein.

12. A stable liquid composition according to any one of claims 1-8 for therapy.

13. A method of treating a TG2-mediated disease or disorder by administering a stable liquid composition according to any one of claims 1-8.

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