High-concentration formulation of factor XII antigen-binding protein

Abstract

The present application relates to a liquid formulation comprising at least 100 mg / ml of a protein comprising an antigen-binding domain that binds to FXII (and / or its activated form), an organic acid buffer, a non-ionic surfactant, and an amino acid stabilizer, wherein the formulation has a pH of 5.0-6.5 and a viscosity of less than 30 mPa*s at 20° C. An exemplary formulation comprises the specific factor XII antibody 3F7 in a formulation comprising a histidine buffer, a polysorbate 80 surfactant, and the amino acid stabilizers arginine and proline.

Description

[Technical Field]

[0001] This disclosure relates to high-concentration protein preparations and their use. [Background technology]

[0002] Normal blood clotting is a highly conserved process in mammalian biology, involving a complex physiological and biochemical process that includes the activation of a clotting factor (or agglutination factor) cascade, which ultimately leads to fibrin formation and platelet aggregation. The blood clotting cascade includes “exogenous” pathways, primary means of coagulation initiation, and “endogenous” pathways that contribute to the stabilization of fibrin blocks.

[0003] Most coagulation factors involved in the coagulation cascade are precursors of proteolytic enzymes, also known as enzyme precursors. These enzymes circulate in the blood in an inactive form and only participate in the coagulation cascade when they are activated (e.g., by proteolytic cleavage).

[0004] Factor XII (FXII, Hagemann factor) is an essential coagulation protein for the initiation of the endogenous coagulation cascade. Activation of FXII, which produces activated FXII (FXIIa), leads to the conversion of factor XI to factor XIa, and to the activation of C1 esterases (C1r, C1s), C1, and the first component of the macromolecular complex of the classical complement pathway. Activation of FXII leads to a series of proteolytic reactions resulting in thrombin generation and hemostatic pathways, while activation of the complement system leads to increased vascular permeability, phagocytic chemotropy of phagocytic cells, activation of inflammatory cells, opsonization of foreign particles, direct cell killing, and tissue damage.

[0005] Despite its role in activating the endogenous coagulation cascade and the classical complement pathway, FXII deficiency is not associated with bleeding abnormalities. However, dysregulation of these pathways can lead to serious conditions.

[0006] While antibodies and inhibitors against FXII and / or FXIIa exist, drug manufacturers face increasing challenges in formulation development. For example, an ideal inhibitor of FXII / FXIIa should not increase the risk of bleeding, be non-immunogenic, and be administered in the lowest possible dose. Furthermore, subcutaneous administration is rapidly becoming the preferred method, replacing intravenous administration. Subcutaneous injection of drugs using self-administering delivery devices such as auto-injectors, pens, or pre-filled syringes is not only more convenient for patients but also reduces healthcare costs by minimizing hospital visits.

[0007] However, formulating high-concentration antibody preparations suitable for subcutaneous administration (e.g., ≥100 mg / ml protein) presents numerous challenges. Subcutaneous formulations typically require higher concentrations to achieve smaller injection volumes, but increasing protein concentration often negatively impacts protein aggregation and degradation, solubility, stability, and viscosity. In addition to changes in endogenous protein properties, manufacturing challenges exist, including difficulties in processing and storage, changes in formulation composition, and the rheological properties and syringe passability of the final formulation. For example, viscous solutions typically require higher injection force for administration, and therefore prolonged injection time, which can contribute to patient pain and discomfort.

[0008] Various solutions for producing high-concentration antibody preparations include lyophilized preparations for reconstitution, unbuffered preparations, and the addition of high concentrations of salts or other excipients to reduce aggregation and / or viscosity of the preparations. However, the use of excessive amounts of such excipients can lead to changes in the ionic strength of hypertonic preparations or formulations and associated protein aggregation problems. [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] Therefore, there is a need for protein-based therapeutic agents, such as formulations containing antibodies that target the coagulation pathway, which are stable over long periods and do not aggregate at high antibody concentrations. [Means for solving the problem]

[0010] This disclosure is based on the identification of pharmaceutical formulations for proteins containing antigen-binding domains that bind to or specifically bind to factor XII and / or its activated form (i.e., activated FXII; FXIIa).

[0011] The inventors have found that in the production of a suitable formulation, it is possible to produce a pharmaceutical formulation, such as a liquid pharmaceutical formulation, that contains a high concentration of protein (i.e., a concentration of at least about 100 mg / ml) including antigen-binding domains that bind to or specifically bind to FXII and / or FXIIa, remains soluble, and maintains a viscosity suitable for injection (e.g., by subcutaneous administration). The high-concentration formulations of the present disclosure comprise organic acids, nonionic surfactants, amino acid stabilizers, and optionally polyols. Notably, the inventors have found that the production of the formulations of the present disclosure does not require additional salts and / or stabilizers. Furthermore, the inventors have found that increasing the protein concentration of the antibody in the formulation (e.g., from about 100 mg / ml to about 170 mg / ml) does not necessarily require a change in the concentration of excipients (i.e., an increase or decrease).

[0012] These findings by the inventors provide a basis for a pharmaceutical formulation comprising at least about 100 mg / ml of protein containing an antigen-binding domain that binds to or specifically binds to factor XII and / or its activated form, and having a dynamic (i.e., absolute) viscosity of less than about 30 mPa*s at 20°C. These findings by the inventors also provide a basis for a method of treating a condition or disorder in a subject, such as a thrombotic disorder, an inflammatory disorder, and / or a thromboinflammatory disorder.

[0013] This disclosure provides a liquid pharmaceutical formulation comprising at least about 100 mg / ml of protein containing an antigen-binding domain that binds to or specifically binds to factor XII and / or its activated form, an organic acid buffer, a nonionic surfactant, and an amino acid stabilizer, wherein the liquid pharmaceutical formulation has a pH of 5.0 to 6.5 and a dynamic viscosity of less than about 30 mPa*s at 20°C.

[0014] In one example, the protein of this disclosure is present in the formulation at a concentration of at least 100 mg / ml. For example, the protein is present in the formulation at a concentration of 100 mg / ml to 200 mg / ml. For example, the protein is present in the formulation at a concentration of approximately 100 mg / ml, or approximately 110 mg / ml, or approximately 120 mg / ml, or approximately 130 mg / ml, or approximately 140 mg / ml, or approximately 150 mg / ml, or approximately 160 mg / ml, or approximately 170 mg / ml, or approximately 180 mg / ml, or approximately 190 mg / ml, or approximately 200 mg / ml. In one example, the protein is present in the formulation at a concentration of approximately 100 mg / ml, or approximately 120 mg / ml, or approximately 150 mg / ml, or approximately 170 mg / ml. In one example, the protein is present in the formulation at a concentration of approximately 100 mg / ml. In one example, the protein is present in the formulation at a concentration of approximately 150 mg / ml. In one example, the protein is present in the formulation at a concentration of at least approximately 150 mg / ml. In another example, the protein is present in the formulation at a concentration of approximately 160 mg / ml to approximately 180 mg / ml. For example, the protein is present in the formulation at a concentration of approximately 170 mg / ml. In yet another example, the protein is present in the formulation at a concentration higher than 200 mg / ml. For example, the protein is present in the formulation at a concentration of approximately 200 mg / ml, or approximately 210 mg / ml, or approximately 220 mg / ml.

[0015] For example, a protein containing an antigen-binding domain that binds to or specifically binds to factor XII and / or its activated form binds to factor XII or activated factor XII (factor XIIa). For example, a protein that binds to factor XII / XIIa binds to or specifically binds to factor XII. For example, a protein that binds to factor XII / XIIa binds to or specifically binds to activated factor XII (factor XIIa).

[0016] In one example, a protein binds to or specifically binds to factor XII and antagonizes the activity of factor XII and / or factor XIIa. In another example, a protein binds to or specifically binds to factor XII and antagonizes the activation of factor XII and / or factor XIIa. References herein to proteins or antibodies that "bind" to factor XII provide literal support for proteins or antibodies that "specifically bind" to factor XII.

[0017] In one example, a protein binds to or specifically binds to activated factor XII (factor XIIa) and antagonizes the activity of factor XII and / or factor XIIa. In another example, a protein binds to or specifically binds to activated factor XII and antagonizes the activation of factor XII and / or factor XIIa. The references herein to proteins or antibodies that "bind" to activated factor XIIa provide literal support for proteins or antibodies that "specifically bind" to activated factor XIIa.

[0018] For example, a protein contains the antigen-binding domain of an antibody. For instance, a protein has at least a heavy chain variable region (V H ) and light chain variable region (V L ) including V H and V L It binds to form an Fv containing an antigen-binding domain.

[0019] For example, V H and V Lis within a single polypeptide chain. For example, the protein is as follows: (i) single-chain Fv fragment (scFv); (ii) dimeric scFv (di-scFv); or (iii) a constant region of an antibody, Fc or heavy-chain constant domain (C H )C H 2 and / or C H 3 linked to at least one of (i) and / or (ii).

[0020] In one example, V L and V H are in separate polypeptide chains. For example, the protein is as follows: (i) diabody; (ii) triabody; (iii) tetrabody; (iv) Fab; (v) F(ab’)2; (vi) Fv; or (vii) a constant region of an antibody, Fc or C H 2 and / or C H 3 linked to one of (i)-(vi).

[0021] <> The aforementioned proteins (described in the previous two lists) can also be referred to as antigen-binding domains of an antibody.

[0022] In one example, the protein is an antibody or an antigen-binding fragment thereof (e.g., scFv containing the variable region of an antibody). Exemplary antibodies are described, for example, in WO2013 / 01409 and WO2017 / 173494, which are incorporated herein by reference.

[0023] In one example, the protein comprises an scFv. In one example, the protein comprises an scFv that binds or specifically binds to factor XII and / or factor XIIa (and, for example, antagonizes the activity of factor XII and / or factor XIIa, or antagonizes the activation of factor XII and / or factor XIIa).

[0024] In one example, a protein containing an antigen-binding domain that binds to factor XII and / or factor XIIa is an antibody, i.e., a full-length antibody. For example, the antibody is an anti-FXII antibody. In another example, the antibody is an anti-FXIIa antibody.

[0025] In one example, the protein is recombinant, chimeric, deimmunized, humanized, human, or primate-like. In another example, the protein or antibody is human. In yet another example, the protein or antibody is humanized.

[0026] In one example, the antibody is a monoclonal antibody.

[0027] In one example, the antibody is an IgG antibody. For instance, the antibody could be IgG1, IgG2, IgG3, or IgG4.

[0028] In one example, the antibody is an IgG4 antibody.

[0029] In one example, the antibody is a monoclonal IgG4 antibody.

[0030] In one example, the protein contains an Fc region. For example, the Fc region is a human IgG1Fc region, a human IgG4Fc region, or a stabilized human IgG4Fc region. For example, the Fc region is a human IgG4Fc region. In one example, the antibody Fc region is modified to inhibit dimerization (for example, as discussed herein).

[0031] In one example, the antibody or its antigen-binding fragment contains the IgG4 constant region.

[0032] For example, the IgG4 constant region is a stabilized IgG4 constant region. For instance, the IgG4 constant region includes a stabilized hinge region. For example, the stabilized IgG4 constant region includes proline at position 241 of the hinge region according to Kabat's system (Kabat et al., Sequences of Proteins of Immunological Interest, Washington DC, United States Department of Health and Human Services, 1987 and / or 1991).

[0033] For example, a protein includes an antigen-binding domain that binds to or specifically binds to at least factor XII and / or its activated form. This does not mean that the proteins of this disclosure do not bind to other proteins, but merely that the protein (or a part thereof) is specific to factor XII and / or activated factor XII and does not bind to proteins in general. Furthermore, this term does not exclude, for example, a bispecific antibody or a protein containing an antigen-binding domain that can specifically bind to factor XII and / or activated factor XII by one (or more) binding domains and specifically bind to another protein by another binding domain.

[0034] For example, a protein can be monospecific, bispecific, or multispecific. For instance, a protein may contain an antigen-binding domain that can specifically bind to factor XII and / or activated factor XII, as well as an antigen-binding domain that can specifically bind to another protein.

[0035] For example, the antigen-binding domain can be monospecific, bispecific, or multispecific.

[0036] In one example, the antigen-binding domain exhibits single-specificity.

[0037] In one example, the antigen-binding domain is not bispecific.

[0038] For example, a protein has a heavy chain variable region (V) containing the sequence shown in Sequence ID No. 1. H ) and the light chain variable region (V) containing the sequence shown in Sequence ID No. 2 L It contains an antibody variable region that competitively inhibits the binding of antibody 3F7, which contains ), to factor XII.

[0039] For example, a protein has a heavy chain variable region (V) containing the sequence shown in Sequence ID No. 3. H ) and the light chain variable region (V) containing the sequence shown in Sequence ID No. 4 L It contains an antibody variable region that competitively inhibits the binding of germline antibody 3F7 (3F7G), which includes ), to factor XII.

[0040] For example, a protein has a heavy chain variable region (V) containing the sequence shown in Sequence ID No. 5. H ) and the light chain variable region (V) containing the sequence shown in Sequence ID No. 6 L ) Affinity matured antibody 3F7 (3F7 aff It contains an antibody variable region that competitively inhibits the binding of ) to factor XII.

[0041] In one example, the protein contains an antibody-variable region that competitively inhibits the binding of galadasimab to factor XII and / or activated factor XII.

[0042] For example, a protein has a heavy chain variable region (V) containing the amino acid sequence shown in SEQ ID NO: 1. H ) and the light chain variable region (V) containing the amino acid sequence shown in SEQ ID NO: 2 L ) includes.

[0043] For example, a protein contains the amino acid sequence shown in SEQ ID NO: 1. H The heavy chain variable region (V) includes the complementarity determination region (CDR). H ), and V containing the amino acid sequence shown in Sequence ID No. 2 L The light chain variable region (V) including the CDR LIt is an antibody containing ) or an antigen-binding fragment thereof.

[0044] For example, proteins include: (i) V including the following H : (a) CDR1 containing the sequence shown at amino acids 25-34 of SEQ ID NO: 1; (b) CDR2 containing the sequence shown in amino acids 49-65 of SEQ ID NO: 1; and (c) CDR3 containing the sequence shown at amino acids 98-108 of SEQ ID NO: 1; and / or (ii) V including the following L : (a) CDR1 containing the sequence shown in amino acids 23-33 of SEQ ID NO: 2; (b) CDR2 containing the sequence shown at amino acids 49-55 of SEQ ID NO: 2; and (c) CDR3 containing the sequence shown in amino acids 88-96 of SEQ ID NO: 2.

[0045] For example, proteins include: (i) V including the following H : (a) The sequence shown in Sequence ID 1; or (b) CDR1 containing the sequence shown in SEQ ID NO: 7; CDR2 containing the sequence shown in SEQ ID NO: 8; and CDR3 containing the sequence shown in SEQ ID NO: 9; or (c) CDR1 containing the sequence shown in SEQ ID NO: 7; CDR2 containing the sequence shown in SEQ ID NO: 10; and CDR3 containing the sequence shown in SEQ ID NO: 11; and / or (ii) V including the following L : (a) The sequence shown in Sequence ID 2; or (b) CDR1 containing the sequence shown in SEQ ID NO: 12; CDR2 containing the sequence shown in SEQ ID NO: 13; and CDR3 containing the sequence shown in SEQ ID NO: 14; or (c) CDR1 containing the sequence shown in SEQ ID NO: 12; CDR2 containing the sequence shown in SEQ ID NO: 13; and CDR3 containing the sequence shown in SEQ ID NO: 15.

[0046] For example, the protein is an antibody that includes the following: (i) V including the following H : (a) CDR1 containing the sequence shown in Sequence ID No. 7; (b) CDR2 containing the sequence shown in Sequence ID No. 8; and (c) CDR3 containing the sequence shown in Sequence ID 9; and / or (ii) V including the following L : (a) CDR1 containing the sequence shown in sequence number 12; (b) CDR2 containing the sequence shown in Sequence ID No. 13; and (c) CDR3 containing the sequence shown in Sequence ID No. 14.

[0047] For example, the protein is an antibody that includes the following: (i) V including the following H : (a) CDR1 containing the sequence shown in Sequence ID No. 7; (b) CDR2 containing the sequence shown in Sequence ID No. 10; and (c) CDR3 containing the sequence shown in Sequence ID No. 11; and / or (ii) V including the following L : (a) CDR1 containing the sequence shown in sequence number 12; (b) CDR2 containing the sequence shown in Sequence ID No. 13; and (c) CDR3 containing the sequence shown in sequence number 15.

[0048] For example, the protein contains V, which includes CDR2 as shown in SEQ ID NO: 10. H Includes.

[0049] For example, V HThe amino acid sequence of CDR2 includes arginine (R), asparagine (N), or aspartic acid (D) at position 3, and / or proline (P), valine (V), isoleucine (I), or methionine (M) at position 4, and / or serine (S), proline (P), or alanine (A) at position 5, and / or glycine (G), leucine (L), valine (V), or threonine (T) at position 6, and / or any amino acid at position 7, and / or threonine (T), glycine (G), or serine (S) at position 8.

[0050] For example, V H The amino acid sequence of CDR2 includes asparagine (N) at position 3, valine (V) at position 4, proline (P) at position 5, leucine (L) at position 6, tyrosine (Y) at position 7, and glycine (G) at position 8.

[0051] For example, V H The amino acid sequence of CDR2 includes asparagine (N) at position 3, valine (V) at position 4, proline (P) at position 5, valine (V) at position 6, glutamine (Q) at position 7, and glycine (G) at position 8.

[0052] For example, V H The amino acid sequence of CDR2 includes aspartic acid (D) at position 3, isoleucine (I) at position 4, proline (P) at position 5, threonine (T) at position 6, lysine (K) at position 7, and glycine (G) at position 8.

[0053] For example, V H The amino acid sequence of CDR2 includes aspartic acid (D) at position 3, methionine (M) at position 4, proline (P) at position 5, threonine (T) at position 6, lysine (K) at position 7, and glycine (G) at position 8.

[0054] For example, the protein is an antibody that includes the following: (i) V including the following H : (a) CDR1 shown in Sequence ID 7; (b) CDR2 shown in Sequence ID 10, where the 3rd X is D, the 4th X is I, the 5th X is P, the 6th X is T, the 6th X is T, the 7th X is K, and the 8th X is G; and (c) CDR3 shown in Sequence ID No. 9; and / or (ii) V including the following L : (a) CDR1 shown in Sequence ID No. 12; (b) CDR2 shown in Sequence ID No. 13; and (c) CDR3 shown in Sequence ID No. 14.

[0055] For example, the protein contains V, which is shown in SEQ ID NO: 11. H Includes.

[0056] For example, V H The amino acid sequence of CDR3 includes isoleucine (I), methionine (M), or valine (V) at position 9, and / or serine (S) or lysine (K) at position 10, and / or proline (P), lysine (K), threonine (T), or histidine (H) at position 11, and / or histidine (H), asparagine (N), glycine (G), or glutamine (Q) at position 12.

[0057] For example, the protein contains V, which is shown in SEQ ID NO: 15. L Includes.

[0058] For example, V LThe amino acid sequence of CDR3 is: alanine (A) or serine (S) at position 2, and / or aspartic acid (D), tyrosine (Y), glutamic acid (E), threonine (T), tryptophan (W) or serine (S) at position 4, and / or alanine (A), asparagine (N), isoleucine (I), leucine (L), valine (V), proline (P), glutamine (Q) or glutamic acid (E) at position 5, and / or It contains serine (S), aspartic acid (D), proline (P), glutamic acid (E), glutamine (Q), or arginine (R) at position 6, and / or leucine (L) or valine (V) at position 7, and / or glycine (G), leucine (L), or lysine (K) at position 9, and / or valine (V), alanine (A), aspartic acid (D), threonine (T), methionine (M), or glycine (G) at position 10.

[0059] For example, a protein has a heavy chain variable region (V) containing the amino acid sequence shown in SEQ ID NO: 3. H ) and the light chain variable region (V) containing the amino acid sequence shown in SEQ ID NO: 4 L ) includes.

[0060] For example, the protein contains the amino acid sequence shown in SEQ ID NO: 3. H V including the complementarity determination region (CDR) H、 and V containing the amino acid sequence shown in SEQ ID NO: 4 L V including CDR L It is an antibody or its antigen-binding fragment containing the following. For example, the protein contains: (i) V including the following H : (a) CDR1 containing the sequence shown at amino acids 25-34 of SEQ ID NO: 3; (b) CDR2 containing the sequence shown at amino acids 49-65 of SEQ ID NO: 3; and (c) CDR3 containing the sequence shown at amino acids 98-108 of SEQ ID NO: 3; and / or (ii) V including the following L : (a) CDR1 containing the sequence shown in amino acids 23-33 of SEQ ID NO: 4; (b) CDR2 containing the sequence shown at amino acids 49-55 of SEQ ID NO: 4; and (c) CDR3 containing the sequence shown in amino acids 88-96 of SEQ ID NO: 4.

[0061] In one example, the protein of this disclosure is an affinity-matured, chimeric, CDR-transplanted, or humanized antibody, or an antigen-binding fragment thereof. In one example, the protein is an affinity-matured form of antibody 3F7. In another example, the protein is galadasimab.

[0062] For example, a protein has a heavy chain variable region (V) containing the amino acid sequence shown in SEQ ID NO: 5. H ) and the light chain variable region (V) containing the amino acid sequence shown in SEQ ID NO: 6 L ) includes.

[0063] For example, the protein contains the amino acid sequence shown in SEQ ID NO: 5. H V including the complementarity determination region (CDR) H , and V containing the amino acid sequence shown in Sequence ID No. 6 L V including CDR L It is an antibody containing or an antigen-binding fragment thereof.

[0064] For example, proteins include: (i) V including the following H : (a) CDR1 containing the sequence shown at amino acids 25-34 of SEQ ID NO: 5; (b) CDR2 containing the sequence shown in amino acids 49-65 of SEQ ID NO: 5; and (c) CDR3 containing the sequence shown at amino acids 98-108 of SEQ ID NO. 5; and / or (ii) V including the following L : (a) CDR1 containing the sequence shown at amino acids 23-33 of SEQ ID NO: 6; (b) CDR2 containing the sequence shown at amino acids 49-55 of SEQ ID NO: 6; and (c) CDR3 containing the sequence shown in amino acids 88-96 of SEQ ID NO: 6.

[0065] For example, proteins include: (i) V including the following H : (a) CDR1 containing the sequence shown in Sequence ID No. 7; (b) CDR2 containing the sequence shown in Sequence ID No. 16; and (c) CDR3 containing the sequence shown in Sequence ID No. 9; and / or (ii) V including the following L : (a) CDR1 containing the sequence shown in sequence number 12; (b) CDR2 containing the sequence shown in Sequence ID No. 13; and (c) CDR3 containing the sequence shown in Sequence ID No. 14.

[0066] For example, a protein, antibody, or its antigen-binding fragment is any form of a protein, antibody, or its functional fragment encoded by a nucleic acid that encodes one of the aforementioned proteins, antibodies, or functional fragments.

[0067] In one example, the organic acid buffer is selected from the group consisting of histidine buffer, glutamate buffer, succinate buffer, and citrate buffer. In another example, the organic acid buffer is selected from the group consisting of histidine buffer and glutamate buffer.

[0068] For example, an organic acid buffer is an amino acid buffer. For instance, an amino acid buffer is selected from the group consisting of histidine buffers and glutamate buffers.

[0069] Advantageously, histidine buffers and glutamate buffers have higher thermal stability and agglomeration stability (i.e., reduced tendency to aggregate) compared to citrate buffers and / or succinate buffers.

[0070] In one example, the organic acid buffer is a histidine buffer. Suitable histidine buffers for use in this disclosure will become apparent to those skilled in the art and include, for example, histidine chloride, histidine acetate, histidine phosphate, and histidine sulfate. In one example, the histidine buffer is L-histidine.

[0071] For example, an organic acid buffer is a glutamate buffer. Suitable glutamate buffers for use in this disclosure will become apparent to those skilled in the art, and include, for example, monosodium glutamate.

[0072] For example, an organic acid buffer is a succinic acid buffer. Suitable succinic acid buffers for use in this disclosure will become apparent to those skilled in the art and include, for example, succinic acid-monosodium succinate mixtures, succinic acid-sodium hydroxide mixtures, and succinic acid-disodium succinate mixtures.

[0073] For example, an organic acid buffer is a citrate buffer. Suitable citrate buffers for use in this disclosure will become apparent to those skilled in the art and include, for example, monosodium citrate-disodium citrate mixtures, citrate-trisodium citrate mixtures, and citrate-monosodium citrate mixtures.

[0074] It will be apparent to those skilled in the art that buffers suitable for use in this disclosure provide sufficient buffering capacity to maintain a desired pH over a range of conditions to which the product is exposed during formulation and storage. In one example, the formulations of this disclosure have a pH of about 5.0 to about 7.0. For example, the formulations have a pH of about 5.0 to about 6.5 or about 5.8 to about 6.4. For example, an organic acid buffer is a histidine buffer having a pH of about 5.5 to about 5.7. In one example, an organic acid buffer is a glutamate buffer having a pH of about 5.5. In one example, an organic acid buffer is a succinate buffer having a pH of about 5.5. In one example, an organic acid buffer is a citrate buffer having a pH of about 5.5. In one example, an organic acid buffer has a pH of about 5.5 to about 6.5, or about 5.6 to about 6.4, or about 5.8 to about 6.4. In one example, the formulations have a pH of 5.8 to 6.4. For example, the formulation has a pH of approximately 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, or 6.4. In one example, the organic acid buffer is a histidine buffer, and the formulation has a pH of approximately 5.8 to 6.4. In another example, the organic acid buffer is a glutamate buffer, and the formulation has a pH of approximately 5.8 to 6.4. In yet another example, the organic acid buffer is a succinate buffer, and the formulation has a pH of approximately 5.8 to 6.4. In yet another example, the organic acid buffer is a citrate buffer, and the formulation has a pH of approximately 5.8 to 6.4.

[0075] In one example, the concentration of the organic acid buffer in the pharmaceutical formulation of this disclosure is approximately 2 mM to 120 mM. In one example, the organic acid buffer is present at a concentration of at least 2 mM. For example, the organic acid buffer is present at a concentration of approximately 2 mM to approximately 10 mM. For example, the organic acid buffer is present at a concentration of approximately 2 mM, or approximately 3 mM, or approximately 4 mM, or approximately 5 mM, or approximately 6 mM, or approximately 7 mM, or approximately 8 mM, or approximately 9 mM, or approximately 10 mM. In one example, the organic acid buffer is present at a concentration of at least approximately 10 mM. For example, the organic acid buffer is present at a concentration of approximately 10 mM to approximately 30 mM. For example, the organic acid buffer is present at a concentration of approximately 10 mM, or approximately 12 mM, or approximately 14 mM, or approximately 16 mM, or approximately 18 mM, or approximately 20 mM, or approximately 25 mM, or approximately 30 mM. For example, organic acid buffers are present at concentrations of approximately 12 mM to 25 mM. For example, organic acid buffers are present at concentrations of approximately 20 mM. For example, organic acid buffers are present at concentrations of approximately 10 mM to 60 mM. For example, organic acid buffers are present at concentrations of approximately 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, or 60 mM. For example, organic acid buffers are present at concentrations of approximately 20 mM. For example, organic acid buffers are present at concentrations of approximately 60 mM to 120 mM. For example, organic acid buffers are present at concentrations of approximately 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115 mM, or 120 mM. In one example, the organic acid buffer is present at a concentration of approximately 100 mM.

[0076] In one example, the organic acid buffer is L-histidine, present at a concentration of approximately 12 mM to 25 mM. In another example, the organic buffer is L-histidine, present at a concentration of approximately 20 mM.

[0077] For example, nonionic surfactants are selected from the group consisting of polyoxyethylene sorbitan fatty acid esters (e.g., polysorbate 20 and polysorbate 80), polyethylene-polypropylene copolymers, polyethylene-polypropylene glycols, polyoxyethylene stearates, polyoxyethylene alkyl ethers (e.g., polyoxyethylene monolauryl ether, alkylphenyl polyoxyethylene ether (Triton-X)), polyoxyethylene-polyoxypropylene copolymers (poloxamer, pluronic), and sodium dodecyl sulfate (SDS). For example, nonionic surfactants are selected from the group consisting of polyoxyethylene sorbitan fatty acid esters and polyoxyethylene-polyoxypropylene copolymers.

[0078] For example, the nonionic surfactant is selected from the group consisting of polysorbate 20, polysorbate 80, and poloxamer 188. For instance, the nonionic surfactant is polysorbate 80.

[0079] In one example, the concentration of the nonionic surfactant in the pharmaceutical formulation of this disclosure is approximately 0.01% (weight / volume) to approximately 1.00% (weight / volume). In one example, the nonionic surfactant is present at a concentration of at least approximately 0.01% (weight / volume). For example, the nonionic surfactant is present at a concentration of approximately 0.01% (weight / volume) to approximately 0.10% (weight / volume). For example, the nonionic surfactant is present at a concentration of approximately 0.01% (weight / volume), or approximately 0.02% (weight / volume), or approximately 0.03% (weight / volume), or approximately 0.04% (weight / volume), or approximately 0.05% (weight / volume), or approximately 0.06% (weight / volume), or approximately 0.07% (weight / volume), or approximately 0.08% (weight / volume), or approximately 0.09% (weight / volume), or approximately 0.10% (weight / volume). In one example, nonionic surfactants are present at a concentration of approximately 0.02% (weight / volume) or approximately 0.05% (weight / volume). For example, nonionic surfactants are present at a concentration of approximately 0.02% (weight / volume). In another example, nonionic surfactants are present at a concentration of approximately 0.05% (weight / volume). In one example, nonionic surfactants are present at a concentration of approximately 0.01% (weight / volume) to approximately 0.03% (weight / volume). In one example, the nonionic surfactant is polysorbate 80, and it is present at a concentration of approximately 0.01% (weight / volume) to approximately 0.03% (weight / volume). In another example, the nonionic surfactant is polysorbate 80, and it is present at a concentration of approximately 0.02% (weight / volume).

[0080] For example, a pharmaceutical preparation may include an amino acid stabilizer selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, methionine, threonine, phenylalanine, tyrosine, serine, cysteine, histidine, tryptophan, proline, aspartic acid, glutamic acid, arginine, lysine, ornithine, and asparagine. For example, the amino acid stabilizer may be selected from the group consisting of proline, arginine, their salts, and combinations thereof. For example, the amino acid stabilizer may be a salt form of the amino acid discussed herein.

[0081] In one example, the amino acid stabilizer is proline. In another example, the amino acid stabilizer is L-proline.

[0082] In one example, the amino acid stabilizer is arginine. In another example, the amino acid stabilizer is L-arginine. In yet another example, the amino acid stabilizer is L-arginine monohydrochloride.

[0083] For example, the preparation contains proline and arginine. For instance, the preparation contains L-proline and L-arginine or L-arginine monohydrochloride.

[0084] Advantageously, proline exhibits a significant effect on thermal stability and aggregation stability (i.e., a reduced tendency towards aggregation) compared to phenylalanine, arginine, and sorbitol.

[0085] In one example, the concentration of the amino acid stabilizer in the pharmaceutical formulation of this disclosure is approximately 50 mM to approximately 250 mM. In another example, the amino acid stabilizer is present at a concentration of approximately 90 mM to approximately 200 mM. For example, the amino acid stabilizer is present at a concentration of approximately 90 mM, or approximately 100 mM, or approximately 110 mM, or approximately 120 mM, or approximately 130 mM, or approximately 140 mM, or approximately 150 mM, or approximately 160 mM, or approximately 170 mM, or approximately 180 mM, or approximately 190 mM, or approximately 200 mM. In one example, the amino acid stabilizer is present at a concentration of approximately 100 mM to approximately 160 mM. In yet another example, the amino acid stabilizer is present at a concentration of approximately 90 mM to approximately 150 mM. For example, the amino acid stabilizer is present at a concentration of approximately 140 mM. In yet another example, the amino acid stabilizer is present at a concentration of approximately 150 mM.

[0086] The aforementioned discussion regarding concentrations also applies to the salt forms of amino acid stabilizers; the concentrations listed herein are not the concentrations of the amino acids themselves, but rather the concentrations of the amino acid salt forms.

[0087] In one example, the formulation contains proline at a concentration of 110 mM to 170 mM, for example, about 140 mM or about 150 mM. In another example, the formulation contains proline at a concentration of 90 mM to 150 mM, for example, about 140 mM.

[0088] In one example, the formulation further comprises arginine at a concentration of 110 mM to 170 mM, for example, about 150 mM. In another example, the formulation further comprises arginine at a concentration of 100 mM to 160 mM, for example, about 150 mM. In one example, the arginine is in the form of a salt of arginine, for example, arginine monohydrochloride, and the concentrations listed herein are the concentrations of the salt form of arginine, not the concentration of arginine itself.

[0089] For example, the formulation contains proline at a concentration of 90 mM to 150 mM and arginine at a concentration of 100 mM to 160 mM. For instance, the formulation contains 140 mM L-proline and 150 mM L-arginine monohydrochloride.

[0090] In one example, the pharmaceutical preparation further contains a polyol. For instance, the polyol is selected from sugars, sugar alcohols, and sugar acids (i.e., aldaric acids).

[0091] For example, a polyol is a sugar. For example, a sugar can be a reducing sugar or a non-reducing sugar. For example, a polyol is a non-reducing sugar selected from the group consisting of fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose, and glucose. For example, a polyol is a reducing sugar selected from the group consisting of sucrose, trehalose, sorbose, melegitose, and raffinose.

[0092] In one example, a polyol is a sugar alcohol. For example, sugar alcohols are selected from the group consisting of mannitol, xylitol, erythritol, threitol, sorbitol, and glycerol. In one example, the sugar alcohol is sorbitol.

[0093] For example, polyols are sugar acids such as L-gluconic acid and its metal salts.

[0094] In one example, the concentration of polyol in the pharmaceutical formulation of this disclosure is approximately 50 mM to approximately 250 mM. In one example, the polyol is present at a concentration of approximately 60 mM to approximately 140 mM. For example, the polyol is present at a concentration of approximately 60 mM, or approximately 70 mM, or approximately 80 mM, or approximately 90 mM, or approximately 100 mM, or approximately 110 mM, or approximately 120 mM, or approximately 130 mM, or approximately 140 mM. In one example, the polyol is present at a concentration of approximately 80 mM.

[0095] For example, the formulation does not contain polyols. For instance, the formulation does not contain sugars, sugar alcohols, or sugar acids.

[0096] For example, the formulation does not contain salts. For instance, the formulation does not contain, for example, sodium chloride, calcium chloride, and / or potassium chloride. The above discussion is not related to the salt forms of amino acids disclosed herein.

[0097] In one example, the formulation has a dynamic (i.e., absolute) viscosity of less than approximately 30 mPa*s at 20°C. For example, the formulation has a dynamic viscosity of approximately 30 mPa*s, or approximately 28 mPa*s, or approximately 26 mPa*s, or approximately 24 mPa*s, or approximately 22 mPa*s, or approximately 20 mPa*s at 20°C. In one example, the formulation has a dynamic (i.e., absolute) viscosity of less than approximately 20 mPa*s at 20°C. For example, the formulation has a dynamic viscosity of approximately 20 mPa*s, or approximately 19 mPa*s, or approximately 18 mPa*s, or approximately 17 mPa*s, or approximately 16 mPa*s, or approximately 15 mPa*s at 20°C. In one example, the formulation has a dynamic viscosity of less than approximately 15 mPa*s at 20°C. For example, the formulation has a dynamic viscosity of approximately 15 mPa*s, or approximately 14 mPa*s, or approximately 13 mPa*s, or approximately 12 mPa*s, or approximately 11 mPa*s, or approximately 10 mPa*s at 20°C. In one example, the formulation has a dynamic viscosity of less than approximately 10 mPa*s at 20°C. For example, the formulation has a dynamic viscosity of approximately 10 mPa*s, or approximately 9 mPa*s, or approximately 8 mPa*s, or approximately 7 mPa*s, or approximately 6 mPa*s, or approximately 5 mPa*s, or approximately 4 mPa*s, or approximately 3 mPa*s, or approximately 2 mPa*s at 20°C. In one example, the formulation has a dynamic viscosity of approximately 3.0 mPa*s to approximately 4.0 mPa*s at 20°C. For example, the formulation has a dynamic viscosity of approximately 3.3 mPa*s at 20°C. In another example, the formulation has a dynamic viscosity of approximately 8.0 mPa*s to approximately 10.0 mPa*s at 20°C. For example, the formulation has a dynamic viscosity of approximately 8.9 mPa*s at 20°C. In one example, the formulation has a dynamic viscosity of approximately 9 mPa*s at 20°C.

[0098] For example, a formulation containing approximately 100 mg / ml of protein or antibody has a dynamic viscosity of less than approximately 10.0 mPa*s at 20°C. For instance, such a formulation has a dynamic viscosity of approximately 3.3 mPa*s at 20°C.

[0099] For example, a formulation containing approximately 170 mg / ml of protein or antibody has a dynamic viscosity of less than approximately 10.0 mPa*s at 20°C. For instance, such a formulation has a dynamic viscosity of approximately 8.9 mPa*s at 20°C.

[0100] In one example, the formulation has a dynamic (i.e., absolute) viscosity of less than approximately 30 mPa*s at 25°C. For example, the formulation has a dynamic viscosity of approximately 30 mPa*s, or approximately 28 mPa*s, or approximately 26 mPa*s, or approximately 24 mPa*s, or approximately 22 mPa*s, or approximately 20 mPa*s at 25°C. In one example, the formulation has a dynamic (i.e., absolute) viscosity of less than approximately 20 mPa*s at 25°C. For example, the formulation has a dynamic viscosity of approximately 20 mPa*s, or approximately 19 mPa*s, or approximately 18 mPa*s, or approximately 17 mPa*s, or approximately 16 mPa*s, or approximately 15 mPa*s at 25°C. In one example, the formulation has a dynamic viscosity of less than approximately 15 mPa*s at 25°C. For example, the formulation has a dynamic viscosity of approximately 15 mPa*s, or approximately 14 mPa*s, or approximately 13 mPa*s, or approximately 12 mPa*s, or approximately 11 mPa*s, or approximately 10 mPa*s at 25°C. In one example, the formulation has a dynamic viscosity of less than approximately 10 mPa*s at 25°C. For example, the formulation has a dynamic viscosity of approximately 10 mPa*s, or approximately 9 mPa*s, or approximately 8 mPa*s, or approximately 7 mPa*s, or approximately 6 mPa*s, or approximately 5 mPa*s, or approximately 4 mPa*s, or approximately 3 mPa*s, or approximately 2 mPa*s at 25°C. In one example, the formulation has a dynamic viscosity of approximately 2 mPa*s to approximately 9 mPa*s at 25°C. In one example, the formulation has a dynamic viscosity of approximately 1.0 mPa*s to approximately 3.0 mPa*s at 25°C. For example, the formulation has a dynamic viscosity of approximately 2.8 mPa*s at 25°C. In another example, the formulation has a dynamic viscosity of approximately 7.0 mPa*s to approximately 8.0 mPa*s at 25°C. For example, the formulation has a dynamic viscosity of approximately 7.5 mPa*s at 25°C.

[0101] For example, a formulation containing approximately 100 mg / ml of protein or antibody has a dynamic viscosity of less than approximately 10.0 mPa*s at 25°C. For instance, such a formulation has a dynamic viscosity of approximately 2.8 mPa*s at 25°C.

[0102] For example, a formulation containing approximately 170 mg / ml of protein or antibody has a dynamic viscosity of less than approximately 10.0 mPa*s at 25°C. For instance, such a formulation has a dynamic viscosity of approximately 7.5 mPa*s at 25°C.

[0103] Methods for investigating viscosity will be apparent to those skilled in the art and / or are described herein. For example, viscosity can be investigated by using a microviscometer, such as a falling ball viscometer. In a falling ball viscometer, the time it takes for a ball to fall through clear and opaque liquids is measured according to Heppler's falling ball principle. An example of a falling ball viscometer is the Anton Par Lovis 2000M microviscometer.

[0104] For example, the formulation is approximately 1.00 to 1.10 g / cm³ at 20°C. 3 It has a density of approximately 1.01 g / cm³ at 20°C. For example, the density of the formulation is approximately 1.01 g / cm³ at 20°C. 3 , or approximately 1.02 g / cm³ 3 , or approximately 1.03 g / cm³ 3 , or approximately 1.04 g / cm³ 3 , or approximately 1.05 g / cm³ 3 , or approximately 1.06 g / cm³ 3 , or approximately 1.07 g / cm³ 3 , or approximately 1.08 g / cm³ 3 , or approximately 1.09 g / cm³ 3 , or approximately 1.10 g / cm³ 3 For example, the density of the formulation is approximately 1.04 g / cm³. 3 , or approximately 1.05 g / cm³ 3 , or approximately 1.06 g / cm³ 3 , or approximately 1.07 g / cm³ 3 For example, the density of the formulation is approximately 1.06 g / cm³. 3 Methods for determining the density of a formulation will be apparent to those skilled in the art and / or are described herein. In one example, the density is determined using a densimeter, for example, a Mettler Toledo DA-100M densimeter.

[0105] In one example, the present disclosure provides a pharmaceutical formulation comprising a protein containing an antigen-binding domain that binds to or specifically binds to factor XII and / or its activated form, an organic acid buffer selected from the group consisting of histidine buffers and glutamate buffers, an amino acid stabilizer selected from the group consisting of proline, arginine, salts thereof and combinations thereof, and polysorbate 80 as a nonionic surfactant, the pharmaceutical formulation having a viscosity of less than about 10 mPa*s at 20°C.

[0106] In one example, the formulation comprises a protein containing an antigen-binding domain that binds to or specifically binds to factor XII and / or its activated form, a histidine buffer or glutamate buffer, proline, and polysorbate 80. For example, the formulation comprises an antibody or its antigen-binding fragment, a histidine buffer, proline, and polysorbate 80. In another example, the formulation comprises an antibody or its antigen-binding fragment, a glutamate buffer, proline, and polysorbate 80. The formulation may further contain arginine or sorbitol.

[0107] This disclosure provides a liquid pharmaceutical formulation comprising approximately 170 mg / ml of antibody or its antigen-binding fragment, a histidine buffer, polysorbate 80, and proline as a stabilizer, having a pH of 5.5 to 6.5 and a viscosity of less than approximately 10 mPa*s at 20°C. For example, the formulation comprises approximately 20 mM L-histidine buffer, 0.02% (weight / volume) of polysorbate 80, and 140 mM or 150 mM L-proline having a pH of 5.8 to 6.4. In one example, the formulation further comprises 150 mM L-arginine monohydrochloride. In another example, the formulation further comprises 80 mM sorbitol.

[0108] This disclosure provides a liquid pharmaceutical formulation comprising approximately 100 mg / ml to approximately 110 mg / ml of protein containing the antigen-binding domain of this disclosure, an L-histidine buffer, polysorbate 80, and L-proline and L-arginine monohydrochloride as stabilizers, having a pH of 5.5 to 6.5 and a viscosity of less than approximately 10 mPa*s at 20°C.

[0109] This disclosure provides a liquid pharmaceutical formulation comprising approximately 160 mg / ml to approximately 180 mg / ml of protein containing the antigen-binding domain of this disclosure, an L-histidine buffer, polysorbate 80, and L-proline and L-arginine monohydrochloride as stabilizers, having a pH of 5.5 to 6.5 and a viscosity of less than approximately 10 mPa*s at 20°C.

[0110] This disclosure also provides a pharmaceutical formulation comprising approximately 170 mg / ml of the antibody or its antigen-binding fragment as described herein, a glutamate buffer, polysorbate 80, and proline as a stabilizer, having a pH of 5.5 to 6.5 and a viscosity of less than approximately 10 mPa*s at 20°C. For example, the formulation comprises approximately 100 mM glutamate buffer, 0.05% (weight / volume) polysorbate 80, and 150 mM proline, with a pH of 5.5.

[0111] This disclosure provides a pharmaceutical formulation having a pH of 5.5 to 6.5 and comprising approximately 160 mg / ml to approximately 180 mg / ml of the antibody or its antigen-binding fragment as described herein, a histidine buffer, polysorbate 80, and proline and arginine monohydrochloride as stabilizers, and having a viscosity of less than approximately 10 mPa*s at 20°C and 25°C. For example, the formulation has a pH of 5.8 to 6.4 and contains approximately 12 to approximately 25 mM of L-histidine buffer, 0.01% to 0.03% (weight / volume) of polysorbate 80, 110 mM to 170 mM of L-proline, and 110 mM to 170 mM of L-arginine. In one example, the formulation contains 12-25 mM L-histidine buffer with a pH of 5.8-6.4, 0.01%-0.03% (weight / volume) of polysorbate 80, 90 mM-150 mM L-proline, and 100 mM-160 mM L-arginine. In one example, the osmotic pressure of the formulation is approximately 430-530 mOsm / kg, for example, approximately 450 mOsm / kg. In one example, the antibody contains the amino acid sequence shown in SEQ ID NO: 5. H and V containing the amino acid sequence shown in SEQ ID NO: 6 L Includes.

[0112] This disclosure provides a pharmaceutical formulation having a pH of 5.5 to 6.5 and comprising approximately 170 mg / ml of the antibody or its antigen-binding fragment as described herein, a histidine buffer, polysorbate 80, and proline and arginine as stabilizers, and having a viscosity of less than approximately 10 mPa*s at 20°C and 25°C. For example, the formulation has a pH of 5.8 to 6.4 and contains approximately 20 mM L-histidine buffer, 0.02% (weight / volume) polysorbate 80, 140 mM L-proline, and 150 mM L-arginine. In one example, the osmotic pressure of the formulation is approximately 450 mOsm / kg. In one example, the antibody contains the amino acid sequence shown in SEQ ID NO: 5. H and V containing the amino acid sequence shown in SEQ ID NO: 6 L Includes.

[0113] This disclosure provides a pharmaceutical formulation having a pH of 5.5 to 6.5 and comprising approximately 100 mg / ml of the antibody or its antigen-binding fragment as described herein, a histidine buffer, polysorbate 80, and proline and arginine monohydrochloride as stabilizers, and having a viscosity of less than approximately 10 mPa*s at 20°C and 25°C. For example, the formulation has a pH of 5.8 to 6.4 and contains 12 to 25 mM histidine buffer, 0.01% to 0.03% (weight / volume) of polysorbate 80, 110 mM to 170 mM proline, and 110 mM to 170 mM arginine. In one example, the osmotic pressure of the formulation is approximately 430 mOsm / kg. In one example, the antibody contains the amino acid sequence shown in SEQ ID NO: 5. H and V containing the amino acid sequence shown in SEQ ID NO: 6 L Includes.

[0114] The present disclosure provides a pharmaceutical formulation comprising an antibody or an antigen-binding fragment thereof described herein at about 100 mg / ml, a histidine buffer having a pH of 5.5 to 6.5, polysorbate 80, and proline and arginine as stabilizers, the pharmaceutical formulation having a viscosity of less than about 5 mPa*s at 20°C and 25°C. For example, the formulation comprises about 20 mM histidine buffer having a pH of 5.8 to 6.4, 0.02% (weight / volume) polysorbate 80, 140 mM proline, and 150 mM arginine. In one example, the osmotic pressure of the formulation is about 430 mOsm / kg. In one example, the antibody comprises V H comprising the amino acid sequence shown in SEQ ID NO: 5 and V L comprising the amino acid sequence shown in SEQ ID NO: 6.

[0115] The present disclosure provides a pharmaceutical formulation comprising a protein at about 100 mg / ml to about 170 mg / ml having a pH of 5.5 to 6.5 and comprising an antigen-binding domain described herein, a histidine buffer, polysorbate 80, and proline and arginine hydrochloride as stabilizers, the formulation having a viscosity of less than about 30 mPa*s at 20°C, and the protein comprising V H comprising the amino acid sequence shown in SEQ ID NO: 5 and V L comprising the amino acid sequence shown in SEQ ID NO: 6. For example, the formulation has a pH of 5.8 to 6.4 and comprises about 20 mM L-histidine buffer, 0.02% (weight / volume) polysorbate 80, 140 mM L-proline, and 150 mM L-arginine hydrochloride. In one example, the osmotic pressure of the formulation is about 430 to about 450 mOsm / kg.

[0116] The present disclosure provides a pharmaceutical formulation comprising a protein at about 100 mg / ml to about 170 mg / ml having a pH of 5.5 to 6.5 and comprising an antigen-binding domain described herein, a histidine buffer, polysorbate 80, and proline and arginine hydrochloride as stabilizers, the formulation having a viscosity of less than about 30 mPa*s at 20°C, and the protein is (i) A V comprising a CDR1 containing the sequence shown in SEQ ID NO: 7; a CDR2 containing the sequence shown in SEQ ID NO: 16; and a CDR3 containing the sequence shown in SEQ ID NO: 9 H ; and (ii) A V comprising a CDR1 containing the sequence shown in SEQ ID NO: 12; a CDR2 containing the sequence shown in SEQ ID NO: 13; and a CDR3 containing the sequence shown in SEQ ID NO: 14 L Also provided is a pharmaceutical formulation comprising the same.

[0117] In one example, the high-concentration formulation of the present disclosure is a stable formulation. For example, the formulation is physically and / or thermally stable.

[0118] In one example, the formulation of the present disclosure is a liquid formulation. For example, the formulation is an aqueous formulation.

[0119] In one example, the formulation has not been lyophilized previously. In one example, the formulation is not a reconstituted formulation. For example, the formulation is a liquid formulation.

[0120] Methods for investigating thermal stability will be apparent to those skilled in the art and / or are described herein. In one example, the thermal aggregation stability is determined by differential scanning fluorimetry (DSF). For example, changes in intrinsic protein fluorescence are monitored over a series of temperatures (e.g., 20-95 °C; the temperature increase is, for example, at a rate of 0.5 °C / min) to determine the midpoint of the thermal transition (T m ; i.e., the melting temperature) and the onset temperature of melting (T onset ). Also, static light scattering is monitored at 266 nm and 473 nm to determine the onset temperature of aggregation (T agg ).

[0121] In one example, the T m of the formulation, determined by differential scanning fluorimetry, is about 55.0 °C to about 70.0 °C, such as about 59 °C to about 67 °C. For example, the T m of the formulation is about 59.0 °C, or about 60.0 °C, or about 61.0 °C, or about 62.0 °C, or about 63.0 °C, or about 64.0 °C, or about 65.0 °C, or about 66.0 °C, or about 67 °C.

[0122] For example, the T of the formulation onset The T of a pharmaceutical product is determined by differential scanning fluorescence quantification and is approximately 58.0°C to 63.0°C, or approximately 55.0°C to 70.0°C. onset The temperature is approximately 58.0°C, or approximately 59.0°C, or approximately 60.0°C, or approximately 61.0°C, or approximately 62.0°C, or approximately 63.0°C.

[0123] For example, the T of the formulation at 266 nm agg The T of the formulation at 266 nm was determined by differential scanning fluorescence quantification and is approximately 56.0°C to 65.0°C. agg The temperature is approximately 56.0°C, or approximately 57.0°C, or approximately 58.0°C, or approximately 60.0°C, or approximately 61.0°C, or approximately 62.0°C, or approximately 63.0°C, or approximately 64.0°C, or approximately 65.0°C.

[0124] For example, the T of the formulation at 473 nm agg The T of the formulation at 266 nm was determined by differential scanning fluorescence quantification and is approximately 58.0°C to 64.0°C. agg The temperature is approximately 58.0°C, or approximately 60.0°C, or approximately 61.0°C, or approximately 62.0°C, or approximately 63.0°C, or approximately 64.0°C.

[0125] In one example, the formation of aggregates of antibodies or their antigen-binding fragments (i.e., particle size distribution) is investigated using dynamic light scattering (DLS). For example, variations in light intensity are measured using a digital correlator (e.g., Malvern Zetasizer software) to determine the Z-mean hydrodynamic diameter and polydispersity index (e.g., using cumulant analysis). In one example, organic acid buffers do not significantly alter (i.e., increase or decrease) the Z-mean hydrodynamic diameter of antibodies or antigen-binding fragments.

[0126] The stability of a formulation can also be investigated by measuring the total aggregate and / or monomer content. Methods for investigating aggregate accumulation and monomer content of a formulation will be apparent to those skilled in the art and / or are described herein. In one example, the total aggregate percentage of the antibody or its antigen-binding fragments in a formulation is determined by size exclusion chromatography (SEC or SE-HPLC). In another example, the monomer percentage of the antibody or its antigen-binding fragments in a formulation is determined by size exclusion chromatography (SEC or SE-HPLC). For example, a formulation of the present disclosure contains at least 90% monomeric antibody or its antigen-binding fragment, and / or less than 10% (i.e., at most 10%) aggregates and / or degraded (e.g., fragmented) antibody or its antigen-binding fragment. In another example, a formulation of the present disclosure contains at least 95% monomeric antibody or its antigen-binding fragment, and / or less than 5% (i.e., at most 5%) aggregates and / or degraded (e.g., fragmented) antibody or its antigen-binding fragment.

[0127] For example, a formulation may contain less than 10% total antibody aggregates. For instance, a formulation may contain less than 10%, or less than 9%, or less than 8%, or less than 7%, or less than 6%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1% total aggregates. For example, the aggregates in the composition may be high molecular weight species and / or degraded antibodies or their antigen-binding fragments.

[0128] For example, after storing the formulation at 5°C for 4-5 weeks, it contains approximately 0.5% to 3.0% total aggregates. For example, after storing the formulation at 5°C for 4-5 weeks, it contains approximately 0.5%, or approximately 1.0%, or approximately 1.5%, or approximately 2.0%, or approximately 2.5%, or approximately 3.0% total aggregates. For example, after storing the formulation at 5°C for 4-5 weeks, it contains approximately 1.5% to 2.0% total aggregates. For example, after storing the formulation at 5°C for 4-5 weeks, it contains approximately 1.7% total aggregates.

[0129] In one example, the formulation contains less than 10% total antibody aggregates after being stored at 5°C for 24 months. For example, the formulation contains less than 10%, or less than 9%, or less than 8%, or less than 7%, or less than 6%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1% total aggregates after being stored at 5°C for 24 months. In one example, the formulation contains about 1.5% to 2.0% total aggregates after being stored at 5°C for 24 months. For example, the formulation contains about 2.0% total aggregates after being stored at 5°C for 24 months. For example, the aggregates in the composition are high molecular weight species and / or degraded antibodies or their antigen-binding fragments.

[0130] For example, after storage at 35-40°C for 4-5 weeks, the formulation contains approximately 2.0% to 7.0% total aggregates.

[0131] In one example, the formulation contains less than 10% total antibody aggregates after being stored at 25°C for 24 months. For example, the formulation contains less than 10%, or less than 9%, or less than 8%, or less than 7%, or less than 6%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1% total aggregates after being stored at 5°C for 24 months. In one example, the formulation contains about 1.5% to 3.5% total aggregates after being stored at 25°C for 24 months. For example, the formulation contains about 3.1% total aggregates after being stored at 25°C for 24 months. For example, the aggregates in the composition are high molecular weight species and / or degraded antibodies or their antigen-binding fragments.

[0132] In one example, the formulation contains less than 10% total antibody aggregates after being stored at 25°C for 36 months. For example, the formulation contains less than 10%, or less than 9%, or less than 8%, or less than 7%, or less than 6%, or less than 5%, or less than 4%, or less than 3% after being stored at 5°C for 36 months. In one example, the formulation contains about 1% to 5% total aggregates after being stored at 25°C for 24 months. For example, the formulation contains about 2.4% total aggregates after being stored at 25°C for 24 months. For example, the aggregates in the composition are high molecular weight species and / or degraded antibodies or their antigen-binding fragments.

[0133] In one example, at least about 90% of the antibodies in a formulation are monomers. For example, at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, or about 99.5% of the antibodies in a formulation are monomers. In one example, at least about 95% of the antibodies in a formulation are monomers.

[0134] In one example, after storage at 5°C for 4-5 weeks, at least approximately 95% of the antibodies in the formulation are monomers. For example, after storage at 5°C for 4-5 weeks, at least approximately 95%, or approximately 95.5%, or approximately 96%, or approximately 96.5%, or approximately 97%, or approximately 97.5%, or approximately 98%, or approximately 98.5%, or approximately 99%, or approximately 99.5% of the antibodies in the formulation are monomers. In one example, after storage at 5°C for 4-5 weeks, approximately 98% to approximately 99% of the antibodies in the formulation are monomers. For example, after storage at 5°C for 4-5 weeks, approximately 98.3% of the antibodies in the formulation are monomers. In one example, after storage at 5°C for 24 months, approximately 98% of the antibodies in the formulation are monomers.

[0135] For example, after storage at 35-40°C for 4-5 weeks, at least approximately 92% of the antibodies in the formulation are monomers.

[0136] For example, after 24 months of storage at 25°C, at least approximately 96% of the antibodies in the formulation are monomers. For example, after 24 months of storage at 25°C, at least approximately 96%, or approximately 96.5%, or approximately 97%, or approximately 97.5%, or approximately 98%, or approximately 98.5%, or approximately 99%, or approximately 99.5% of the antibodies in the formulation are monomers. For example, after 24 months of storage at 25°C, approximately 96.9% of the antibodies in the formulation are monomers.

[0137] For example, the osmotic pressure of a formulation is approximately 150 mOsm / kg to approximately 550 mOsm / kg. For instance, the osmotic pressure of a formulation is approximately 150 mOsm / kg, or approximately 175 mOsm / kg, or approximately 200 mOsm / kg, or approximately 225 mOsm / kg, or approximately 250 mOsm / kg, or approximately 275 mOsm / kg, or approximately 300 mOsm / kg, or approximately 325 mOsm / kg, or approximately 350 mOsm / kg, or approximately 375 mOsm / kg, or approximately 400 mOsm / kg, or approximately 425 mOsm / kg, or approximately 450 mOsm / kg, or approximately 475 mOsm / kg, or approximately 500 mOsm / kg, or approximately 550 mOsm / kg. In one example, the osmotic pressure of the formulation is approximately 400 mOsm / kg to approximately 550 mOsm / kg. For example, the osmotic pressure of the formulation is approximately 400 mOsm / kg, or approximately 410 mOsm / kg, or approximately 420 mOsm / kg, or approximately 430 mOsm / kg, or approximately 440 mOsm / kg, or approximately 450 mOsm / kg, or approximately 460 mOsm / kg, or approximately 470 mOsm / kg, or approximately 480 mOsm / kg, or approximately 490 mOsm / kg, or approximately 500 mOsm / kg, or approximately 550 mOsm / kg. In one example, the osmotic pressure is approximately 400 mOsm / kg to approximately 500 mOsm / kg. For example, the osmotic pressure is approximately 430 mOsm / kg. In one example, the osmotic pressure of the formulation is approximately 450 mOsm / kg.

[0138] For example, the present disclosure provides a pharmaceutical formulation of the present disclosure for use in treating or preventing a disease or condition in a subject.

[0139] In one example, the present disclosure provides a pharmaceutical formulation of the present disclosure for use in antagonizing the activity of factor XII and / or activated factor XII in a subject.

[0140] In one example, the present disclosure provides a pharmaceutical formulation of the present disclosure for use in antagonizing the activation of factor XII and / or activated factor XII in a subject.

[0141] This disclosure also provides methods for treating or preventing a disease or condition in which the subject is administered a high-concentration formulation of the disclosure.

[0142] The disclosure also provides a method for antagonizing the activity of factor XII and / or activated factor XII in a subject, which includes administering a high-concentration formulation of the disclosure.

[0143] This disclosure also provides a method for antagonizing the activation of factor XII and / or activated factor XII in a subject, which includes administering a high-concentration formulation of this disclosure.

[0144] For example, this disclosure provides the use of the pharmaceutical formulations of this disclosure in the manufacture of a pharmaceutical for treating or preventing a disease or condition in the subject.

[0145] For example, the present disclosure provides the use of the pharmaceutical formulations of the present disclosure in the manufacture of a pharmaceutical for antagonizing the activity of factor XII and / or activated factor XII in a subject.

[0146] For example, the present disclosure provides the use of the pharmaceutical formulations of the present disclosure in the manufacture of a pharmaceutical for antagonizing the activation of factor XII and / or activated factor XII in a subject.

[0147] In one example, the subject requires treatment with the pharmaceutical formulation of this disclosure (i.e., is in need of it).

[0148] For example, the disease or condition is a thrombotic disorder, an inflammatory disorder, and / or a thromboinflammatory disorder. For instance, the subject has or is at risk of having a thrombotic disorder, an inflammatory disorder, and / or a thromboinflammatory disorder.

[0149] In one case, the subject suffers from a thrombotic disorder, an inflammatory disorder, and / or a thromboinflammatory disorder. In another case, the subject has been diagnosed with a thrombotic disorder, an inflammatory disorder, and / or a thromboinflammatory disorder. In yet another case, the subject is receiving treatment for a thrombotic disorder, an inflammatory disorder, and / or a thromboinflammatory disorder.

[0150] In any example of the methods described herein, the pharmaceutical formulations of the Disclosure are administered before or after the onset of a disease or condition, such as a thrombotic disorder, an inflammatory disorder, and / or a thromboinflammatory disorder. In any example of the methods described herein, the pharmaceutical formulations of the Disclosure are administered before the onset of a disease or condition. In any example of the methods described herein, the pharmaceutical formulations of the Disclosure are administered after the onset of a disease or condition.

[0151] In one example, a pharmaceutical product is administered before or after the onset of symptoms of a disease or condition, such as a thrombotic disorder, an inflammatory disorder, and / or a thromboinflammatory disorder. In another example, a pharmaceutical product is administered before the onset of symptoms of a disease or condition, such as a thrombotic disorder, an inflammatory disorder, and / or a thromboinflammatory disorder. In yet another example, a pharmaceutical product is administered after the onset of symptoms of a disease or condition, such as a thrombotic disorder, an inflammatory disorder, and / or a thromboinflammatory disorder. In yet another example, a pharmaceutical product is administered in a dose that alleviates or reduces one or more symptoms of a disease or condition, such as a thrombotic disorder, an inflammatory disorder, and / or a thromboinflammatory disorder.

[0152] The symptoms of thrombotic disorders, inflammatory disorders, and / or thromboinflammatory disorders are evident to those skilled in the art and depend on the condition. Illustrative symptoms of a condition or disorder include, for example: • Recurrent infections; ·arthritis; • Muscle weakness; • Skin rash or discoloration; • Edema, especially in the limbs (e.g., feet, hands, legs, or arms) or eyes; ·abdominal pain; • Pain, swelling, and tenderness in the affected area; • Dull or severe pain in the affected area; • Warm skin in the area of ​​the thrombus; ·Red skin; ·Chest pain; • Difficulty breathing (e.g., shortness of breath and / or chest tightness); • Sudden weakness in one arm or leg; ·nausea; ·fatigue; Hematuria; • Partial or complete paralysis; and • Inferior cognitive abilities.

[0153] In one example, thrombotic disorders, inflammatory disorders and / or thromboinflammatory disorders include venous, arterial, or capillary thrombosis (such as stroke, myocardial infarction, deep venous thrombosis (DVT), portal vein thrombosis, renal vein thrombosis, jugular vein thrombosis, sinus thrombosis, Budd-Chiari syndrome, Paget-Schlotter disease, or asymptomatic cerebral ischemia), cardiac thrombosis, thromboembolism, thrombosis during and / or after contact between blood and artificial surfaces in human or animal subjects, disseminated intravascular coagulation (DIC), atrial fibrillation, acute coronary syndrome (ACS), atherosclerotic disease, ischemic stroke due to reperfusion, and ischemia-reperfusion injury (I). The group consists of diseases related to radioisotopes (RI), trauma, organ transplantation, etc., neurotraumatic injuries (traumatic brain injury, spinal cord injury, etc.), neuroinflammatory diseases (multiple sclerosis, etc.), interstitial lung diseases (idiopathic pulmonary fibrosis (IPF), etc.), pneumonia, fibrinolysis, diseases related to FXII / FXIIa-induced kinin formation (hereditary angioedema (HAE), etc.), sepsis, diseases related to FXII / FXIIa-mediated complement activation, acute respiratory distress syndrome (ARDS), organ and cell transplantation, sickle cell disease and conditions related to increased vascular permeability, fibrinolysis, and conditions related to increased vascular permeability.

[0154] For example, the disease or condition is venous, arterial, or capillary thrombosis. For instance, venous or arterial thrombosis is associated with a disease or condition selected from the group consisting of stroke, myocardial infarction, deep venous thrombosis (DVT), portal vein thrombosis, thromboembolism, renal vein thrombosis, jugular vein thrombosis, cerebral venous sinus thrombosis, Budd-Chiari syndrome, asymptomatic ischemia (SBI), and Paget-Schröder disease.

[0155] In one example, the disease or condition is thrombus formation during and / or after contact between the subject's blood and an artificial surface. In another example, thrombus formation occurs during and / or after a medical procedure on the subject, during and / or after contact between the blood of a human or animal subject and an artificial surface, and the formulation of the present disclosure is administered before and / or during and / or after such medical procedure. For example, in subjects having valve replacement, stenting, percutaneous coronary intervention (PCI), extracorporeal membrane oxygenation (ECMO), or undergoing cardiopulmonary bypass (CPB) surgery.

[0156] For example, the disease or condition is chronic and / or acute thromboembolism. For instance, chronic and / or acute thromboembolism is pulmonary embolism, cerebral thromboembolism, and thrombus formation induced by atrial fibrillation (e.g., stroke prevention in atrial fibrillation (SPAF)).

[0157] In one example, stroke refers to thrombotic stroke. In another example, stroke refers to stroke prevention in atrial fibrillation (SPAF).

[0158] For example, the disease or condition is ischemic stroke due to reperfusion. For example, the disease or condition is a secondary form of stroke (e.g., a secondary form of ischemic or hemorrhagic stroke).

[0159] In one example, the disease or condition is a neurotraumatic disorder. For example, a neurotraumatic disorder is a traumatic injury of the central nervous system (CNS), including spinal cord injury and traumatic brain injury. In one example, the disease or condition is a spinal cord injury. In another example, the disease or condition is a traumatic brain injury.

[0160] In one example, the disease or condition is ischemia-reperfusion injury (IRI). For example, IRI can be caused by a natural event (e.g., restoration of blood flow after a myocardial infarction), trauma, or one or more surgical procedures (e.g., organ transplantation) or other therapeutic interventions that restore blood flow to tissues or organs that have suffered a reduction in blood supply. Such surgical procedures may include, for example, coronary artery bypass grafting, coronary artery reconstruction, organ transplantation, elective surgery, reconstructive surgery, vascular surgery, cardiac surgery, trauma surgery, crush or bruise surgery, cancer surgery, orthopedic surgery, transplantation, or minimally invasive surgery. In one example, the surgical procedure may include the insertion of instruments for the delivery of pharmacologically active substances, such as thrombolytics or vasodilators, or the insertion of instruments to mechanically remove complete or partial occlusion, such as vascular occlusion.

[0161] In one example, thromboembolism is pulmonary embolism. In another example, thromboembolism is systemic embolism. In yet another example, thromboembolism is chronic thromboembolic pulmonary hypertension.

[0162] In one example, the disease or condition is contact-mediated thromboinflammatory disease.

[0163] In one example, the disease or condition is atrial fibrillation.

[0164] In one example, the disease or condition is acute coronary syndrome (ACS).

[0165] In one example, the disease or condition is interstitial lung disease (ILD). For example, interstitial lung disease is fibroproliferative and / or idiopathic pulmonary fibrosis. In another example, the disease or condition is idiopathic pulmonary fibrosis (IPF). For example, the subject has idiopathic pulmonary fibrosis (IPF).

[0166] In one example, the disease or condition is an inflammatory disorder. For example, the inflammatory disorder is a neurological inflammatory disease (or neuroinflammatory disorder). In one example, a neurological inflammatory disease is spinal cord injury (SCI), stroke, traumatic brain injury (TBI), secondary cerebral hydrops, central nervous system hydrops, multiple sclerosis (MS), transverse myelitis, or neuromyelitis optica (Dovick's disease). In another example, the inflammatory disorder is pneumonia.

[0167] In one example, the disease or condition is fibrinolysis.

[0168] In one example, the disease or disorder is angiogenesis.

[0169] For example, the disease or condition is a disorder associated with FXII / FXIIa-induced kinin formation. For instance, the disease or condition is selected from the group consisting of hereditary angioedema (HAE), bacterial pulmonary infections, trypanosomiatic infections, hypotensive shock, pancreatitis, Chagas disease, arthralgout, arthritis, disseminated intravascular coagulation (DIC), and sepsis.

[0170] For example, the disease or condition is hereditary angioedema (HAE).

[0171] In one example, the formulation of the present disclosure is administered subcutaneously to a subject who requires it. In another example, the formulation of the present disclosure is administered intravenously to a subject who requires it.

[0172] In one example, the formulation of this disclosure is self-administered.

[0173] In one example, the formulation of this disclosure is administered subcutaneously by the patient.

[0174] In one example, the formulations of this disclosure are provided in pre-filled syringes.

[0175] In one example, the formulation of this disclosure is self-administered subcutaneously using a pre-filled syringe.

[0176] In one example of any method described herein, the subject is a mammal, such as a primate like a human.

[0177] The treatment methods described herein may further include administering additional compounds to reduce, treat, or prevent the effects of thrombotic disorders, inflammatory disorders, and / or thromboinflammatory disorders.

[0178] This disclosure provides a kit comprising at least one pharmaceutical formulation of this disclosure, accompanied by instructions for use in antagonizing the activity and / or activation of factor XII and / or

[0179] This disclosure further provides a kit comprising at least one pharmaceutical formulation of this disclosure, packaged together with instructions for use in treating or preventing a disorder in a subject. The kit may optionally further comprise additional therapeutic compounds or drugs.

[0180] The Disclosure also provides a kit comprising at least one pharmaceutical formulation of the Disclosure, packaged together with instructions for administering the conjugate or composition, optionally in combination with further therapeutic compounds or drugs, to subjects suffering from or at risk of suffering from a disorder.

[0181] In one example, the formulation is contained within a vial, syringe, or auto-injector device. For instance, the syringe is a pre-filled syringe.

[0182] This disclosure provides vials containing the pharmaceutical formulations thereof. For example, the vials are single-use vials.

[0183] This disclosure provides syringes containing the pharmaceutical formulations described herein.

[0184] This disclosure also provides auto-injector devices, including the pharmaceutical formulations described herein.

[0185] This disclosure provides a pre-filled syringe containing a pharmaceutical formulation comprising approximately 170 mg / ml of the antibody or its antigen-binding fragment as described herein, a histidine buffer having a pH of 5.5 to 6.5, polysorbate 80, and proline and arginine monohydrochloride as stabilizers, wherein the formulation has a viscosity of less than approximately 10 mPa*s at 20°C and 25°C. For example, the pre-filled syringe contains a formulation comprising 12 mM to 25 mM histidine buffer having a pH of 5.8 to 6.4, 0.01% to 0.03% (weight / volume) of polysorbate 80, 110 mM to 170 mM proline, and 110 mM to 170 mM arginine. In one example, the osmotic pressure of the formulation is approximately 450 mOsm / kg. In one example, the antibody contains the amino acid sequence shown in SEQ ID NO: 5. H and V containing the amino acid sequence shown in SEQ ID NO: 6 L This includes, for example, a pre-filled syringe with a volume of 0.5 ml to 2 ml.

[0186] This disclosure provides a pre-filled syringe containing a pharmaceutical formulation comprising approximately 160 mg / ml to 180 mg / ml of the antibody or its antigen-binding fragment as described herein, a histidine buffer having a pH of 5.8 to 6.4, polysorbate 80, proline and arginine monohydrochloride as stabilizers, wherein the formulation has a viscosity of less than approximately 10 mPa*s at 20°C and 25°C. For example, the pre-filled syringe contains a formulation comprising 12 to 25 mM histidine buffer having a pH of 5.8 to 6.4, 0.01% to 0.03% (weight / volume) of polysorbate 80, 90 mM to 150 mM proline and 100 mM to 160 mM arginine. In one example, the osmotic pressure of the formulation is approximately 430 to 530 mOsm / kg. In one example, the antibody contains the amino acid sequence shown in SEQ ID NO: 5. H and V containing the amino acid sequence shown in SEQ ID NO: 6 L This includes, for example, a pre-filled syringe with a volume of 0.5 ml to 2 ml.

[0187] This disclosure provides a pre-filled syringe containing a pharmaceutical formulation comprising approximately 170 mg / ml of the antibody or its antigen-binding fragment as described herein, a histidine buffer having a pH of 5.5 to 6.5, polysorbate 80, and proline and arginine monohydrochloride as stabilizers, wherein the formulation has a viscosity of less than approximately 10 mPa*s at 20°C and 25°C. For example, the pre-filled syringe contains a formulation comprising approximately 20 mM histidine buffer having a pH of 5.8 to 6.4, 0.02% (weight / volume) of polysorbate 80, 140 mM proline, and 150 mM arginine. In one example, the osmotic pressure of the formulation is approximately 450 mOsm / kg. In one example, the antibody contains the amino acid sequence shown in SEQ ID NO: 5. H and V containing the amino acid sequence shown in SEQ ID NO: 6 L This includes, for example, a pre-filled syringe with a volume of 0.5 ml to 2 ml.

[0188] This disclosure provides a vial containing a pharmaceutical formulation comprising approximately 100 mg / ml of the antibody or its antigen-binding fragment as described herein, a histidine buffer having a pH of 5.5 to 6.5, polysorbate 80, and proline and arginine monohydrochloride as stabilizers, wherein the formulation has a viscosity of less than approximately 10 mPa*s at 20°C and 25°C. For example, the vial contains a formulation comprising 12 to 25 mM histidine buffer having a pH of 5.8 to 6.4, 0.01% to 0.03% (weight / volume) of polysorbate 80, 90 mM to 150 mM proline, and 100 mM to 160 mM arginine. In one example, the osmotic pressure of the formulation is approximately 450 mOsm / kg. In one example, the antibody contains the amino acid sequence shown in SEQ ID NO: 5. H and V containing the amino acid sequence shown in SEQ ID NO: 6 L This includes [the following]. In one example, the volume of the vial is 2 ml.

[0189] The present disclosure provides a vial containing a pharmaceutical formulation comprising an antibody or an antigen-binding fragment thereof described herein at about 100 mg / ml, a histidine buffer having a pH of 5.5 to 6.5, polysorbate 80, and proline and arginine as stabilizers, wherein the formulation has a viscosity of less than about 5 mPa*s at 20 °C and 25 °C. For example, the vial contains a formulation comprising a histidine buffer of about 20 mM having a pH of 5.8 to 6.4, 0.02% (weight / volume) of polysorbate 80, 140 mM of proline and 150 mM of arginine. In one example, the osmotic pressure of the formulation is about 450 mOsm / kg. In one example, the osmotic pressure of the formulation is about 450 mOsm / kg. In one example, the antibody comprises V H having the amino acid sequence shown in SEQ ID NO: 5 and V L having the amino acid sequence shown in SEQ ID NO: 6. In one example, the volume of the vial is 2 ml.

[0190] Exemplary effects of the pharmaceutical formulations of the present disclosure are described herein and can be taken to apply to the examples of the present disclosure shown in the previous paragraph with necessary modifications.

[0191] Key to the Sequence Listing

[0192]

Table 1

Mode for Carrying Out the Invention

[0193] General Throughout this specification, unless specifically stated otherwise or the context otherwise requires, references to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e., one or more) of those steps, compositions of matter, group of steps or group of compositions of matter.

[0194] Those skilled in the art will understand that this disclosure is subject to changes and modifications other than those specifically described herein. It should be understood that this disclosure includes all such changes and modifications. This disclosure includes all processes, configurations, compositions and compounds referred to or indicated in this specification individually or collectively, as well as any combination or any two or more of such processes or configurations.

[0195] This disclosure is not limited in scope by the specific examples described herein, which are for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of this disclosure.

[0196] In this specification, any example in this disclosure should be understood to apply to any other example in this disclosure with necessary modifications unless otherwise specified. In other words, any specific example in this disclosure may be combined with any other specific example in this disclosure (unless they are mutually exclusive).

[0197] Any example in this disclosure disclosing a specific configuration or group of configurations or a method or process is to be taken as providing clear support for rejecting the specific configuration or group of configurations or a method or process.

[0198] Unless otherwise specifically provided, all technical and scientific terms used herein should be understood to have the same meaning as those generally understood by those skilled in the art (e.g., cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0199] Unless otherwise specified, the recombinant proteins, cell cultures, and immunological techniques used in this disclosure are standard methods well known to those skilled in the art. Such techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), TA Brown (ed.), Essential Molecular Biology: A Practical Approach, Vol. 1 and 2, IRL Press (1991), DMGlover and B.D. Hames (eds.), DNA Cloning: A Practical Approach, Vols. 1-4, IRL Press (1995 and 1996), and FMAusubel et al. (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all latest editions to date), Ed Harlow and David Lane (eds.), Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and JEColigan et al. (eds.), Current This is described and explained throughout the literature from sources such as *Protocols in Immunology*, John Wiley & Sons (including all the most recent editions to date).

[0200] The descriptions and definitions of variable regions and their parts, antibodies and their fragments, as used herein can be further clarified by the discussions in Kabat's Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991.

[0201] The term "Kabat's EU numbering system" is understood to mean that the numbering of antibody heavy chains is based on the EU index, as taught by Kabat et al., 1991, *Sequences of Proteins of Immunological Interest*, 5th edition, United States Public Health Service, National Institutes of Health, Bethesda. The EU index is based on the residue numbering of human IgG1 EU antibodies.

[0202] The term "and / or," for example, "X and / or Y," should be understood to mean "X and Y" or "X or Y," and should be taken as providing clear support for both meanings or either meaning.

[0203] Throughout this specification, variations of the word such as “comprise,” “comprises,” or “comprising” imply that the explicitly stated element, integer, or process, or group of elements, integers, or processes, is included, but not that any other element, integer, or process, or group of elements, integers, or processes, is excluded.

[0204] As used herein, the term “derived from” should be understood to mean that the specified integer can be obtained from a specific source, but does not necessarily have to be obtained directly from that source.

[0205] Selected definition Coagulation factor XII, also known as Hagemann factor or FXII, is a plasma protein. It is the enzyme precursor form of factor XIIa, an enzyme of the serine protease (or serine endopeptidase) class. In humans, factor XII is encoded by the F12 gene. For nomenclature purposes only and not for restriction, exemplary sequences of human factor XII are shown in NCBI reference sequence: NP_000496.2; NCPI protein accession number NP_000496, and Sequence ID No. 17. Additional sequences of factor XII can be determined using sequences provided herein and / or in public databases, and / or using standard techniques (e.g., Ausubel et al., (ed.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all latest editions to date) or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)).

[0206] As used herein, the terms “factor XII inhibitor” or “FXII inhibitor” or “inhibitor of FXII” refer to either or both of factor XII (pre-activation, i.e., its enzyme precursor) and activated factor XII (FXIIa), as well as inhibitors of FXII activation. Thus, “inhibitors of FXII” may include either or both of FXII and FXIIa (also called αFXIIa), as well as inhibitors of FXII activation, including the FXIIa cleavage products FXIIa alpha and FXIIa beta (also called FXIIf). FXII inhibitors encompass functional variants and fragments of wild-type inhibitors. A functional variant or fragment is a molecule that retains at least 50% (e.g., about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 99%, or about 100%) of the wild-type molecule's ability to inhibit FXII, FXIIa, or FXII activation. For example, FXII inhibitors are non-endogenous inhibitors; that is, they are not inhibitors that are naturally present in the human or animal body.

[0207] The term "amide solubility" refers to the ability of the proteins in this disclosure to catalyze the hydrolysis of at least one peptide bond in another polypeptide. The term "organic acid buffer" refers to conventional buffers of organic acids and salts.

[0208] The term "nonionic surfactant" as used herein refers to any surfactant having a non-charged head.

[0209] A "stable" formulation is one in which the antibody or its antigen-binding fragment essentially maintains its physical and / or chemical stability and / or biological activity during storage.

[0210] In the context of the present disclosure, the term "monomer" or "monomeric" refers to an antibody that is properly folded or an antigen-binding fragment thereof. For example, the monomer of an antibody according to the present disclosure relates to a standard tetrameric antibody that contains two identical glycosylated heavy chains and light chains, respectively. In that case, an "aggregate" is the non-specific association of two antibody molecules (e.g., high molecular weight species).

[0211] As used herein, the term "amino acid stabilizer" refers to an amino acid or its derivative that improves or otherwise enhances the stability of a formulation.

[0212] As used herein, the term "polyol" refers to a substance having a plurality of hydroxyl groups.

[0213] The term "dynamic viscosity" or "absolute viscosity" refers to the ratio of the shear stress to the shear rate of a fluid's internal resistance to flow at a specific temperature (e.g., 20 °C). When a force of 1 dyne / square centimeter causes two parallel liquid surfaces with an area of 1 square centimeter separated by 1 square centimeter to pass through each other at a speed of 1 cm / second, the liquid has a dynamic viscosity of 1 poise. 1 poise is equal to 100 centipoises (cP), and 1 centipoise is equal to 1 millipascal second (mPa*s) in the International System of Units (SI) units.

[0214] As used herein, the term "density" of a formulation refers to the mass density or mass per unit volume (g / cm 3 ).

[0215] As used herein, the term "osmotic pressure" is a measure of the osmoles (Osm) of solute per kilogram of solvent (Osm / kg or Osm / kg).

[0216] As used herein, the term “binding” in relation to the interaction between an antibody or its antigen-binding fragment and an antigen means that the interaction depends on the presence of a specific structure of the antigen (e.g., an antigenic determinant or epitope). For example, an antibody or its antigen-binding fragment recognizes and binds to a specific protein structure, rather than to the protein in general. If an antibody binds to epitope “A”, the presence of a molecule containing epitope “A” (or free, unlabeled “A”) in the reactant containing labeled “A” and the protein reduces the amount of labeled “A” bound to the antibody.

[0217] As used herein, the terms “specifically bind” or “the binding is specific” should be understood to mean that an antibody or its antigen-binding fragment reacts or accompanies a particular antigen or cell expressing it more frequently, more quickly, for a longer period, and / or with greater affinity than it does with alternative antigens or cells. For example, an antibody or its antigen-binding fragment binds to FXII (or FXIIa) with substantially higher affinity (e.g., 1.5x or 2x or 5x or 10x or 20x or 40x or 60x or 80x to 100x or 150x or 200x) than to other blood coagulation factors or to antigens commonly recognized by polyreactive native antibodies (i.e., naturally occurring antibodies known to bind to a variety of naturally occurring antigens found in humans). Generally, but not always, references to binding should be understood to mean specific binding, and each term should be understood to provide clear support for other terms.

[0218] The term "recombinant" should be understood to mean the product of artificial genetic modification. Therefore, in relation to antibodies or their antigen-binding fragments, this term does not include naturally occurring antibodies within the subject's body, which are products of natural recombination that occur during B cell maturation. However, such antibodies, when isolated, should be considered isolated proteins containing an antibody-antigen-binding domain. Similarly, when a nucleic acid encoding a protein is isolated and expressed using recombinant means, the resulting protein is a recombinant protein containing an antibody-antigen-binding domain. Recombinant proteins also include proteins expressed by artificial recombinant means, for example, if they exist within the cells, tissues, or subject in which they are expressed.

[0219] The term "protein" should be understood to include either a single polypeptide chain, i.e., a series of consecutive amino acids linked by peptide bonds, or a series of polypeptide chains linked to each other covalently or non-covalently (i.e., a polypeptide complex). For example, a series of polypeptide chains may be covalently linked using suitable chemical or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0220] The term "polypeptide" or "polypeptide chain" should be understood to mean a series of consecutive amino acids linked together by peptide bonds, as discussed in the previous paragraph.

[0221] For the purposes of this disclosure, the term “antibody” includes proteins that are capable of specifically binding to one or a few closely related antigens (e.g., blood coagulation factors) thanks to antigen-binding domains contained within the Fv. The term includes four-chain antibodies (e.g., two light chains and two heavy chains), recombinant or modified antibodies (e.g., chimeric antibodies, humanized antibodies, human antibodies, CDR-transplanted antibodies, primate-transplanted antibodies, deimmunized antibodies, synhumanized antibodies, semi-antibodies, bispecific antibodies). Antibodies may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), a class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or a subclass. In one example, the antibody is a murine (mouse or rat) antibody or a primate (human, etc.) antibody. In one example, the antibody heavy chain lacks a C-terminal lysine residue. In one example, the antibody is humanized, synhumanized, chimeric, CDR-transplanted, or deimmunized.

[0222] "Anti-FXII antibodies" include antibodies that bind to and / or inhibit either or both of the enzyme precursors of the activating protein (FXIIa), which includes FXIIa alpha and FXIIa beta cleavage fragments. In some cases, the antibody specifically binds to FXIIa or the alpha or beta chain fragments of FXIIa.

[0223] As used herein, the term “germline” antibody refers to an antibody that reverses some or all somatic mutations that introduce changes to framework residues back into the original sequence present in the genome, e.g., the human genome. In this regard, not all changes necessarily need to be reversed by germline antibodies.

[0224] As used herein, “variable region” refers to a portion of the light and / or heavy chain of an antibody as defined herein that is capable of specifically binding to an antigen, and includes the complementarity-determining region (CDR); i.e., the amino acid sequences of CDR1, CDR2, and CDR3, as well as the framework region (FR).

[0225] As used herein, the term “complementarity-determining regions” (synonym CDR; i.e., CDR1, CDR2, and CDR3) refers to amino acid residues in the antibody variable region, whose presence is a major contributor to specific antigen binding. Each variable region typically has three CDR regions, identified as CDR1, CDR2, and CDR3. For example, the amino acid positions assigned to CDRs and FRs are defined by Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 (also known as the “Kabat numbering system”). According to the Kabat numbering system, V H FR and CDR are arranged as follows: residues 1-30 (FR1), 31-35 (CDR1), 36-49 (FR2), 50-65 (CDR2), 66-94 (FR3), 95-102 (CDR3), and 103-113 (FR4). According to Kabat's numbering system, V L The FR and CDR are arranged as follows: residues 1-23 (FR1), 24-34 (CDR1), 35-49 (FR2), 50-56 (CDR2), 57-88 (FR3), 89-97 (CDR3), and 98-107 (FR4).

[0226] The "framework region" (hereinafter referred to as FR) consists of variable domain residues other than CDR residues.

[0227] Antibody "antigen-binding fragments" contain one or more variable regions of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; and multispecific antibodies formed from single-chain antibody molecules, semi-antibodies, and antibody fragments.

[0228] As used herein, the term "Fv" refers to the variable region (V) of a light chain, whether it consists of multiple polypeptides or a single polypeptide. L ) and the variable region of the heavy chain (V HThis should be interpreted as any protein that has an antigen-binding domain and forms a complex with an antigen-binding domain, that is, any protein that is capable of specifically binding to an antigen. H and V L This may be present in a single polypeptide chain or in different polypeptide chains. Furthermore, the Fv of the present disclosure (and any protein of the present disclosure) may have multiple antigen-binding domains that may or may not bind to the same antigen. This term should be understood to encompass fragments directly derived from antibodies and proteins corresponding to such fragments produced using recombinant means. Exemplary Fv-containing polypeptides or proteins include Fab fragments, Fab' fragments, F(ab') fragments, scFv, diabodies, triabodies, tetrabodies or higher-order complexes, or constant regions or their domains, e.g., C H 2 or C H The Fab fragment consists of one of the above-mentioned structures linked to the 3 domains, for example, a minibody. The Fab fragment consists of a monovalent antigen-binding fragment of immunoglobulin and can be produced by digesting the whole antibody with the enzyme papain to obtain a fragment consisting of intact light and heavy chain portions, or by using recombinant means. The Fab fragment of an antibody is obtained by treating the whole antibody with pepsin and then reducing it to obtain intact light and V H and can be obtained by giving a molecule consisting of a portion of a heavy chain containing a single constant domain. Two Fab' fragments are obtained for each antibody treated in this way. Fab' fragments can also be generated by recombinant means. The "F(ab')2 fragment" of an antibody consists of a dimer of two Fab' fragments joined by two disulfide bonds and can be obtained without further reduction by treating the entire antibody molecule with the enzyme pepsin. The "Fab2" fragment can be obtained, for example, from a leucine zipper or C H This recombinant fragment contains two Fab fragments linked using three domains. A "single-chain Fv" or "scFv" is a recombinant molecule containing an antibody variable region fragment (Fv) in which the variable regions of the light chain and the heavy chain are covalently linked by a suitable flexible polypeptide linker.

[0229] The terms “crystallizable fragment,” “Fc,” “Fc region,” or “Fc moiety” (which may be used interchangeably herein) refer to a region of an antibody that contains at least one constant domain, is generally (but not necessarily) glycosylated, and is capable of binding to one or more Fc receptors and / or components of the complement cascade. The heavy chain constant region can be selected from one of five isotypes: α, δ, ε, γ, or μ. Furthermore, various subclasses of heavy chains (such as IgG subclass heavy chains) are responsible for different effector functions, and therefore, by selecting the desired heavy chain constant region, it is possible to generate proteins with the desired effector function. Exemplary heavy chain constant regions are gamma 1 (IgG1), gamma 2 (IgG2), gamma 3 (IgG3), and gamma 4 (IgG4), or hybrids thereof.

[0230] The term "constant region," as used herein, refers to the portion of the antibody's heavy or light chain other than the variable region. In the heavy chain, the constant region generally includes multiple constant domains and hinge regions. For example, the IgG constant region consists of the following linked components: constant region heavy chain (C H )C H 1. Linker, C H 2 and C H It includes 3. In heavy chains, the steady region includes Fc. In light chains, the steady region generally consists of one steady domain (C). L Includes 1).

[0231] The term "stabilized IgG4 constant region" is understood to mean an IgG4 constant region that has been modified to reduce Fab arm exchange or the tendency to undergo Fab arm exchange, or to reduce the formation of or tendency to form half-antibodies. "Fab arm exchange" refers to a type of protein modification of human IgG4 in which the IgG4 heavy chain and attached light chain (half) are replaced with a heavy-light chain pair derived from another IgG4 molecule. Thus, an IgG4 molecule can acquire two different Fab arms that recognize two different antigens (resulting in a bispecific molecule). Fab arm exchange occurs naturally in vivo and can be induced in vitro by purified blood cells or by reducing agents such as reduced glutathione.

[0232] As used herein, the term “single-specific” refers to a binding domain containing one or more antigen-binding sites, each having the same epitope specificity. Thus, a single-specific binding domain may contain a single antigen-binding site (e.g., Fv, scFv, Fab, etc.) or several antigen-binding sites, e.g., diabodies or antibodies, that recognize the same epitope (e.g., identical to each other). The requirement that a binding domain is “single-specific” does not mean that it binds to only one antigen. This is because multiple antigens may have shared or very similar epitopes to which a single antigen-binding site can bind. A single-specific binding domain that binds to only one antigen is said to “bind exclusively” to that antigen.

[0233] The term "multispecific" refers to a binding domain that contains two or more antigen-binding sites, each binding to a different epitope, for example, each binding to a different antigen. For example, a multispecific binding domain may contain antigen-binding sites that recognize two or more different epitopes on the same protein (e.g., a coagulation factor) or that can recognize two or more different epitopes on different proteins (i.e., different coagulation factors). In one example, the binding domain may be "bispecific," meaning it contains two antigen-binding sites that specifically bind to two different epitopes. For example, a bispecific binding domain specifically binds to or has specificity for two different epitopes on the same protein. In another example, a bispecific binding domain specifically binds to two different epitopes on two different proteins.

[0234] As used herein, the term “binding” means, with respect to the interaction of a compound or its antigen-binding site with an antigen, that the interaction depends on the presence of a specific structure on the antigen (e.g., an antigenic determinant or epitope). For example, an antibody recognizes and binds to a specific protein structure rather than the protein in general. If an antibody binds to epitope “A”, the presence of molecules containing epitope “A” (or free, unlabeled “A”) in a reaction involving labeled “A” and the protein reduces the amount of labeled “A” that binds to the antibody.

[0235] As used herein, the terms “specifically bind” or “specifically conjugate” should be understood to mean that the protein of this disclosure reacts to or binds to a particular antigen or cell expressing it more frequently, more rapidly, for a longer period of time and / or with greater affinity than alternative antigens or cells. For example, a conjugate containing an antibody Fc that binds to factor XII (e.g., human factor XII) with substantially greater affinity (e.g., 20x or 40x or 60x or 80x to 100x, or 150x or 200x) than it binds to antigens commonly recognized by other cytokine receptors or multireactive native antibodies (i.e., by naturally occurring antibodies known to bind to a variety of naturally occurring antigens in humans). In general, but not limited to, references to binding should be understood to mean specific binding, and each term should be understood to provide clear support for other terms.

[0236] The term “competitively inhibiting” should be understood to mean that the protein (or its antigen-binding site) of this disclosure reduces or blocks the binding of the listed antibody or protein to factor XII and / or factor XIIa. This can be due to the binding of the protein (or antigen-binding site) and antibody to the same or overlapping epitopes. From the foregoing, it is clear that the protein does not need to completely inhibit antibody binding; rather, it is sufficient for it to reduce binding by only a statistically significant amount, for example, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. Preferably, the protein reduces antibody binding by at least about 30%, more preferably at least about 50%, more preferably at least about 70%, even more preferably at least about 75%, even more preferably at least about 80% or 85%, and even more preferably at least about 90%. Methods for determining competitive inhibition of binding are known in the art and / or described herein. For example, an antibody is exposed to factor XII in the presence or absence of a protein. If fewer antibodies bind in the presence of the protein than in the absence of the protein, the protein is considered to competitively inhibit antibody binding. In one example, the competitive inhibition is not due to steric hindrance.

[0237] In relation to two epitopes, "duplication" should be understood as meaning that the two epitopes share a sufficient number of amino acid residues to allow a protein (or its antigen-binding site) that binds to one epitope to competitively inhibit the binding of a protein (or its antigen-binding site) that binds to the other epitope. For example, "duplication" epitopes share at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

[0238] The term "conservative amino acid substitution" refers to the replacement or substitution of an amino acid residue with an amino acid residue having a similar side chain and / or being hydrophobic and / or hydrophilic. Families of amino acid residues having similar side chains are defined in the art as including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). The hydrophobic index is described, for example, in Kyte and Doolittle J. Mol. Biol., Vol. 157: pp. 105-132, 1982, and the hydrophilic index is described, for example, in U.S. Patent No. 4554101.

[0239] As used herein, the terms “disease,” “disorder,” or “condition” refer to the disruption or interference with normal function, and include, but are not limited to, any specific condition.

[0240] As used herein, the term “thrombotic disorder” refers to a condition characterized by the formation or potential for the formation of a blood clot. A blood clot is also called a “thrombi,” “thrombus,” or “thrombosis.”

[0241] As used herein, the term “inflammatory disorder” refers to a condition characterized by the occurrence of inflammation or the potential for inflammation.

[0242] As used herein, the term “thromboinflammatory disorder” refers to any condition characterized by exhibiting both inflammatory and thrombotic tendencies.

[0243] As used herein, the phrase “thrombotic disorder, inflammatory disorder and / or thromboinflammatory disorder” refers to a disease or condition that is classified as one or more of the following: thrombotic disorder, inflammatory disorder, or thromboinflammatory disorder.

[0244] As used herein, the terms “to treat,” “to treat,” or “treatment” include administering any of the proteins described herein to reduce or eliminate at least one symptom of a specified disease or condition, or to slow the progression of a disease or condition.

[0245] As used herein, the terms “prevent,” “prevent,” or “prevent” include providing a preventive measure in relation to the occurrence or recurrence of a specified disease or condition in an individual. The individual may have a predisposition or risk of developing the disease or disease recurrence, but has not yet been diagnosed with the disease or recurrence.

[0246] As used herein, a subject “at risk” of developing a disease or condition or its recurrence or relapse may have a detectable disease or symptoms of the disease, and may not have presented a detectable disease or symptoms of the disease prior to treatment according to this disclosure. “At risk” means that the subject has one or more risk factors, which are measurable parameters that correlate with the development of a disease or condition, as is known in the art and / or described herein.

[0247] The “effective dose” refers to the amount that is effective in terms of the dosage and duration required to achieve the desired outcome. For example, the desired outcome may be the result of a treatment or preventive measure. An effective dose may be provided in one or more doses. In some examples of this disclosure, the term “effective dose” means the amount required to achieve treatment for the disease or condition described above herein. In some examples of this disclosure, the term “effective dose” means the amount required to achieve a change in the factors associated with the disease or condition described above herein. The effective dose may also vary depending on the disease or condition to be treated or the factors to be changed, as well as on body weight, age, racial background, sex, health and / or physical condition, and other factors related to the mammal being treated. Typically, the effective dose falls within a relatively broad range (e.g., “dosage” range) that can be determined by routine testing and experimentation by a physician. Therefore, this term should not be construed as limiting this disclosure to a specific amount, such as weight or number. The effective dose may be administered in a single dose or in doses repeated once or several times over a period of treatment.

[0248] The "therapeutic dose" is the minimum concentration required to achieve a measurable improvement in a particular disease or condition. The therapeutic dose as used herein may vary depending on factors such as the patient's disease state, age, sex, and weight, as well as the antibody or its antigen-binding fragment's ability to induce a desired response in the individual. The therapeutic dose is also the amount at which the therapeutically beneficial effect outweighs any toxic or adverse effects of the antibody or its antigen-binding fragment.

[0249] As used herein, the term “subject” should be understood to mean any animal, including humans, e.g., mammals. Exemplary subjects include, but are not limited to, humans and non-human primates. For example, the subject is humans.

[0250] Proteins in pharmaceutical formulations As discussed herein, the disclosure provides a liquid pharmaceutical formulation comprising at least 100 mg / ml of protein containing an antigen-binding domain that binds to or specifically binds to factor XII and / or its activated form (i.e., activated FXII; FXIIa).

[0251] Proteins containing antigen-binding domains This disclosure provides a pharmaceutical formulation comprising a protein containing an antigen-binding domain that binds to or specifically binds to factor XII and / or its activated form. For example, the protein contains at least V H and V L including, V H and V L It binds to form an Fv containing an antigen-binding domain.

[0252] In one example, the antigen-binding domain binds to or specifically binds to factor XII and / or its activated form, antagonizing the activity of factor XII and / or activated factor XII. For example, the protein binds to or specifically binds to factor XII, antagonizing its activity. In another example, the protein binds to or specifically binds to activated factor XII (FXIIa), antagonizing its activity.

[0253] In one example, the antigen-binding domain binds to or specifically binds to factor XII and / or its activated form, antagonizing the activation of factor XII and / or activated factor XII. For instance, the protein binds to or specifically binds to factor XII, inhibiting its activation to factor XIIa.

[0254] Antibody or antigen-binding fragments In one example, a protein containing an antigen-binding domain that binds to or specifically binds to factor XII and / or its activated form is an antibody or antigen-binding fragment. For example, the protein is an antibody or antigen-binding fragment that binds to factor XII and / or activated factor XII (FXIIa). For example, the protein is an antibody or antigen-binding fragment that binds to factor XII. In another example, the protein is an antibody or antigen-binding fragment that binds to activated factor XII (FXIIa).

[0255] Methods for producing antibodies are known in the art and / or described in Harlow and Lane (eds.), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988). Generally, in such methods, factor XII (e.g., hFXII) or its region (e.g., extracellular domain), or its immunogenic fragment or epitope, or cells expressing and presenting it (i.e., immunogen), which are optionally formulated with any suitable or desired carrier, adjuvant, or pharmaceutically acceptable excipient, are administered to non-human animals, such as mice, chickens, rats, rabbits, guinea pigs, dogs, horses, cattle, goats, or pigs. The immunogen can be administered intranasally, intramuscularly, subcutaneously, intravenously, intradermally, intraperitoneally, or by other known routes.

[0256] Monoclonal antibodies are one exemplary form of antibodies as envisioned by this disclosure. The term “monoclonal antibody” or “mAb” refers to a homogeneous population of antibodies capable of binding to the same antigen(s), e.g., the same epitope within an antigen. The term is not limited to the source of the antibody or the method by which it is produced.

[0257] For the production of mAbs, one of several known techniques can be used, for example, any one of the methods exemplified in U.S. Patent No. 4,196,265 above or Harlow and Lane (1988).

[0258] Alternatively, the ABL-MYC technology (NeoClone, Madison WI 53713, USA) is used to produce MAb-secreting cell lines (e.g., Largaespada et al., described in J. Immunol. Methods, Vol. 197: pp. 85-95, 1996).

[0259] Antibodies can also be produced or isolated by screening display libraries, such as phage display libraries, such as those described in U.S. Patent No. 6,300,064 and / or U.S. Patent No. 5,885,793. For example, the inventors isolated fully human antibodies from phage display libraries.

[0260] The antibodies in this disclosure may be synthetic antibodies. For example, the antibody may be a chimeric antibody, a humanized antibody, a human antibody, or a deimmunized antibody.

[0261] The antibodies or antigen-binding fragments of this disclosure may be humanized.

[0262] The term “humanized antibody” should be understood to refer to a protein containing a human-like variable region (this type of antibody is also called a “CDR-transplanted antibody”), which includes a CDR derived from an antibody of a non-human species (e.g., mouse or rat or non-human primate) transplanted or inserted into a FR derived from a human antibody. Humanized antibodies also include antibodies in which one or more residues of a human protein are modified by one or more amino acid substitutions, and / or one or more FR residues of a human antibody are replaced by corresponding non-human residues. Humanized antibodies may also contain residues not found in either human or non-human antibodies. Any additional regions of the antibody (e.g., Fc regions) are generally human. Humanization can be carried out using methods known in the art, for example in U.S. Patent No. 5,225,539, U.S. Patent No. 6,054,297, U.S. Patent No. 7,566,771 or U.S. Patent No. 5,585,089. The term “humanized antibody” also includes, for example, the hyperhumanized antibody described in U.S. Patent No. 7,732,578. The same meaning applies to the term "humanized antigen-binding fragment."

[0263] The antibodies or antigen-binding fragments of the present disclosure may be human antibodies or antigen-binding fragments thereof. The term “human antibody” means, as used herein, an antibody having a variable antibody region and optionally a constant antibody region found in human, e.g., human germline cells or somatic cells, or derived from a library produced using such regions. A “human” antibody may contain amino acid residues not encoded by a human sequence, e.g., mutations introduced by random or site-directed mutations in vitro (in particular, mutations involving a small number of residues in a protein, e.g., one, two, three, four, or five residues of a protein with a conservative substitution or mutation). These “human antibodies” do not necessarily have to be produced as a result of a human immune response, but rather can be produced using recombinant means (e.g., screening of phage display libraries), and / or by transgenic animals (e.g., mice) containing nucleic acids encoding the human antibody constant region and / or variable region, and / or by induced selection (e.g., as described in U.S. Patent No. 5,565,332). The term also encompasses affinity-matured forms of such antibodies. For the purposes of this disclosure, human antibodies are considered to include FRs that are derived from human antibodies or FRs that are derived from consensus sequences of human FRs, and also include proteins in which one or more CDRs are random or semi-random, such as those described in U.S. Patent No. 6,300064 and / or U.S. Patent No. 6,248516. The same meaning applies to the term “human antigen-binding fragment.”

[0264] The antibody or its antigen-binding fragment of the present disclosure may also be a syn-humanized antibody or its antigen-binding fragment. The term "syn-humanized antibody" refers to an antibody prepared by the method described in WO2007019620. The syn-humanized antibody includes a variable region of the antibody, which includes FR derived from the New World primate antibody variable region and CDR derived from the non-New World primate antibody variable region.

[0265] The antibodies or antigen-binding fragments of the present disclosure may be primate-modified. A “primate-modified antibody” includes a variable region derived from an antibody produced after immunization of a non-human primate (e.g., a cynomolgus monkey). Optionally, a primate-modified antibody is produced by ligating the variable region of a non-human primate antibody to a human constant region. An exemplary method for producing a primate-modified antibody is described in U.S. Patent No. 6,113,898.

[0266] In one example, the antibody or its antigen-binding fragment of the present disclosure is a chimeric antibody or fragment. The terms “chimeric antibody” or “chimeric antigen-binding fragment” refer to an antibody or fragment in which one or more variable domains originate from a particular species (e.g., a murid such as mouse or rat) or belong to a particular antibody class or subclass, while the remainder of the antibody or fragment originates from another species (e.g., a human or a non-human primate) or belongs to another antibody class or subclass. In one example, the chimeric antibody is derived from a non-human antibody (e.g., a murid antibody). H and / or V L The antibody contains the antigen-binding fragment, with the remaining region of the antibody being derived from a human antibody. The preparation of such chimeric antibodies and their antigen-binding fragments is known in the art and can be achieved by standard means (see, for example, U.S. Patent No. 6,331,415; U.S. Patent No. 5,807,715; U.S. Patent No. 4,816,567 and U.S. Patent No. 4,816,397).

[0267] This disclosure also intends to include immunized antibodies or their antigen-binding fragments, such as those described in WO2000034317 and WO2004108158. The immunized antibodies and fragments have one or more epitopes, such as B-cell epitopes or T-cell epitopes, removed (i.e., mutated), thereby reducing the likelihood that a target will elicit an immune response to the antibody or protein. For example, one could analyze an antibody of this disclosure to identify one or more B-cell epitopes or T-cell epitopes and mutate one or more amino acid residues within the epitope, thereby reducing the immunogenicity of the antibody.

[0268] Exemplary human antibodies described herein include 3F7, 3F7G, and affinity-mature 3F7, and / or their variable regions. A further exemplary antibody is the anti-FXII antibody galadasimab. These human antibodies offer the advantage of reduced immunogenicity in humans compared to non-human antibodies. Exemplary antibodies are described in WO2013 / 014092 and WO2017 / 173494, which are incorporated herein by reference. Additional antibodies and proteins containing variable regions are described in WO2006 / 066878 and in Ravon et al., Blood Vol. 86: pp. 4134-43 (1995).

[0269] bispecific antibody In one example, the protein of this disclosure may be a bispecific antibody or a fragment thereof. For example, the antibody or fragment may bind to factor XII and / or its activated form as well as to another target. A bispecific antibody is a molecule comprising two antibodies or antibody fragments (e.g., two half-antibodies) having specificity for different antigens or epitopes. An exemplary bispecific antibody binds to two different epitopes of the same protein. Alternatively, a bispecific antibody binds to two different epitopes of two different proteins.

[0270] Exemplary "key-and-hole" or "knob-and-hole" bispecific proteins as described in U.S. Patent No. 5,731,168. In one example, the constant region (e.g., the IgG4 constant region) contains the T366W mutation (or knob), and the constant region (e.g., the IgG4 constant region) contains the T366S, L368A, and Y407V mutations (or holes). In another example, the first constant region contains the T350V, T366L, K392L, and T394W mutations (or knobs), and the second constant region contains the T350V, L351Y, F405A, and Y407V mutations (or holes).

[0271] Methods for generating bispecific antibodies are known in the art, and exemplary methods are described herein.

[0272] In one example, IgG-type bispecific antibodies are secreted by hybrid hybridomas (quadromas) formed by fusing two types of hybridomas that produce IgG antibodies (Milstein C et al., Nature 1983, Vol. 305: pp. 537-540). In another example, antibodies can be secreted by introducing the genes for the light and heavy chains that make up the two target IgGs to be co-expressed into cells (Ridgway, JB et al., Protein Engineering 1996, Vol. 9: pp. 617-621; Merchant, AM et al., Nature Biotechnology 1998, Vol. 16: pp. 677-681).

[0273] In one example, bispecific antibody fragments are prepared by chemically crosslinking Fab' derived from different antibodies (Keler T et al., Cancer Research 1997, Vol. 57: pp. 4008-4014).

[0274] In one example, leucine zippers derived from Fos and Jun are used to form bispecific antibody fragments (Kostelny SA et al., J. of Immunology, 1992, Vol. 148: pp. 1547-1553).

[0275] In one example, a bispecific antibody fragment is prepared in the form of a diabody containing two crossover scFv fragments (Holliger P et al., Proc. of the National Academy of Sciences of the USA 1993, Vol. 90: pp. 6444-6448).

[0276] antibody fragment As described herein, the proteins of this disclosure are the constant region Fc or heavy chain constant domain C of the antibody. H 2 and / or C H It includes an antigen-binding fragment linked to 3. An example antigen-binding fragment for use in this disclosure is described below.

[0277] Single-domain antibody In some examples, the antigen-binding fragment of the antibody of this disclosure is or contains a single-domain antibody (which is used interchangeably with the terms “domain antibody” or “dAb”). A single-domain antibody is a single polypeptide chain that contains all or part of the heavy chain variable domain of the antibody.

[0278] Diabody, Triabody, Tetrabody In some examples, the antigen-binding fragments of this disclosure are or include diabodies, triabodies, tetrabodies, or higher-order protein complexes, such as those described in WO98 / 044001 and / or WO94 / 007921.

[0279] For example, a diabody is a protein containing two accompanying polypeptide chains, and each polypeptide chain is structure V L -XV H or V H -XV L It contains, and in the structure, X is V in a single polypeptide chain. H and V L The linker contains insufficient residues to allow for the attachment (or formation of Fv) of one polypeptide chain, or it is absent, and the V of one polypeptide chain H The V of the other polypeptide chain. L It binds to form an antigen-binding site, that is, it forms an Fv molecule that can specifically bind to one or more antigens. L and V H V may be the same in each polypeptide chain, or L and V H These may differ in each polypeptide chain so as to form a bispecific diabody (i.e., containing two Fv with different specificities).

[0280] Single chain Fv (scFv) fragment A person skilled in the art is a V in a single polypeptide chain. H and V LThe region, as well as the scFv, allows the scFv to form the desired structure for antigen binding (i.e., the V of a single polypeptide chain). H and V L (In order for them to combine with each other to form Fv), V H and V L It is known that polypeptide linkers are included between them. For example, the linker contains more than 12 amino acid residues, and (Gly4Ser)3 is one of the more favorable linkers for scFv.

[0281] In one example, the linker contains the sequence SGGGGSGGGGSGGGGS.

[0282] In this disclosure, a single cysteine ​​residue is V H FR and V L It is introduced into the FR, and a stable Fv is obtained by linking cysteine ​​residues by disulfide bonds, and a disulfide-stabilized Fv (or diFv or dsFv) is also intended.

[0283] Alternatively, the present disclosure may encompass a protein comprising a dimeric scFv, i.e., two scFv molecules linked by non-covalent or covalent linkage, for example, by a leucine zipper domain (e.g., derived from Fos or Jun). Alternatively, the two scFvs may be linked by a peptide linker of sufficient length to allow both scFvs to form and bind to an antigen, as described, for example, in U.S. Patent Application Publication No. 20060263367.

[0284] Heavy chain antibodies In some cases, the antigen-binding fragments of this disclosure are or contain heavy chain antibodies. Heavy chain antibodies are structurally different from many other forms of antibodies, in that they contain a heavy chain but not a light chain. Thus, these antibodies are also called “heavy chain only antibodies.” Heavy chain antibodies are found, for example, in camels and cartilaginous fish (also called IgNARs). General descriptions of heavy chain antibodies and their variable regions from camels, as well as methods for their preparation and / or isolation and / or use, can be found, in particular, in the following references WO94 / 04678, WO97 / 49805 and WO97 / 49805. General descriptions of heavy chain antibodies and their variable regions from cartilaginous fish, as well as methods for their preparation and / or isolation and / or use, can be found, in particular, in WO2005 / 118629.

[0285] half antibody In some examples, the antigen-binding fragments of this disclosure are half-antibodies or half-bodies. Those skilled in the art will recognize that a half-antibody refers to a protein comprising a single heavy chain and a single light chain. The term “half-antibody” also encompasses proteins comprising an antibody light chain and an antibody heavy chain that has been mutated to prevent co-occurrence with another antibody heavy chain. In one example, a half-antibody is formed when an antibody dissociates to form two molecules, each comprising a single heavy chain and a single light chain.

[0286] Methods for generating half-antibodies are known in the art, and exemplary methods are described herein.

[0287] In one example, a semi-antibody can be secreted by introducing the genes for a single heavy chain and a single light chain that constitute the target IgG to be expressed into a cell. In one example, the constant region (e.g., the IgG4 constant region) contains a "key or hole" (or "knob or hole") mutation to prevent heterodimerization. In another example, the constant region (e.g., the IgG4 constant region) contains the T366W mutation (or knob). In yet another example, the constant region (e.g., the IgG4 constant region) contains the T366S, L368A, and Y407V mutations (or holes). In yet another example, the constant region contains the T350V, T366L, K392L, and T394W mutations (nobs). In yet another example, the constant region contains the T350V, L351Y, F405A, and Y407V mutations (holes). Exemplary constant region amino acid substitutions are numbered according to the EU numbering system.

[0288] Other antibodies and antibody fragments This disclosure also intends to cover other antibodies and antibody fragments, such as: (i) For example, the minibody described in U.S. Patent No. 5837821; (ii) Heteroconjugate proteins, for example, as described in U.S. Patent No. 4676980; (iii) heteroconjugate proteins prepared using chemical crosslinking agents, for example, those described in U.S. Patent No. 4676980; and (iv) Fab3 (for example, as described in EP19930302894).

[0289] Stabilizing protein The proteins of this disclosure may include an IgG4 constant region or a stabilized IgG4 constant region. The term “stabilized IgG4 constant region” is understood to mean an IgG4 constant region modified to undergo Fab arm exchange, or to reduce the tendency to undergo Fab arm exchange or the formation of half-antibodies. “Fab arm exchange” refers to a type of protein modification of human IgG4 in which the IgG4 heavy chain and the bound light chain (half-chain) are replaced with a heavy-light chain pair from another IgG4 molecule. Thus, an IgG4 molecule can acquire two different Fab arms that recognize two different antigens (resulting in a bispecific molecule). Fab arm exchange is naturally occurring in vivo and can be induced in vitro by purified blood cells or by reducing agents such as reduced glutathione.

[0290] For example, the stabilized IgG4 constant region contains a proline at position 241 of the hinge region according to Kabat's system (Kabat et al., Sequences of Proteins of Immunological Interest, Washington DC, United States Department of Health and Human Services, 1987 and / or 1991). This position corresponds to position 228 of the hinge region according to the EU numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, Washington DC, United States Department of Health and Human Services, 2001 and Edelman et al., Proc. Natl. Acad. USA, Vol. 63, pp. 78-85, 1969). In human IgG4, this residue is typically serine. After the substitution of serine with proline, the IgG4 hinge region contains the sequence CPPC. In this regard, those skilled in the art will know that the "hinge region" is the high-proline portion of the antibody heavy chain constant region that links the Fc and Fab regions and confers mobility to the two Fab arms of the antibody. The hinge region contains cysteine ​​residues involved in the inter-heavy-chain disulfide bond. It is generally defined as extending from Glu226 to Pro243 in human IgG1 according to the Kabat numbering system. Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form the inter-heavy-chain disulfide (SS) bond at the same positions (see, e.g., WO2010080538).

[0291] Preparation of pharmaceutical formulations This disclosure provides a liquid pharmaceutical formulation comprising at least 100 mg / ml of protein containing an antigen-binding domain that binds to or specifically binds to factor XII and / or its activated form (i.e., activated FXII; FXIIa), an organic acid buffer, a nonionic surfactant, and an amino acid stabilizer, having a pH of 5.0 to 6.5 and a viscosity of less than about 30 mPa*s at 20°C. The preparation of pharmaceutical formulations is carried out in accordance with standard methods known in the art and / or in accordance with the methods described herein.

[0292] Organic acid buffer In one example, the present disclosure provides a pharmaceutical formulation comprising at least 100 mg / ml of the present disclosure protein and an organic acid buffer having a pH of 5.0 to 6.5.

[0293] Those skilled in the art will understand that organic acid buffers suitable for use in this disclosure comprise one or more carboxylic acid groups or acidic phenol groups and have no basic amino groups. In addition to the buffering capacity provided by the acidic groups, such organic buffers used herein may also comprise additional ionizable functional groups, such as amino groups.

[0294] It will be apparent to those skilled in the art that buffers suitable for use in this disclosure are stable and effective at the desired pH and provide sufficient buffering capacity to maintain the desired pH over the range of conditions to which the product is exposed during formulation and storage. For example, a stable buffer provides thermal agglomeration stability (e.g., during freezing / thawing or at high temperatures), is unaffected by oxidation of physical decomposition (e.g., insoluble particle formation), and provides the desired polydispersity (i.e., particle distribution). A suitable buffer does not form harmful complexes with metal ions, is not toxic, and does not excessively penetrate, dissolve, or absorb onto membranes or other surfaces. Furthermore, those skilled in the art will recognize that such buffers should not interact with other components of the composition in any way that reduces their availability or effectiveness. In addition, buffers in pharmaceutical formulations must be safe for administration, compatible with other components of the composition over the shelf life of the product, and tolerable for administration to the subject.

[0295] Suitable organic acid buffers for use in this disclosure will become apparent to those skilled in the art, for example, histidine buffers (e.g., histidine chloride, histidine acetate, histidine phosphate, histidine sulfate, etc.), glutamate buffers (e.g., monosodium glutamate, etc.), citrate buffers (e.g., monosodium citrate-disodium citrate mixture, citrate-trisodium citrate mixture, citrate-monosodium citrate mixture, etc.), succinate buffers (e.g., succinate-monosodium succinate mixture, succinate-sodium hydroxide mixture, succinate-disodium succinate mixture, etc.), tartaric acid buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), These include fumarate buffers (e.g., fumarate-monosodium fumarate mixture, fumarate-disodium fumarate mixture, monosodium fumarate-disodium fumarate mixture, etc.), gluconate buffers (e.g., gluconate-sodium gluconate mixture, gluconate-sodium hydroxide mixture, gluconate-potassium gluconate mixture, etc.), oxalate buffers (e.g., oxalate-sodium oxalate mixture, oxalate-sodium hydroxide mixture, oxalate-potassium oxalate mixture, etc.), lactic acid buffers (e.g., lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate mixture, etc.), and acetate buffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture, etc.).

[0296] In one example of this disclosure, the organic acid buffer is selected from the group consisting of histidine buffers, glutamate buffers, succinate buffers, and citrate buffers. For example, the organic acid buffer is a glutamate buffer. For example, the organic acid buffer is a histidine buffer. For example, the organic acid buffer is L-histidine.

[0297] Methods for investigating the suitability of buffers are apparent to those skilled in the art and / or are described herein, and include, for example, differential scanning fluorescence quantification and dynamic light scattering.

[0298] Nonionic surfactants In one example, the present disclosure provides a pharmaceutical formulation comprising at least 100 mg / ml of the present protein and a nonionic surfactant.

[0299] The amount of surfactant added to a pharmaceutical formulation will be apparent to those skilled in the art and is such that it inhibits aggregation (e.g., by preventing surface denaturation), increases stability (e.g., during thermal and / or physical stress), minimizes particle formation in the formulation (e.g., formation of invisible particles), reduces surface adsorption, and / or assists protein refolding.

[0300] Suitable nonionic surfactants for use in this disclosure will become apparent to those skilled in the art and include, for example, polyoxyethylene sorbitan fatty acid esters (e.g., polysorbate 20 and polysorbate 80), polyethylene-polypropylene copolymers, polyethylene-polypropylene glycols, polyoxyethylene stearates, polyoxyethylene alkyl ethers, for example, polyoxyethylene monolauryl ethers, alkylphenyl polyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymers (poloxamer, pluronic), and sodium dodecyl sulfate (SDS).

[0301] In one example of this disclosure, the nonionic surfactant is selected from the group consisting of polyoxyethylene sorbitan fatty acid esters and polyoxyethylene-polyoxypropylene copolymers. For example, the polyoxyethylene sorbitan fatty acid ester is polyoxyethylene sorbitan monooleate (i.e., polysorbate 80) or polyoxyethylene sorbitan monolaurate (polysorbate 20).

[0302] Amino acid stabilizer In one example, the present disclosure provides a pharmaceutical formulation comprising at least 100 mg / ml of the protein and amino acid stabilizers of the present disclosure.

[0303] The amount of amino acid stabilizer added to a pharmaceutical preparation will be apparent to those skilled in the art, and is such that it reduces thermal stress and / or physical stress (e.g., freezing / thawing or stirring), and / or imparts or enhances protein stability.

[0304] Suitable amino acids for use in this disclosure will become apparent to those skilled in the art and include, for example, glycine, alanine, valine, leucine, isoleucine, methionine, threonine, phenylalanine, tyrosine, serine, cysteine, histidine, tryptophan, proline, aspartic acid, glutamic acid, arginine, lysine, ornithine, and asparagine and their salts.

[0305] In one example of this disclosure, the amino acid is selected from the group consisting of proline, arginine, and methionine. For example, the amino acid stabilizer is proline or a salt thereof. For example, the amino acid stabilizer is arginine or a salt thereof. For example, the amino acid stabilizers are proline and arginine or salts thereof.

[0306] polyol For example, the present disclosure provides a pharmaceutical formulation comprising at least 100 mg / ml of the present protein and polyol.

[0307] The amount of polyol added to a pharmaceutical formulation will be apparent to those skilled in the art and is an effective amount for reducing aggregation of the antibody or its fragments and increasing stability. For example, the polyol used in this disclosure helps the antibody or its fragments maintain a compact state (e.g., by reducing unfolding and / or aggregation).

[0308] Polyols suitable for use in this disclosure will become apparent to those skilled in the art and include, for example, sugars (reducing or non-reducing sugars), sugar alcohols, and sugar acids. "Reducing sugars" means sugars capable of reducing metal ions, or sugars containing hemiacetal groups capable of covalently reacting with lysine and other amino groups in proteins. Examples of reducing sugars include, for example, fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose, and glucose. "Non-reducing sugars" do not possess such properties. Examples of non-reducing sugars include, for example, sucrose, trehalose, sorbose, melegitose, and raffinose. Examples of sugar alcohols include mannitol, xylitol, erythritol, treitol, sorbitol, and glycerol. In one example of this disclosure, the polyol is sorbitol. Examples of sugar acids include L-gluconic acid and its metal salts.

[0309] Testing the pharmaceutical formulations and proteins of this disclosure The high-concentration pharmaceutical formulations and proteins of this disclosure can be easily screened for physical and biological activity and / or stability using methods known in the art and / or the methods described below.

[0310] Binding to Factor XII and / or Factor XIIa It will be apparent to those skilled in the art from the disclosure herein that the proteins of this disclosure bind (or specifically bind) to the ligand-binding domain of factor XII and / or activated factor XII (i.e., factor XIIa). Methods for investigating binding to proteins are known in the art, for example, as described in Scopes (source: Protein purification: principles and practice, 3rd edition, Springer Verlag, 1994). Such methods generally involve labeling the protein and contacting it with an immobilized compound. After washing to remove non-specifically bound proteins, the amount of the labeled and, as a result, the bound protein is detected. Naturally, the protein can be immobilized and labeled with a compound that binds to factor XII and / or factor XIIa. Panning-type assays can also be used. Alternatively, surface plasmon resonance assays can be used.

[0311] The assay described above can also be used to detect the level of binding of the proteins of this disclosure to factor XII and / or factor XIIa or their ligand-binding domains. Methods for detecting the binding level will be apparent to those skilled in the art and / or are described herein. For example, the binding level can be determined using a biosensor.

[0312] Measuring the activity of factor XII / XIIa Methods for investigating the antagonistic activity of proteins are known in the art, including, for example, chromogenic assays. Chromogenic assays for measuring inhibitory activity are known in the art (e.g., Chromogenix S-2302®; Diapharma).

[0313] In one example, an assay buffer is premixed with factor XIIa. The conjugate of this disclosure is added, followed by the chromogenic substrate. After the chromogenic reaction is stopped, the inhibitory activity of the conjugate is investigated.

[0314] Determine competitive coupling Assays for determining proteins that competitively inhibit the binding of antibodies 3F7 and / or 3F7G (or any other antibodies described herein) will become apparent to those skilled in the art. For example, 3F7 or 3F7G is conjugated with a detectable label, such as a fluorescent or radioactive label. The labeled antibody and the test protein are then mixed and brought into contact with factor XII or its region, or with cells expressing it. The level of labeled 3F7 or 3F7G is then determined and compared to the level determined when the labeled antibody is brought into contact with factor XII, the region, or the cell in the absence of the protein. If the level of labeled 3F7 or 3F7G is reduced in the presence of the test protein compared to the absence of the protein, the protein is considered to competitively inhibit the binding of 3F7 or 3F7G to factor XII.

[0315] In some cases, the test protein is conjugated to different labels, either 3F7 or 3F7G. This alternative labeling allows for the detection of the test protein's binding level to factor XII or its region or cells.

[0316] In another example, the protein is bound to factor XII or its region or cells expressing it before contacting factor XII, its region, or cells with 3F7 or 3F7G. A reduction in the amount of bound 3F7 or 3F7G in the presence of the protein compared to the absence of the protein indicates that the protein competitively inhibits the binding of 3F7 or 3F7G to factor XII. The reverse assay can also be performed by using a labeled protein and first binding 3F7 or 3F7G to factor XII. In this case, a reduction in the amount of labeled protein bound to factor XII in the presence of 3F7 or 3F7G compared to the absence of 3F7 or 3F7G indicates that the protein competitively inhibits the binding of 3F7 or 3F7G to factor XII.

[0317] FXIIa amide solubility For example, the protein of this disclosure inhibits the amide-soluble activity of human factor XIIa. Methods for determining the amide-soluble activity of the conjugate of this disclosure are apparent to those skilled in the art and / or are described herein.

[0318] In one example, an in vitro assay is used to determine the level of FXIIa amide-solubility activity. For example, amide-solubility activity can be measured by an assay of cleavage of FXII in the presence of the conjugate and buffer of the Disclosure. For example, FXII is incubated in or in the presence of the conjugate or control of the Disclosure. After incubation and addition of the detection substrate, amide-solubility activity is determined by spectrophotometric method as a change in optical density (i.e., a change in color). Proteins found to effectively inhibit amide-solubility activity are identified as proteins that inhibit FXII activity.

[0319] Visual appearance The pharmaceutical formulations included in this disclosure are examined for their visual appearance, for example, to determine their color and transparency.

[0320] Dynamic light scattering In one example, the particle size distribution is investigated using dynamic light scattering (DLS). DLS measures the light scattered from particles based on Brownian motion and depends on the difference in refractive index between the particles and the formulation. For example, a digital correlator is used to measure the variation in light intensity. The correlation function is fitted to an analysis program (e.g., Malvern Zetasizer software) to calculate the particle size distribution. To determine the Z-mean hydrodynamic diameter, for example, cumulant analysis and the Stokes-Einstein equations are performed using the viscosity of water at 25°C (0.8872 mPa*s). The polydispersion index can also be obtained from the same cumulant analysis. The modality of the fitting is evaluated based on the plot of size distribution versus intensity. The modality can be described as unimodal (i.e., one peak) or multimodal (i.e., two or more peaks).

[0321] Microflow imaging In one example, invisible particles are investigated using microflow imaging (MFI). For instance, digital images of particles suspended in a fluid are captured, and particle parameters such as aspect ratio (AR) and intensity are automatically analyzed. Size (e.g., in μm) and count (i.e., number of particles per ml) can also be obtained. This method allows for morphological classification of the data into proteinaceous (i.e., non-round) and non-proteinaceous (i.e., non-proteinaceous particles such as bubbles or silicone oil droplets), and the ratio of non-proteinaceous particles to proteinaceous particles (i.e., roundness ratio) can be determined. A low roundness ratio indicates that the test specimen contains mainly non-round, possibly proteinaceous, particles.

[0322] Size exclusion chromatography In one example, soluble aggregates are investigated using size exclusion chromatography (SEC or SE-HPLC) to separate low and high molecular weight variants of proteins as well as any impurities. According to this method, the results are described as the sum of aggregate peaks (AP) and the sum of degraded peaks (DP). For example, the identity of the pharmaceutical formulations of this disclosure is determined by comparing the chromatographic retention time of the major peak with the retention time of the major peak of a reference standard.

[0323] Differential scanning fluorescence (DSF) In one example, the thermal stability of the pharmaceutical formulations of this disclosure is investigated using differential scanning fluorescence (DSF). DSF is a fluorescence-based assay using real-time PCR to monitor thermally induced protein denaturation by measuring the fluorescence change of a dye that preferentially binds to unfolded proteins. For example, thermal unfolding and aggregation are monitored as a function of temperature by changes in endogenous protein fluorescence and static light scattering, respectively. According to this method, by monitoring endogenous fluorescence, the midpoint of the thermal transition (T) can be determined. m ) and melting onset temperature (T onset The aggregation start temperature (T) is determined. aggThis is determined by monitoring static light scattering, for example, at 266 nm and 473 nm. Samples of pharmaceutical formulations can be investigated by increasing the temperature over a temperature range (e.g., 20–95°C) at a rate of, for example, 0.5°C / min.

[0324] Capillary gel electrophoresis In one example, the pharmaceutical formulations of this disclosure are investigated for stability and / or total impurity accumulation using capillary gel electrophoresis (CGE). For example, both reducing CGE (R-CGE) and non-reducing CGE (NR-CGE) can be performed. In one example, R-GCE and NR-CGE are performed using a capillary electrophoresis system (e.g., Beckman P / ACE MDQ or PA800) having capillary lengths of, for example, 20.2 cm and 10 cm from the inlet to the detection window, temperature control of, for example, 20–40°C (±2°C), and a detector excited at, for example, 488 nm.

[0325] Cation exchange chromatography In one example, the pharmaceutical formulations of this disclosure are investigated for total charge variants using cation exchange (CEX) chromatography. In CEX chromatography, proteins are separated according to their overall charge under natural conditions. The purity of the product is determined by separating acidic and basic variants using CEX analysis. The protein of interest must have a charge opposite to the charge of the functional group attached to the resin of the column in order to bind. Protein elution is achieved by increasing the ionic strength to disrupt the ionic interaction between the protein and the resin. In this chromatographic technique, acidic, neutral, and basic variants of the sample are separated based on ionic strength. The peak of interest is observed by UV detection at 280 nm, with acidic variants eluting first, followed by neutral and basic variants. In one example, CEX chromatography is performed using a high-performance liquid chromatography (HPLC) system (e.g., Dionex UltiMate 3000 BioRS(U)HPLC).

[0326] Gibbs free energy (ΔG) trend ;HUNK) For example, the chemical stability and aggregation behavior of the pharmaceutical formulations of this disclosure are determined by the Gibbs free energy or ΔG trend Evaluated by changes in (HUNK) analysis. ΔG trend The analysis measures the relationship between the ΔG of protein unfolding and protein aggregation as a function of protein concentration. If aggregation is absent, the ΔG of protein unfolding is a unimolecule process independent of protein concentration. If a change in ΔG is observed as a function of protein concentration, it indicates the presence of aggregation. According to this method, if aggregation occurs, two relationships are possible between the ΔG of protein unfolding and protein concentration. 1. ΔG trend ΔG increases with protein concentration: this relationship indicates the presence of native aggregates, and ΔG of protein unfolding increases as a function of protein concentration (becoming more positive) (i.e., the concentration of native protein aggregates increases as a function of protein concentration); or 2. ΔG trend ΔG decreases with protein concentration: this relationship indicates the presence of denatured aggregates, and the ΔG of protein unfolding decreases as a function of protein concentration (becoming a less positive value) (i.e., the concentration of denatured protein aggregates increases as a function of protein concentration).

[0327] In the HUNK experiment, the ΔG of protein unfolding isothermally determined by measuring the change in the endogenous fluorescence spectrum (i.e., emission from tryptophan residues) of the protein during unfolding in the presence of gradually increasing amounts of denaturant.

[0328] In one example, ΔG trendThe ΔG of unfolded protein is determined by measuring the ΔG of unfolded protein in various concentrations (e.g., 0.25, 0.6, 2.5, 6.0, 25.0 mg / ml) diluted to target concentrations in the buffer of the pharmaceutical formulation of this disclosure. Each concentration level is titrated with gradually increasing denaturant concentrations while measuring the fluorescence spectrum at 300–500 nm (excitation at 280 nm) with a slit width of 10 nm (e.g., a 32-point curve in the range of urea concentrations from 2.00 to 8.74 M). For each sample concentration level, the emission spectral wavelength ratio at 350 nm / 330 nm is plotted against the urea concentration, and the ΔG of protein unfolding is determined using a two-state (i.e., one-transition) model fitting. The determined ΔG values ​​are plotted against the sample concentration, and the ΔG trend To decide.

[0329] Osmotic pressure In one example, the osmotic pressure of the pharmaceutical formulation described herein is investigated.

[0330] Turbidity investigated by absorbance at 550 nm In one example, the turbidity of the pharmaceutical formulation of this disclosure is investigated. For example, turbidity is investigated by measuring the absorbance at 550 nm using a spectrophotometer.

[0331] Syringe passage characteristics In one example, the syringe passage characteristics of the pharmaceutical formulations of this disclosure are investigated. For example, the formulation is dispensed from 2 ml syringes, 10 ml syringes, or left untreated as a pre-dispensing control. According to this method, the syringe plunger is pushed at a linear velocity of 0.2 inches / min for the 2 ml syringe and 0.6 inches / min for the 10 ml syringe until the plunger reaches the bottom and the force reaches 30 N. Break-loose force (BF) and sliding force (GF) are measured during dispensing and used to investigate applicability. Break-loose force describes the force required to initiate the plunger's movement (the first 0.3 mm for the 2 ml syringe and 0.5 mm for the 10 ml syringe). Maximum sliding force refers to the maximum frictional force required to maintain the plunger's movement. The maximum force value is measured from the edge of the brake-loose area to the edge of the sliding force area just before the point where the force reaches 30N (26mm for a 2ml syringe, 24mm for a 10ml syringe).

[0332] Use of pharmaceutical preparations As discussed herein, the Disclosure provides a method for treating or preventing a disease or condition in a subject, which includes administering a pharmaceutical formulation of the Disclosure to the subject. For example, the Disclosure provides a method for treating or preventing a disease or condition in a subject requiring such treatment or prevention.

[0333] This disclosure also provides the use of the pharmaceutical formulations disclosed to treat or prevent a disease or condition in a subject, including administering the pharmaceutical formulations disclosed to that subject. For example, the disclosure provides pharmaceutical formulations disclosed to treat or prevent a disease or condition in a subject that requires it.

[0334] This disclosure provides a method for antagonizing the activity of factor XII and / or activated factor XII in a subject, including a method for administering a high-concentration formulation of this disclosure to the subject. For example, this disclosure provides a method for antagonizing the activity of factor XII and / or activated factor XII in a subject that requires it.

[0335] For example, the disease or condition is a thrombotic disorder, an inflammatory disorder, and / or a thromboinflammatory disorder.

[0336] In one example, the subject suffers from a thrombotic disorder, an inflammatory disorder, and / or a thromboinflammatory disorder. The thrombotic disorder, an inflammatory disorder, and / or a thromboinflammatory disorder may be hereditary or acquired. For example, a subject suffering from a thrombotic disorder, an inflammatory disorder, and / or a thromboinflammatory disorder exhibits symptoms of the thrombotic disorder, an inflammatory disorder, and / or a thromboinflammatory disorder.

[0337] In one example, thrombotic disorders, inflammatory disorders, and / or thromboinflammatory disorders include venous, arterial, or capillary thrombosis (such as stroke, myocardial infarction, deep venous thrombosis (DVT), portal vein thrombosis, renal vein thrombosis, jugular vein thrombosis, cerebral venous sinus thrombosis, Budd-Chiari syndrome, Paget-Schlotter disease, or asymptomatic cerebral ischemia), cardiac thrombosis, thromboembolism, thrombosis during and / or after contact between blood and artificial surfaces in human or animal subjects, disseminated intravascular coagulation (DIC), atrial fibrillation, acute coronary syndrome (ACS), atherosclerotic disease, and ischemic cerebral ischemia due to reperfusion. The group consists of conditions related to stroke, ischemia-reperfusion injury (IRI, trauma, organ transplantation, etc.), neurotraumatic injuries (traumatic brain injury, spinal cord injury, etc.), neuroinflammatory diseases (multiple sclerosis, etc.), interstitial lung diseases (idiopathic pulmonary fibrosis (IPF), etc.), pneumonia, fibrinolysis, diseases related to FXII / FXIIa-induced kinin formation (hereditary angioedema (HAE), etc.), sepsis, diseases related to FXII / FXIIa-mediated complement activation, acute respiratory distress syndrome (ARDS), organ and cell transplantation, sickle cell disease, and conditions related to increased vascular permeability.

[0338] For example, the pharmaceutical formulations of this disclosure are administered to a subject in a dose that reduces the severity of the subject's disease or condition.

[0339] For example, a subject is at risk of developing a thrombotic disorder, an inflammatory disorder, and / or a thromboinflammatory disorder. A subject is at risk if their risk of developing a thrombotic disorder, an inflammatory disorder, and / or a thromboinflammatory disorder is higher than that of the control group. The control group may include one or more subjects randomly selected (e.g., matched for age, sex, race, and / or ethnicity) from the general population who do not have a history of or have no family history of a thrombotic disorder, an inflammatory disorder, and / or a thromboinflammatory disorder. A subject can be considered at risk of a disease or condition if “risk factors” associated with a thrombotic disorder, an inflammatory disorder, and / or a thromboinflammatory disorder are found to be associated with that subject. Risk factors may include, for example, any activity, trait, event, or characteristic associated with a given disorder in statistical or epidemiological studies of the subject population. Thus, a subject can be classified as at risk of a thrombotic disorder, an inflammatory disorder, and / or a thromboinflammatory disorder even if the subject was not specifically included in a study that identified underlying risk factors.

[0340] In one example, the subject is at risk of developing a thrombotic disorder, an inflammatory disorder, and / or a thromboinflammatory disorder, and the pharmaceutical formulation of this disclosure is administered before or after the onset of symptoms of a thrombotic disorder, an inflammatory disorder, and / or a thromboinflammatory disorder. In one example, the pharmaceutical formulation is administered before the onset of symptoms of a thrombotic disorder, an inflammatory disorder, and / or a thromboinflammatory disorder. In one example, the pharmaceutical formulation of this disclosure is administered in a dose that alleviates or reduces one or more symptoms of a thrombotic disorder, an inflammatory disorder, and / or a thromboinflammatory disorder in a subject at risk.

[0341] The method of this disclosure can be readily applied to any form of thrombotic disorders, inflammatory disorders and / or thromboinflammatory disorders.

[0342] For example, the method of this disclosure reduces any symptoms of thrombotic disorders, inflammatory disorders and / or thromboinflammatory disorders known in the art and / or described herein.

[0343] As will be apparent to those skilled in the art, “reduction” of the symptoms of a disorder in a subject is a comparison with another subject who has a similar disorder but has not been treated by the method described herein. This does not necessarily require a parallel comparison of two subjects. Rather, population data can be relied upon. For example, a population of subjects with thrombotic disorders, inflammatory disorders and / or thromboinflammatory disorders who have not been treated by the method described herein (possibly a population of subjects similar to the treated subjects, for example, in age, weight, and race) may be investigated, and their mean values ​​may be compared with the results of the subjects or population of subjects treated by the method described herein.

[0344] Furthermore, the method of this disclosure may include co-administration of the pharmaceutical formulation according to this disclosure in conjunction with the administration of another therapeutically effective agent for preventing or treating thrombotic disorders, inflammatory disorders and / or thromboinflammatory disorders.

[0345] For example, the pharmaceutical formulations of this disclosure may be used in combination with at least one additional known compound or therapeutic protein currently in use or under development to prevent or treat thrombotic disorders, inflammatory disorders and / or thromboinflammatory disorders, or to antagonize the activity of factor XII and / or its activated form. Compounds currently used in the treatment of thrombotic disorders, inflammatory disorders and / or thromboinflammatory disorders are known in the art, for example, vitamin K antagonists (e.g., warfarin), heparin (e.g., unfractionated heparin or low molecular weight heparin), synthetic pentasaccharides (e.g., fondaparinux and hydraparinux), direct inhibitors of factor Xa and thrombin (e.g., rivaroxaban, repiridine, decilidine and dabigatran), cyclooxygenase inhibitors (e.g., aspirin, rofecoxib and valdecoxib), ADP receptor antagonists (clopidogrel, prasugrel), protease-activated receptor-1 inhibitors (i.e., PAR1), α IIbThis includes β3-integrin inhibitors (e.g., absiximab and eptifavatide) and statins (e.g., lovastatin, pravastatin, rosuvastatin, simvastatin, atorvastatin, and fluvastatin).

[0346] As will be apparent from the foregoing, the Disclosure provides a method for a combination therapeutic treatment of a subject, comprising administering an effective amount of a first agonist and a second agonist to a subject in need thereof, wherein the first agonist is a pharmaceutical formulation of the Disclosure, and the second agonist is also for the prevention or treatment of thrombotic disorders, inflammatory disorders and / or thromboinflammatory disorders.

[0347] As used herein, the term “simultaneous” as in the phrase “simultaneous therapeutic procedure” includes administering a first drug in the presence of a second drug. A simultaneous therapeutic procedure method includes a method in which a first, second, third, or further drug is administered simultaneously. A simultaneous therapeutic procedure method also includes a method in which a first or further drug is administered in the presence of a second or further drug, where the second or further drug may have been administered previously, for example. A simultaneous therapeutic procedure may be performed stepwise by different actors. For example, one actor may administer a first drug to a subject, and a second actor may administer a second drug to the subject, and the administration steps may be performed simultaneously, nearly simultaneously, or at different times, as long as the first drug (and / or further drug) has been administered in the presence of the second drug (and / or further drug). The actors and the subject may be the same entity (e.g., a human).

[0348] Kits of substances and other compositions Another example of this disclosure is providing a kit containing a pharmaceutical formulation of this disclosure that is useful for treating or preventing the diseases or conditions described above.

[0349] For example, the kit includes (a) a container containing the pharmaceutical formulation of the present disclosure; and (b) a package insert containing instructions for treating or preventing a disease or condition in the subject.

[0350] For example, the kit may include (a) at least one pharmaceutical formulation of the present disclosure; (b) instructions for using the kit in treating or preventing a disease or condition in a subject; and (c) optionally at least one further therapeutic compound or drug.

[0351] In this example of the Disclosure, the accompanying information is on or attached to the container. Suitable containers include, for example, bottles, vials, syringes, etc. Containers can be formed from a variety of materials, such as glass or plastic. The container holds or contains a composition effective for the treatment or prevention of blood coagulation disorders and may have a sterile access port (for example, the container may be an intravenous solution bag or vial with a stopper that can be punctured with a subcutaneous needle). The label or accompanying information indicates that the composition is used to treat subjects eligible for treatment, e.g., subjects having or predisposed to developing thrombotic disorders, inflammatory disorders and / or thromboinflammatory disorders, along with specific guidance regarding the dosage and interval of the pharmaceutical formulation and any other pharmaceuticals provided. The kit may further include other substances desirable from a commercial and user standpoint, including filters, needles, and syringes. In some examples of the Disclosure, the formulation may be contained within an injectable device (e.g., an injectable syringe, e.g., an injectable pre-filled syringe). The syringe can be adapted for individual doses, for example, as a single vial system including an auto-injector (e.g., a pen-type injection device). In one example, the injectable device is a pen-type or other suitable pre-filled auto-injectable device, which may include instructions for use and administration.

[0352] The kit may further include a container containing a second pharmaceutical agent, which is the first pharmaceutical agent, and the kit further includes instructions in its package insert for treating the subject with an effective dose of the second pharmaceutical agent. The second pharmaceutical agent may be the therapeutic protein described above.

[0353] In one example, the Disclosure provides a pre-filled syringe or auto-injector containing the formulation of the Disclosure. In one example, the pre-filled syringe is a glass Luer syringe having a plunger.

[0354] For example, this disclosure provides a vial containing the formulation of the Disclosure.

[0355] This disclosure includes the following non-limiting embodiments. [Examples]

[0356] Physical and chemical stability of excipients in pharmaceutical formulations To investigate the formulation excipients, biophysical and excipient screening studies, as well as solubility studies, were conducted. Monoclonal anti-factor XII antibody (affinity-mature 3F7 or 3F7 as described herein) aff Pre-formulation experiments were conducted using ).

[0357] Baseline biophysical screening study The physical and thermal stability of anti-factor XII antibodies was evaluated in 10 buffer types (F1-F10, Table 1). A target concentration of 100 mg / ml was achieved for each formulation using volume dilution and 320 nm baseline correction.

[0358] Absorbance (A 280 / 320 The protein concentration of the formulation was determined by measuring the pH, and the pH was investigated using a calibrated pH meter.

[0359] When the buffer was exchanged with 20 mM glutamate buffer (F1) at pH 5.5, it was observed that the antibody solution formulated at 100 mg / ml did not reach the pH target even after thorough buffer exchange (the measured pH was 5.65).

[0360] The particle distribution and thermal stability of antibody solutions were studied using dynamic light scattering (DLS) and differential scanning fluorescence (DSF).

[0361] Dynamic light scattering was performed in optical-quality plastic cuvettes using at least 100 μl of undiluted sample. Five consecutive scans were acquired at 25°C for each sample measurement (using Malvern's auto-decay selection setting), with a 3-minute equilibration period at the start of each measurement. A protein analysis algorithm (Malvern Zetasizer software) was used as the model for data processing. The Z-mean hydrodynamic diameter was calculated from cumulant analysis and the Stokes-Einstein equation using the viscosity of water at 25°C (0.8872 mPa*s). The polydispersity index (PDI) was obtained from the same cumulant analysis. The modality of the fitting was evaluated based on the size distribution versus intensity plot. The modality can be described as unimodal (i.e., one peak) or multimodal (i.e., two or more peaks).

[0362] In the DLS study, formulations were compared with respect to Z-mean diameter and PDI (Table 1). A clear trend in Z-mean with respect to pH was observed in succinate buffer (F2–F4) and citrate buffer (F8–F10), with the Z-mean increasing with increasing pH. However, the trend observed in Z-mean did not correlate with changes in PDI. This suggests that the increase in Z-mean is related to stronger, nonspecific electrostatic interactions between antibody molecules at higher pH. In contrast to the samples formulated with succinate and citrate buffers, the PDI of histidine buffer (F5–F7) showed a slight upward trend as a function of pH, with no change in the Z-mean. The Z-mean was lower compared to the succinate and citrate formulations. Similar results were observed with glutamate buffer (F1), with increased PDI (compared to succinate and citrate buffers) and a lower Z-mean.

[0363] DSF experiments were conducted using the Unchained Labs UNit. Test formulations were screened for thermal stability and soluble aggregate formation. Thermal unfolding and aggregation were monitored as functions of temperature by changes in endogenous protein fluorescence and static light scattering, respectively. By monitoring endogenous fluorescence, the midpoint of the thermal transition (T) was identified. m ) and melting onset temperature (T onset The aggregation onset temperature (T) was determined by monitoring static light scattering at, for example, 266 nm and 473 nm. agg The following was determined. Each DSF analysis was performed in triple duplication using 8.8 μl at a protein concentration of 100 mg / ml. Samples were analyzed while increasing the temperature at a rate of 0.5 °C / min over a series of temperatures (20 to 95 °C).

[0364] DSF analysis evaluated thermal stability and soluble aggregate formation. T was determined for all test formulations. m , T onset , and T agg The values ​​are shown in Table 1.

[0365] [Table 2]

[0366] T obtained with glutamate buffer and histidine buffer onset and T m The values ​​were slightly higher compared to succinate and citrate. This indicates that formulations F1 and F5-F7 have higher thermal stability. No significant differences in thermal stability were observed at different pH values. The onset aggregation temperature and static light scattering intensity at 266 nm and 473 nm were higher for glutamate buffer (F1) and histidine buffer (F5-F7) compared to succinate (F2-F4) and citrate (F8-F10). agg The antibody exhibited higher thermal agglutination stability, showing lower SLS intensities and values. No significant difference in thermal agglutination stability was observed at different pH values ​​of the given buffer.

[0367] Based on DLS and DSF results, formulations containing glutamate and histidine at pH 5.5 showed the most positive effect on the thermal stability and agglutination stability of the anti-FXII antibody.

[0368] Excipient screening Based on baseline biophysical screening, twelve formulations were evaluated to investigate the effect of four different excipients (phenylalanine, proline, arginine, and sorbitol) in three different pH 5.5 buffers (glutamate, succinate, and histidine) on the stability of antibodies at a concentration of 100 mg / ml (F11-F22, Table 2). Furthermore, formulations containing glutamate and histidine at pH 5.5 were evaluated (F23). The effect of five different excipients on antibody stability was analyzed by DLS and DSF using the methods described above.

[0369] In the DLS study, no significant differences were observed in the Z-mean and PDI between formulations with and without excipients (±) (Table 2). Similarly, no significant differences were observed among the tested buffers. The most pronounced effects on the Z-mean and PDI were observed with arginine in succinate buffer (F17), which reduced both parameters compared to other excipients. Overall, no significant differences were observed in the particle size distribution among the tested formulations.

[0370] T was determined for the test formulation by DSF analysis. onset and T m This is shown in Table 2. onset and T mA comparison of the values ​​showed that all formulations containing phenylalanine, proline, and sorbitol exhibited comparable thermal stability. Similar stability was observed for formulation F23, which contains histidine and glutamate. The formulation containing arginine showed the lowest thermal stability. A comparison of the results showed that all formulations containing succinate and / or arginine exhibited increased antibody thermal agglutination compared to the other test formulations. Similar results were obtained for formulation F23, which contains histidine and glutamate.

[0371] [Table 3]

[0372] Based on DLS and DSF results, succinate buffer was excluded from further study due to an increased tendency for unfolding protein aggregation at high temperatures. The presence of sorbitol and phenylalanine in the test formulation did not have a significant effect on antibody stability compared to proline, therefore both excipients were excluded from further study.

[0373] Solubility test Based on the results of an excipient screening study, six formulations were evaluated to investigate the solubility of antibodies at concentrations of ≥200 mg / ml. Two of the test formulations contained 150 mM glutamate buffer at pH 5.5, one containing 20 mM histidine (F23) and the other without 20 mM histidine (F24). The four other formulations contained glutamate buffer or histidine buffer at pH 5.5, with proline or arginine as excipients (F12, F13, F20, F21; Table 3). Two sets of samples were prepared and evaluated for appearance. Six control samples from the first set were concentrated to 100 mg / ml. Six solubility samples from the second set were concentrated to antibody concentrations of ≥200 mg / ml.

[0374] The visual appearance (i.e., color, clarity, and particulate matter) was investigated according to GTM-0033-03 "Visual Appearance Evaluation". The type of particles observed was determined to be product-related or non-product-related. The control solution (i.e., 100 mg / ml) was slightly yellow, while the concentrated solubility solution (i.e., 200 mg / ml) was slightly brown due to its higher protein concentration. The control solution and concentrated solubility solution were slightly cloudy and cloudy liquids, respectively, and no visible product-related particles were present. After the visual examination, the concentrated solubility solution (200 mg / ml) was diluted to 100 mg / ml with the corresponding buffer (solubility solution) and tested against the control sample by the DLS method described above and the SEC method described below.

[0375] DLS analysis was performed on each set of control and solubility solutions. The results are shown in Table 3. All control and solubility solutions showed comparable Z-mean and PDI values ​​(≤0.13).

[0376] Size exclusion chromatography (SEC) was performed using a Dionex UltiMate 3000 BioRS HPLC system to separate high molecular weight species, major monomers, and fragments under natural conditions. As shown in Table 3, there was a slight increase of 0.4–1.0% in high molecular weight species in the solubility solution compared to the control. No significant changes in fragment percentage were observed between the solubility samples and the control samples.

[0377] In summary, the concentrated samples (≥200 mg / ml) remained soluble, and no negative effects on antibody stability were observed in any of the test formulations.

[0378] [Table 4]

[0379] Surfactant screening The protective effect of nonionic surfactants against soluble antibody and insoluble aggregate formation was evaluated for polysorbate 80 (PS-80), polysorbate 20 (PS-20), and poloxamer 188 using freeze-thaw and stirring tests. A total of 14 formulations with an antibody concentration of 100 mg / ml were screened using freeze-thaw and stirring tests (F21 and F24-F36; Table 4). In the freeze-thaw study, all formulations were subjected to three freeze-thaw cycles at -75±10°C. Samples were stored at -75±10°C for a minimum of 1 hour, followed by thawing at room temperature for 1 hour. Each control sample was prepared by incubation at 5±3°C. In the stirring study, formulations were stirred at room temperature for approximately 50 hours using a laboratory rotary mixer (approximately 50 rpm). Each control sample was prepared by incubation at 5±3°C. All samples were analyzed by appearance, size exclusion chromatography, and differential light scattering as described above.

[0380] In both freeze-thaw and agitation tests, all stressed samples remained unchanged compared to the control sample in terms of color (i.e., slightly yellow), transparency (i.e., clear), and particle content (i.e., no visible particles were present).

[0381] DLS results showed that histidine and glutamate formulations without surfactants, subjected to F / T stress and stirring stress, exhibited increased PDI compared to control samples. The highest increase in PDI (0.09) compared to control was observed in the F21 stirred sample. This may suggest the presence of particles in the stressed samples. Similar differences were not observed in either the freeze-thaw or stirred samples containing surfactants. The Z-mean and PDI values ​​measured in the stressed samples were comparable to those of the control.

[0382] No significant changes in aggregate concentration were detected in the SEC of freeze-thawed and stirred samples compared to the corresponding control. The area percentage of monomer peaks ranged from 97.5% to 98.4%, the area percentage of high molecular weight species ranged from 1.0% to 1.9%, and the area percentage of low molecular weight species ranged from 0.6% to 0.8%.

[0383] To characterize the size, concentration, and morphological features of particles present in the sample, invisible particles were measured by microflow imaging (MFI) using an MFI5200 with an Automated Pipet System. Analysis was performed in single measurements using 700 or 1000 μl neat samples to determine the cumulative count per ml of particles ≥2 μm, ≥5 μm, ≥10 μm, and ≥25 μm in size (for particles between 1 and 100 μm). Furthermore, particles ≥5 μm in size with an aspect ratio (AR) ≥0.85 were processed using morphological classification parameters within the MFI software (MVAS) to determine the roundness percentage. A low roundness percentage indicates that the test specimen contains primarily non-round, possibly proteinaceous, particles.

[0384] The absence of surfactants in the control samples (F21 and F24) subjected to freeze-thaw stress and stirring stress was considered to have a significant impact on the formation of invisible particles. An increase in particle count was observed in both surfactant-deficient formulations. The only exception was formulation F24, subjected to stirring stress, which exhibited a particle count comparable to the control samples. In contrast to the results obtained for the control samples, all types of surfactants present in the samples subjected to freeze-thaw stress and stirring stress significantly suppressed the formation of invisible particles. The inhibitory effect was similar for all test surfactants, but the particle reduction observed with the formulation containing PS-80 was the most beneficial. Furthermore, the observed protective effect was slightly better at a PS-80 concentration of 0.05% than at 0.02%.

[0385] [Table 5] [Examples]

[0386] Design of experimental research Formulation buffers for this study were designed using the results of baseline biophysical screening, excipient screening, solubility, and surfactant screening studies. A total of 30 formulations were tested for accelerated antibody stability (Table 5). Formulation F DOE 12 was selected as the center point (100 mg / ml antibody, 20 mM glutamate, 100 mM arginine, 0.05% PS-80, pH 5.5). Furthermore, the original formulation (F original A control was used. Samples were stored at 5°C and 40°C and analyzed at time zero, 2 weeks, and 4 weeks.

[0387] [Table 6]

[0388] The following analyses were performed on the selected samples. • Time zero: Osmotic pressure, pH, UV, appearance and ΔG trend HUNK analysis • Week 2: Size exclusion chromatography (SEC), capillary gel electrophoresis (reducing (R-CGE) and non-reducing (NR-CGE)) • Week 4: Dynamic light scattering (DLS), microflow imaging (MFI), SEC, cation exchange chromatography (CEX), pH, R-CGE, NR-CGE, and appearance

[0389] Selected preparation (F DOE 9, F DOE 15, F DOE 19, and F DOE 25) The osmotic pressure was determined before storage. The osmotic pressure of the selected formulation was 296 mOsm / kg (F DOE 9), 435 mOsm / kg(F DOE 15), 154 mOsm / kg(F DOE 19) and 240 mOsm / kg(F DOE 25)

[0390] Different formulations (F DOE 3, F DOE 6, F DOE 9, F DOE 12 and F DOE 15) The chemical stability and aggregation behavior of antibodies in ΔG should also be checked before storage. trend The study was conducted using (HUNK) analysis.

[0391] By measuring the ΔG of unfolding at concentrations of 0.25, 0.6, 2.5, 6.0, and 25.0 mg / ml, ΔG trend The following was determined. All samples were diluted to the target concentration with formulation buffer. Each concentration level was titrated with gradually increasing denaturant concentrations while measuring the fluorescence spectrum at 300-500 nm (excitation 280 nm) with a 10 nm slit width (32-point curve in the range of urea concentrations from 2.00 to 8.74 M). The gain was adjusted according to the sample concentration to provide the maximum signal without saturating the detector (100 for 0.25 and 0.6 mg / ml; 10 for 2.5 and 6.0 mg / ml; 1 for 25.0 mg / ml). For each sample concentration level, the emission spectral wavelength ratio at 350 nm / 330 nm was plotted against the urea concentration, and the ΔG of protein unfolding was determined using a two-state (i.e., one-transition) model fitting. The determined ΔG values ​​were plotted against the sample concentration, and the ΔG trend This was determined. Positive ΔG trend (That is, ΔG increases with sample concentration) indicates a natural self-announcement, and negative ΔG trend (That is, the decrease in ΔG with increasing sample concentration) indicates aggregation from a denatured state.

[0392] F DOE ΔG performed on 3 trend The analysis showed that ΔG gradually increased with antibody concentration, suggesting the formation of aggregates of native proteins in the test sample. The observed effect was that proline was converted to F DOE When added to 3, it was significantly reduced (F DOE 6 and F DOE 9) Formulation F DOE6 (containing 100 mM proline) and FDOE9 ΔG observed (with 200 mM proline) trend It showed no aggregate formation, independent of antibody concentration. Interestingly, F DOE When arginine is added to 3 (F DOE 12 and F DOE 15) It was shown that ΔG gradually decreases with increasing antibody concentration. The reduction effect was F DOE The reaction was dramatic at 15 (containing 200 mM arginine), which is evidence of the formation of denatured protein aggregates in the test sample. This result is in good agreement with DSF data obtained in an excipient screening study in which thermal aggregation of antibodies was observed with all formulations containing arginine.

[0393] The pH of the samples was measured at the initial stage (i.e., before storage) and at 4 weeks. For samples stored for 4 weeks at 5±3°C and 40°C / 75% relative humidity (RH), the measured pH was within 0.11 pH units of the initial value for each formulation.

[0394] The appearance of the samples at the initial and 4-week mark was evaluated for color, clarity, and particle content as described above. Samples held at 5±3°C and 40°C / 75%RH for 4 weeks showed no difference compared to the initial sample. Each sample appeared as a slightly yellowish, clear liquid, and no visible protein-related particles were present.

[0395] MFI was performed as described above. The results obtained from samples stored at 5±3°C and 40°C / 75%RH did not reveal any clear trend in cumulative particle count with respect to storage temperature, buffer, or excipient type. A significant tendency for particle accumulation as a function of pH was observed in all test formulations. In general, the minimum difference in particle count between samples stored at 5±3°C and 40°C / 75%RH was observed at pH 5.5 for glutamate buffer and at pH 5.5 and pH 5.7 for histidine buffer for all particle sizes.

[0396] The results obtained from R-CGE, NR-CGE, SEC, DLS, and CEX were statistically analyzed using Stat-Ease, Inc. Design-Expert® version 7.0.3. The significance of the responses was assessed using the ANOVA method with a 95% confidence interval (p-values ​​less than 0.05 indicated a statistically significant correlation). Responses meeting this significance threshold were analyzed in the linear design mode of the software. Calculations were performed using three factors: pH, proline concentration, and arginine concentration, and four responses: total impurity accumulation (R-CGE), total impurity accumulation (SEC), PDI difference (DLS), and total charge variant accumulation (CEX). The p-values ​​obtained by ANOVA for the total impurity percentage determined by NR-CGE were not statistically significant (p-value = 0.34 for glutamate buffer, 0.47 for histidine buffer), and therefore such data were excluded from further analysis.

[0397] The generated model for the accumulation of total impurities detected by R-CGE and SEC analysis of glutamate buffer ± proline (difference between total impurities obtained from samples stored at 5±3°C and 40°C / 75%RH) showed a trend of progressive decrease in impurities as a function of pH. No differences in impurity accumulation were observed between glutamate buffer ± proline at all tested pH levels.

[0398] In R-CGE analysis, the model generated for histidine buffer ± proline showed a slight reduction in impurities as a function of pH, whereas in SEC analysis, the model generated for histidine buffer ± proline did not show a reduction in impurities as a function of pH. In contrast to glutamate, the presence of proline in histidine buffer slightly reduced impurity accumulation. This was mainly observed at pH 5.5 and pH 5.7. At pH 5.5, impurity accumulation was comparable between glutamate buffer with and without proline and histidine buffer, respectively.

[0399] R-CGE and SEC analyses revealed that the generated models for the accumulation of total impurities in glutamate buffer ± arginine showed a tendency for impurities to gradually decrease as a function of pH, similar to the effect observed with glutamate buffer ± proline. Compared to proline, the presence of arginine in glutamate buffer increased the accumulation of total impurities at all tested pH levels.

[0400] R-CGE analysis revealed a small pH-dependent trend in impurity reduction for histidine buffer ± arginine. No significant difference in impurity accumulation was observed between histidine buffer ± arginine at all tested pH levels. Impurity accumulation at pH 5.5 was similar between glutamate buffer and histidine buffer with and without arginine, respectively. In contrast, SEC analysis did not show a pH-dependent trend in impurity accumulation in the generated models for histidine buffer ± arginine. In contrast to proline, the presence of arginine in histidine buffer increased total impurity accumulation at all tested pH levels.

[0401] The accumulation of impurities at pH 5.5 was comparable between glutamate buffers with and without arginine, and histidine buffers, respectively.

[0402] The generated model for PDI determined by DLS analysis of glutamate buffer ± proline (difference between PDI values ​​obtained from samples stored at 5±3°C and 40°C / 75%RH) showed a slight decreasing trend in PDI values ​​as a function of pH. No significant difference in PDI values ​​was observed between glutamate buffer ± proline at all tested pH levels. Similarly, the generated model for PDI in glutamate buffer ± arginine also showed a slight decreasing trend in PDI values ​​as a function of pH. In contrast to proline, the presence of arginine in glutamate buffer significantly increased PDI values ​​at all tested pH levels.

[0403] The models generated for histidine buffer ± proline showed a slight increasing trend in PDI values ​​as a function of pH. No significant difference in PDI values ​​was observed between histidine buffer ± proline at all tested pH levels. Similarly, the models generated for PDI in histidine buffer ± arginine also showed a slight increasing trend in PDI values ​​as a function of pH. In contrast to proline, the presence of arginine in histidine buffer significantly increased PDI values ​​at all tested pH levels.

[0404] At pH 5.5, the PDI values ​​were comparable not only between glutamate buffers with and without arginine, but also between glutamate buffers with and without proline, respectively.

[0405] The generated model for the accumulation of total charged mutants detected by CEX analysis of glutamate buffer ± proline (differences between total charged mutants obtained from samples stored at 5±3°C and 40°C / 75%RH) showed a gradual decrease in charged mutants as a function of pH. A slight beneficial effect of proline on the reduction of charged mutants was observed at all tested pH levels. Similarly, the generated model for the accumulation of charged mutants in glutamate buffer ± arginine showed a gradual decrease in impurities as a function of pH.

[0406] The models generated for histidine buffer ± proline showed a tendency for the accumulation of charged mutants to increase as a function of pH. Similar to glutamate buffer, the presence of proline in histidine buffer reduced the accumulation of charged mutants at all tested pH levels. Similar to proline, but more importantly, the presence of arginine reduced the accumulation of total charged mutants at all tested pH levels. The models generated for histidine buffer ± arginine showed a tendency for the accumulation of charged mutants to increase as a function of pH. No difference in impurity accumulation was observed between histidine buffer ± arginine at all tested pH levels.

[0407] The lowest accumulation of charge variants was observed at pH 5.5 for all test formulations. No significant differences were observed at pH 5.5 between glutamate buffers containing and without proline, and between glutamate buffers containing and without arginine, and between glutamate buffers containing and without arginine, and between glutamate buffers containing and without arginine, and between glutamate buffers containing and without arginine, and between glutamate buffers containing and without arginine, and between glutamate buffers containing and without arginine, and without arginine. The only exception was the glutamate buffer without arginine, which had significantly higher concentrations of charged species compared to the histidine buffer containing arginine.

[0408] The results of this study showed that the beneficial effect of proline on antibody stability was highest in glutamate buffer at pH 5.5 and histidine buffer at pH 5.5–5.7. The protective effect of arginine was observed only by CEX analysis. DLS, SEC, and R-CGE results indicated that the presence of arginine in glutamate buffer and histidine buffer (within the tested pH range) may reduce protein stability. [Examples]

[0409] Determining the syringe passage characteristics of high-concentration formulations. The suitability of antibody formulations F37 and F38 for manufacturing and clinical application at a liquid formulation concentration of 100 mg / ml was investigated. Placebo solutions (formulations F37 and F38 without the active pharmaceutical ingredient) were simultaneously tested as part of fluid delivery, filter compatibility, and syringe passability studies.

[0410] Formulation 37: 100 mg / ml antibody; 100 mM glutamate at pH 5.6; 150 mM proline and 0.05% PS-80.

[0411] Preparation 38: 100 mg / ml antibody; 20 mM histidine at pH 5.8; 150 mM proline, 80 mM sorbitol, and 0.05% PS-80.

[0412] Fluid delivery research Fluid delivery studies were conducted to evaluate whether changes related to aggregation or degradation could occur during the physical process of delivering antibody-containing substances when they are handled in a specific manufacturing process. To assess the stability of each formulation, an initial time point (T0) was collected and compared with samples taken after 60 minutes and 120 minutes of fluid delivery in a series of analytical tests.

[0413] exterior The appearance of both placebo and antibody-containing samples was investigated by color, clarity, and the relative number of visible particles. All antibody-containing samples evaluated at 60 and 120 minutes showed no difference in color or clarity compared to the initial samples. Each placebo sample appeared as a clear, colorless liquid, while each antibody-containing sample appeared as a slightly yellowish, slightly opaque liquid. No particles or non-product-related particles were observed in any of the placebo or antibody-containing samples.

[0414] Turbidity investigated using absorbance at 550 nm The turbidity of antibody-containing and placebo formulations was evaluated by measuring the absorbance at 550 nm. The turbidity values ​​of all antibody-containing and placebo samples showed no difference compared to the initial control. A550 was less than 0.07 AU for all samples at all time points.

[0415] Size exclusion chromatography The effect of delivery on the formation of soluble high and low molecular weight species in antibody-containing samples of each formulation was evaluated using SEC as described above. No significant changes were observed in the total peak area percentage of higher molecular weight or lower molecular weight species between the initial and 120-minute time points for any of the formulation samples. Formulation F38 contained a larger proportion of high molecular weight species than formulation F37 (2.5% vs. 1.5%), and the proportion of low molecular weight species did not differ significantly between the two formulation samples (0.7% vs. 0.7%). These differences in the total peak area of ​​high molecular weight species were consistent and observed in all samples for delivery, filter compatibility, and syringe passage characteristics studies.

[0416] Dynamic light scattering To evaluate the effects of fluid delivery stress, the particle distribution of antibody-containing samples was investigated using DLS as described above. The formulations were compared with respect to the Z-mean hydrodynamic diameter, mean polydispersity (PDI), and fitting modality. Changes were observed in the fitting modality (evaluated based on plots of size distribution by intensity). While both formulations were unimodal at T0, the F38 sample was multimodal at 60 minutes, and both the F37 and F38 samples were multimodal at 120 minutes. The Z-mean diameter values ​​were similar for all test samples of both formulations, showing a slight upward trend as a function of stress time. Similar, but more pronounced, increasing trends were observed in the PDI values ​​obtained from the samples of both test formulations. The PDI of formulation F37 increased from 0.087 (at T0) to 0.120 and 0.136 after 60 and 120 minutes of fluid delivery (approximately 1.4 and 1.6 times increases, respectively). The PDI obtained for formulation F38 increased from 0.129 (at T0) to 0.238 and 0.242 after 60 and 120 minutes of infusion (approximately 1.8 and 1.9 times increases, respectively). In summary, infusion stress had a slight effect on the particle distribution of F37 and F38.

[0417] MFI experiments were conducted to characterize the size and concentration of particles present in antibody-containing samples throughout the delivery study. No clear trend in the accumulation of invisible particles was observed over time in the F37 and F38 placebo groups. In contrast, a gradual increase in invisible particle concentration was observed over the delivery period in both the F37 and F38 formulations. The increase in invisible particles was similar in both formulations.

[0418] Overall, the results indicate that there was no increase in either visible or invisible particles in F37 and F38 when subjected to fluid delivery stress.

[0419] Filter compatibility research A filter suitability study was designed to investigate the suitability and performance of filter materials for the final API formulation. Samples of placebo and antibody-containing formulations were passed through a 0.22 μm PES filter, a 0.22 μm PVDF filter, or left unfiltered as a control. Analytical tests were then performed to evaluate the suitability and performance of the filters.

[0420] exterior The appearance of the samples was investigated in terms of physical state, color, clarity, and the relative number of visible particles, as described above. Samples filtered with PES or PVDF filters showed no difference in color or clarity compared to unfiltered control samples. Each placebo sample appeared as a clear, colorless liquid, while each antibody-containing sample appeared as a slightly yellowish, slightly opaque liquid. No particles or non-product-related particles were observed in any of the samples.

[0421] Turbidity Sample turbidity was investigated as described above. The turbidity values ​​of all antibody-containing and placebo samples showed no significant difference compared to the initial control. A550 was less than 0.05 AU for all samples at all time points. No clear trends were observed in the placebo samples with respect to time points or formulation composition.

[0422] Size exclusion chromatography The effect of filtration on the formation of soluble high and low molecular weight species in antibody-containing samples of each formulation was evaluated using SEC as described above. In all formulations, no significant changes were observed in the peak area percentage of high or low molecular weight species between the unfiltered control and the membrane-filtered samples.

[0423] Dynamic light scattering and microflow imaging DLS was performed as described above. The results showed that, regardless of the filter material, filtration did not change the fitting modality for any of the formulations, and the difference in the mean Z diameter between the control sample and the filtered sample was not significant. The PDI value of formulation F37 was reduced from 0.106 for the control to 0.049 and 0.050 for the samples filtered with PES and PVDF, respectively. The PDI value of formulation F38 was reduced from 0.087 for the control sample to 0.081 and 0.063 for the samples filtered with PES and PVDF, respectively.

[0424] MFI experiments were conducted as described above to characterize the size and concentration of particles present in antibody-containing samples taken from filter suitability tests. No significant difference in cumulative particle count was observed between filtered samples and control placebo samples for both formulations. Both antibody samples in formulation F37 and formulation F38 showed an overall reduction in cumulative particle count of all sizes in filtered samples compared to the control samples. The particle reduction observed in formulation F37 was greater with PVDF filtration than with PES filtration. In contrast, in formulation F38, the overall particle reduction in antibody-containing samples was not significantly greater with one filter material than with the other.

[0425] The overall changes in particle distribution observed in DLS and MFI analyses demonstrate that antibody filtration is beneficial in reducing potential aggregation during material processing, as simulated by filter suitability studies. Overall, similar reductions were observed with both formulations and both filtration materials.

[0426] Syringe passage characteristics study A syringe passage characteristics study was designed to simulate the application of the final formulation as a drug product and to determine the suitability of each formulation for DP application using two different sizes of Injekt Solo syringes. Samples of the placebo formulation and the antibody-containing formulation were dispensed using 2 ml syringes, 10 ml syringes, or left untreated as a pre-dispensing control. To achieve this, the syringe plunger was pushed in at a linear velocity of 0.2 inches / min for the 2 ml syringe and 0.6 inches / min for the 10 ml syringe until the plunger reached the bottom and the force reached 30 N.

[0427] force Break-loose force (BF) and sliding force (GF) were measured during dispensing and used to evaluate applicability. Break-loose force describes the force required to initiate plunger movement (the first 0.3 mm for a 2 ml syringe, and 0.5 mm for a 10 ml syringe). Maximum sliding force refers to the maximum frictional force required to maintain plunger movement. The maximum force value is measured from the end of the break-loose region to the end of the sliding force region before the point where the force reaches 30 N (26 mm for a 2 ml syringe, and 24 mm for a 10 ml syringe). Under all test conditions, the peak BF and maximum GF required to dispense the antibody-containing sample or placebo sample were less than 12N, and the peak BF was less than the maximum GF. These indicate that both formulations may be suitable for application in either syringe, as simulated in the syringe passage characteristics study. Comparing the test conditions, BF and GF values ​​were higher when dispensed from a 10ml syringe compared to a 2ml syringe, regardless of the formulation.

[0428] exterior The appearance of both placebo and antibody-containing samples before and after dispensing was examined for color, clarity, and the relative number of visible particles. No discernible differences were observed between the control sample and the dispensed sample. All placebo samples were clear and colorless liquids, while all antibody-containing samples were slightly yellowish and slightly opaque liquids. No particles or non-product-related particles were observed in any of the samples.

[0429] Turbidity Sample turbidity was investigated as described above. The absorbance at 550 nm was less than 0.05 AU for all samples, except for the control sample containing antibody in formulation F37, which had an absorbance of 0.137 AU. No clear trends regarding conditions or formulation composition were observed in the placebo and antibody-containing samples.

[0430] Size exclusion chromatography The effect of syringe passage of antibody-containing samples on the formation of soluble high and low molecular weight species was evaluated using SEC. No significant changes were observed in the total peak area percentage of high or low molecular weight species between the control sample and the dispensed sample for any of the formulations.

[0431] Dynamic light scattering Using DLS, the particle distribution of antibody-containing samples before and after dispensing with syringes of different sizes (2 ml and 10 ml) was evaluated. Samples were compared with respect to Z-mean diameter, mean polydispersity (PDI), and fitting modality. Syringe dispensing did not alter the fitting modality for any formulation, and there were no significant differences in Z-mean diameter and PDI between the control sample and the syringe sample for any formulation.

[0432] Microflow imaging MFI experiments were conducted to characterize the size and concentration of particles present in the samples. Placebo samples of formulations F37 and F38 showed an increase in the cumulative particle count in the post-dispensing sample compared to the control sample, with the increase being greater in the 10 ml syringe sample compared to the 2 ml syringe sample. No significant differences were observed between the antibody control and the 2 ml syringe sample in formulation F37, but the 10 ml syringe sample showed a slight overall increase in the cumulative particle count.

[0433] The antibody samples in formulation F38 showed a more pronounced response to syringe application. Samples that passed through a 2 ml syringe showed a 2.7–3.6-fold increase in cumulative count compared to the control, while samples that passed through a 10 ml syringe showed a 10–12-fold increase in cumulative count.

[0434] Results from syringe passage characteristics studies showed that shear stress did not affect antibody aggregation. The increase in the concentration of invisible particles was detected only by MFI of F38. [Examples]

[0435] Preparation of high-concentration formulations Using tangential flow filtration (TFF), an antibody preparation containing approximately 116 mg / ml of antibody, 20 mM histidine, 140 mM proline, 150 mM arginine, and 0.02% wt / volt polysorbate 80 was concentrated to a preparation containing approximately 200 mg / ml of antibody ("High Concentration 2"). A moderately high concentration preparation of approximately 150 mg / ml was prepared in TFF ("High Concentration 1"). The High Concentration 2 preparation was diluted to prepare a preparation containing 100 mg / ml of antibody ("DP Test"). Additionally, the original starting material was diluted to prepare a preparation containing 100 mg / ml of antibody ("DP Control").

[0436] After concentration, the concentration, density, and viscosity of each formulation were investigated as shown in Table 6. Viscosity was compared to purified antibody preparations without polysorbate 80, using 20 mM histidine, 140 mM proline, and 150 mM arginine.

[0437] [Table 7]

[0438] The stress stability of high-concentration formulations (DP control; DP test; high concentration 1; and high concentration 2) was investigated by exposing the formulations to 35°C for up to 5 weeks, both in glass vials and pre-filled glass syringes (PFS). After 5 weeks at 35°C, both protein concentration and pH (pH 6.1) remained unaffected in both the syringes and vials.

[0439] Microflow imaging (MFI) was used as described above to characterize the size, concentration, and morphological characteristics of particles present in the samples. After 5 weeks at 35°C, no significant differences were observed in the size, number, and / or morphological characteristics of the particles present between formulations of different concentrations.

[0440] The percentages of high molecular weight species and monomers were determined using SEC as described above. The formulations were examined at time zero and after being held at 5°C or 35°C for 5 weeks. The results are shown in Table 7.

[0441] [Table 8]

[0442] As shown above, the high-concentration antibody preparation exhibited long-term thermal stability. [Examples]

[0443] Long-term stability of high-concentration formulations in vials The long-term stability of the high-concentration DP control formulation of Example 4 was investigated by maintaining the formulations at 5°C (±3°C) or 25°C (±2°C) for 36 months and 24 months, respectively, using the method described above. The results are shown in Tables 8 and 9.

[0444] [Table 9-1] [Table 9-2]

[0445] [Table 10-1] [Table 10-2] [Examples]

[0446] Stability of high-concentration formulations in pre-filled syringes Using the method described above, the stability of the high-concentration DP control formulation (Example 4) in pre-filled syringes was investigated at 3, 6, and 12 months by maintaining the formulation at 5°C (±3°C) or 25°C (±2°C). The results are shown in Tables 10-15.

[0447] [Table 11]

[0448] [Table 12]

[0449] [Table 13]

[0450] [Table 14]

[0451] [Table 15]

[0452] [Table 16]

[0453] [Table 17]

[0454] [Table 18] [Examples]

[0455] Example formulation Based on the data presented herein, two stable high-concentration formulations are shown in Table 16. The acceptance criteria for these formulations are shown in Table 17.

[0456] [Table 19]

[0457] [Table 20] [Examples]

[0458] Single-dose pharmacokinetic study of high-concentration formulation after subcutaneous administration in rabbits. The objective of this study was to investigate the pharmacokinetic (PK) characteristics of two high-concentration formulations (100 and 170 mg / ml, as shown in Example 7) after a single subcutaneous administration to rabbits.

[0459] Blood samples from New Zealand white rabbits were processed for plasma and analyzed. Analysis of the plasma samples showed a similar increase in the drug concentration profile of anti-FXII antibody concentrations in both groups. The levels of anti-FXII antibody antigen in rabbit plasma samples were determined using a validated assay (ELISA). The maximum plasma concentration (C) of anti-FXII antibody was determined. ma C) was generally observed 24 or 48 hours after administration. C in Group 1 and Group 2 max The area under the curve (AUC) increased to approximately the same extent on day 1 of the trial. The mean terminal plasma elimination half-life of the anti-FXII antibody was also measured.1 / 2 The results were comparable across all treatments. No significant sex differences were observed for any of the pharmacokinetic parameters. The few minor differences in pharmacokinetic parameters observed among the various treatments tested are all considered to be within the normal range of variation for this sample size (1 sex and 3 animals per time point). The results of the pharmacokinetic evaluation of CSL312 in rabbit serum are summarized in Table 18.

[0460] [Table 21]

[0461] Overall, both high-concentration formulations showed similar PK profiles after SC administration. [Examples]

[0462] Local tolerance of repeated doses of high-concentration formulations after subcutaneous administration in rabbits. The local tolerance of two high-concentration formulations (100 and 170 mg / mL, as shown in Example 7) was investigated after subcutaneous administration to rabbits on two separate days separated by a one-week interval. Subcutaneous injections into the dorsal skin of New Zealand white rabbits were visually inspected at 1, 2, 6, 24, 48, and 72 hours after administration. After the general observation period (11 days after the first administration), all animals were sacrificed and the injection sites were examined visually and under a microscope.

[0463] During the observation period, no signs of toxicity were observed in any of the rabbits, and no effects on body weight were observed. No animals died prematurely. No local irritation was observed at any application site at any point in time during the study period after administration of the two high-concentration formulations. No changes were revealed at autopsy. Histopathological examination of rabbit skin treated subcutaneously with the two formulations revealed no drug-related morphological changes after subcutaneous administration. Only a few mild to moderate morphological changes were observed in the drug-treated skin on the right side and the 0.9% NaCl solution-treated negative control on the left side. All of these changes are thought to be procedure-related, caused by the technical application, and not drug-related.

[0464] Overall, subcutaneous administration of two high-concentration formulations over two days was well-tolerated, and no drug-related changes were observed at the injection site.

Claims

1. A liquid pharmaceutical formulation comprising at least 100 mg / ml of protein containing an antigen-binding domain that binds to or specifically binds to factor XII and / or its activated form, an organic acid buffer, a nonionic surfactant, and an amino acid stabilizer, wherein the pH is 5.0 to 6.5 and the viscosity is less than 30 mPa*s at 20°C, and the protein is an antibody, and the antibody is (i) VH containing the amino acid sequence shown in SEQ ID NO: 5 and VL containing the amino acid sequence shown in SEQ ID NO: 6; or (ii) (a) CDR1 containing the sequence shown in SEQ ID NO: 7, CDR2 containing the sequence shown in SEQ ID NO: 10, and CDR3 containing the sequence shown in SEQ ID NO: 9, or (b) V H containing CDR1 containing the sequence shown in SEQ ID NO: 7, CDR2 containing the sequence shown in SEQ ID NO: 16, and CDR3 containing the sequence shown in SEQ ID NO: 9 V L containing CDR1 containing the sequence shown in SEQ ID NO: 12, CDR2 containing the sequence shown in SEQ ID NO: 13, and CDR3 containing the sequence shown in SEQ ID NO: 14 Includes, The organic acid buffer is selected from the group consisting of histidine buffers and glutamate buffers. The nonionic surfactant is selected from the group consisting of polysorbate 80, polysorbate 20, and poloxamer 188. The liquid pharmaceutical preparation wherein the amino acid stabilizer is selected from the group consisting of proline, arginine, salts thereof, and combinations thereof.

2. The formulation according to claim 1, wherein the protein is present in the formulation at a concentration of at least 150 mg / ml.

3. The formulation according to claim 1 or 2, wherein the protein is present in the formulation at a concentration of 160 mg / ml to 180 mg / ml.

4. The formulation according to any one of claims 1 to 3, which is an aqueous formulation.

5. The formulation according to any one of claims 1 to 4, wherein the organic acid buffer is a histidine buffer.

6. The formulation according to any one of claims 1 to 5, wherein the nonionic surfactant is polysorbate 80.

7. The preparation according to any one of claims 1 to 6, wherein the amino acid stabilizer is proline.

8. A formulation according to any one of claims 1 to 7, further comprising a polyol.

9. A formulation according to any one of claims 1 to 8, comprising a histidine buffer, proline, and polysorbate 80.

10. The formulation according to claim 9, further comprising arginine monohydrochloride.

11. A pharmaceutical preparation comprising approximately 100 mg / ml to 110 mg / ml of protein containing an antigen-binding domain that binds to or specifically binds to factor XII and / or its activated form, a histidine buffer, polysorbate 80, and proline and arginine monohydrochloride as stabilizers, wherein the preparation has a pH of 5.5 to 6.5 and a viscosity of less than approximately 10 mPa*s at 20°C, and the protein is an antibody, and the antibody is (i) VH containing the amino acid sequence shown in SEQ ID NO: 5 and VL containing the amino acid sequence shown in SEQ ID NO: 6; or (ii) (a) CDR1 containing the sequence shown in SEQ ID NO: 7, CDR2 containing the sequence shown in SEQ ID NO: 10, and CDR3 containing the sequence shown in SEQ ID NO: 9, or (b) V H containing CDR1 containing the sequence shown in SEQ ID NO: 7, CDR2 containing the sequence shown in SEQ ID NO: 16, and CDR3 containing the sequence shown in SEQ ID NO: 9 V L containing CDR1 containing the sequence shown in SEQ ID NO: 12, CDR2 containing the sequence shown in SEQ ID NO: 13, and CDR3 containing the sequence shown in SEQ ID NO: 14 The pharmaceutical preparation comprising the above.

12. A pharmaceutical formulation comprising approximately 160 mg / ml to 180 mg / ml of protein containing an antigen-binding domain that binds to or specifically binds to factor XII and / or its activated form, a histidine buffer, polysorbate 80, and proline and arginine monohydrochloride as stabilizers, wherein the formulation has a pH of 5.5 to 6.5 and a viscosity of less than approximately 10 mPa*s at 20°C, and the protein is an antibody, and the antibody is (i) VH containing the amino acid sequence shown in SEQ ID NO: 5 and VL containing the amino acid sequence shown in SEQ ID NO: 6; or (ii) (a) CDR1 containing the sequence shown in SEQ ID NO: 7, CDR2 containing the sequence shown in SEQ ID NO: 10, and CDR3 containing the sequence shown in SEQ ID NO: 9, or (b) V H containing CDR1 containing the sequence shown in SEQ ID NO: 7, CDR2 containing the sequence shown in SEQ ID NO: 16, and CDR3 containing the sequence shown in SEQ ID NO: 9 V L containing CDR1 containing the sequence shown in SEQ ID NO: 12, CDR2 containing the sequence shown in SEQ ID NO: 13, and CDR3 containing the sequence shown in SEQ ID NO: 14 The pharmaceutical preparation comprising the above.

13. A formulation according to any one of claims 1 to 12, having a pH of 5.8 to 6.4 and containing 12 mM to 25 mM L-histidine buffer, 0.01% to 0.03% (weight / volume) of polysorbate 80, 90 mM to 150 mM L-proline, and 100 mM to 160 mM L-arginine monohydrochloride.

14. A formulation according to any one of claims 1 to 13, having a pH of 5.8 to 6.4 and comprising approximately 20 mM L-histidine buffer, 0.02% (weight / volume) polysorbate 80, 140 mM L-proline, and 150 mM L-arginine monohydrochloride.

15. The formulation according to any one of claims 1 to 14, wherein the viscosity of the formulation is less than approximately 9 mPa*s at 20°C.

16. Approximately 1.00 to 1.10 g / cm³ at 20°C ３ A formulation according to any one of claims 1 to 15, having the density of [a certain value].

17. A formulation according to any one of claims 1 to 16, comprising total aggregates of less than approximately 10% of the protein.

18. The preparation according to any one of claims 1 to 17, wherein at least 90% of the protein in the preparation is monomers.

19. The protein contains V, which includes CDR2 as shown in Sequence ID No.

10. Ｈ A formulation according to any one of claims 1 to 18, comprising, in the sequence, X at position 3 is D, X at position 4 is I, X at position 5 is P, X at position 6 is T, X at position 7 is K, and X at position 8 is G.

20. Proteins are IgG ４ A formulation according to any one of claims 1 to 19, comprising a steady-state region.

21. IgG ４ The steady-state region is stabilized IgG ４ The formulation according to claim 20, which is in the steady-state region.

22. A pharmaceutical formulation comprising approximately 100 mg / ml to approximately 170 mg / ml of protein containing an antigen-binding domain that binds to or specifically binds to factor XII and / or its activated form, a histidine buffer, polysorbate 80, and proline and arginine monohydrochloride as stabilizers, wherein the formulation has a pH of 5.5 to 6.5 and a viscosity of less than approximately 30 mPa*s at 20°C, and the protein contains the amino acid sequence shown in SEQ ID NO:

5. Ｈ and V containing the amino acid sequence shown in Sequence ID No. 6 Ｌ The pharmaceutical preparation comprising the above.

23. A pharmaceutical formulation comprising approximately 100 mg / ml to approximately 170 mg / ml of protein containing an antigen-binding domain that binds to or specifically binds to factor XII and / or its activated form, a histidine buffer, polysorbate 80, and proline and arginine monohydrochloride as stabilizers, wherein the formulation has a pH of 5.5 to 6.5 and a viscosity of less than approximately 30 mPa*s at 20°C, and the protein is (i) V containing the sequence shown in Sequence ID No. 7; CDR1 containing the sequence shown in Sequence ID No. 16; and CDR3 containing the sequence shown in Sequence ID No.

9. Ｈ ; and (ii) A V comprising a CDR1 containing the sequence shown in SEQ ID NO: 12; a CDR2 containing the sequence shown in SEQ ID NO: 13; and a CDR3 containing the sequence shown in SEQ ID NO: 14 Ｌ The pharmaceutical preparation comprising the above.

24. A pharmaceutical preparation according to any one of claims 1 to 23, for use in antagonizing the activity of factor XII and / or its activated form and / or antagonizing its activation.

25. Use of a pharmaceutical preparation according to any one of claims 1 to 23 in the manufacture of a pharmaceutical preparation for antagonizing the activation of factor XII and / or its activated form in a subject.

26. A pharmaceutical preparation according to any one of claims 1 to 23, for use in treating or preventing a disease or condition in a target.

27. Use of a pharmaceutical preparation according to any one of claims 1 to 23 in the manufacture of a pharmaceutical product for treating or preventing a disease or condition in a target area.

28. The preparation according to claim 26, wherein the disease or condition is a thrombotic disorder, an inflammatory disorder and / or a thromboinflammatory disorder.

29. The use according to claim 27, wherein the disease or condition is a thrombotic disorder, an inflammatory disorder and / or a thromboinflammatory disorder.

30. Diseases or conditions include venous, arterial, or capillary thrombosis (such as stroke, myocardial infarction, deep vein thrombosis (DVT), portal vein thrombosis, renal vein thrombosis, jugular vein thrombosis, sinus thrombosis, Budd-Chiari syndrome, Paget-Schlotter disease, or asymptomatic cerebral ischemia), cardiac thrombosis, thromboembolism, thrombosis during and / or after contact between blood and artificial surfaces in human or animal subjects, disseminated intravascular coagulation (DIC), atrial fibrillation, acute coronary syndrome (ACS), atherosclerotic disease, ischemic stroke due to reperfusion, ischemic-reperfusion injury (IRI, trauma). The preparation according to claim 28, selected from the group consisting of diseases related to organ transplantation, etc., neurotraumatic injuries (traumatic brain injury, spinal cord injury, etc.), neuroinflammatory diseases (multiple sclerosis, etc.), interstitial lung diseases (idiopathic pulmonary fibrosis (IPF), etc.), pneumonia, fibrinolysis, diseases related to FXII / FXIIa-induced kinin formation (hereditary angioedema (HAE), etc.), sepsis, diseases related to FXII / FXIIa-mediated complement activation, acute respiratory distress syndrome (ARDS), organ and cell transplantation, sickle cell disease, and conditions related to increased vascular permeability.

31. Diseases or conditions include venous, arterial, or capillary thrombosis (such as stroke, myocardial infarction, deep vein thrombosis (DVT), portal vein thrombosis, renal vein thrombosis, jugular vein thrombosis, sinus thrombosis, Budd-Chiari syndrome, Paget-Schlotter disease, or asymptomatic cerebral ischemia), cardiac thrombosis, thromboembolism, thrombosis during and / or after contact between blood and artificial surfaces in human or animal subjects, disseminated intravascular coagulation (DIC), atrial fibrillation, acute coronary syndrome (ACS), atherosclerotic disease, ischemic stroke due to reperfusion, ischemic-reperfusion injury (IRI, trauma). The use according to claim 29, selected from the group consisting of diseases associated with organ transplantation, neurotraumatic injuries (traumatic brain injury, spinal cord injury, etc.), neuroinflammatory diseases (multiple sclerosis, etc.), interstitial lung diseases (idiopathic pulmonary fibrosis (IPF), etc.), pneumonia, fibrinolysis, diseases associated with FXII / FXIIa-induced kinin formation (hereditary angioedema (HAE), etc.), sepsis, diseases associated with FXII / FXIIa-mediated complement activation, acute respiratory distress syndrome (ARDS), organ and cell transplantation, sickle cell disease, and conditions associated with increased vascular permeability.

32. A kit for use in antagonizing the activity and / or activation of factor XII and / or its activated form in a subject, (a) at least one pharmaceutical formulation according to any one of claims 1 to 23; (b) Instructions for using the kit to antagonize and / or antagonize the activation of factor XII and / or its activated form in a subject; and (c) Depending on the case, at least one further therapeutic compound or drug The kit includes the above.

33. A kit for use in treating or preventing a disease or condition in a subject, (a) at least one pharmaceutical formulation according to any one of claims 1 to 23; (b) Instructions for using the kit in treating or preventing a disease or condition in a subject; and (c) Depending on the case, at least one further therapeutic compound or drug The kit includes the above.

34. The kit according to claim 32 or 33, wherein the formulation is present in a vial, a pre-filled syringe, or an auto-injector device.

35. A pre-filled syringe comprising a pharmaceutical preparation according to any one of claims 1 to 23.

36. A self-injector device comprising a pharmaceutical preparation according to any one of claims 1 to 23.

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