Anti-connexin antibody formulations

Abstract

To provide anti-connexin antibody formulations.SOLUTION: The disclosure relates to pharmaceutical compositions and methods for treating diseases or conditions associated with the insufficient opening of Cx43 hemichannel in osteocytes, optionally for treating cancer metastasis, osteosarcoma, osteoporosis, or osteopenia. In an aspect, the disclosure relates to a pharmaceutical formulation comprising an anti-Cx43 antibody or antigen binding fragment thereof, a buffer, a surfactant and a stabilizer, where the formulation has a pH of from about 5 to about 6, where the anti-Cx43 antibody or antigen binding fragment thereof comprises first, second and third heavy chain complementarity determining region (CDR) sequences having amino acid sequences of SEQ ID NOs:1, 2 and 3, and first, second and third light chain complementarity determining region (CDR) sequences having amino acid sequences of SEQ ID NOs: 4, 5 and 6, respectively.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and benefit of U.S. Provisional Patent Application No. 62 / 909,267, filed October 2, 2019, the entire disclosure of which is incorporated herein by reference. Sequence Listing

[0002] The ASCII text file having a size of 43,785 bytes submitted herewith via EFS-Web entitled "172628_020501_sequence.txt", created on October 2, 2020, is hereby incorporated by reference in its entirety. Field

[0003] The present disclosure relates generally to stable aqueous pharmaceutical compositions comprising anti-connexin (Cx) 43 antibodies. [Background technology]

[0004] background Antibodies are used in the treatment of various diseases and conditions due to the specificity of target recognition, which results in highly selective results after systemic administration. For antibodies to remain effective, they must maintain their biological activity during antibody production, purification, transportation and storage. New production and purification techniques have been developed to produce large amounts of highly purified monoclonal antibodies. However, there are still challenges to stabilize these antibodies for transportation and storage, and even more challenges to provide antibodies in a suitable dosage form for administration.

[0005] Denaturation, aggregation, contamination and particle formation can be serious obstacles in antibody formulation and storage.Because of antibody diversity, there is no universal formulation or condition suitable for the storage of all antibodies.The optimal formulation and condition suitable for the storage of one antibody is often specific to that antibody.Therefore, the formulation and method of antibody storage is often an important part of the research and development process for commercial antibodies.

[0006] Various methods have been proposed to overcome the challenges associated with antibody stability. For example, in some instances, antibodies are often lyophilized and then reconstituted immediately prior to administration. However, reconstitution is generally not ideal, as it adds an additional step to the administration process and may introduce contaminants into the formulation. Furthermore, even reconstituted antibodies can suffer from aggregation and particle formation. Thus, there is a need to provide a stable aqueous antibody formulation, particularly an anti-Cx43 antibody formulation, that can overcome the challenges associated with transportation and storage. Summary of the Invention [Means for solving the problem]

[0007] overview The present disclosure provides, in one aspect, a pharmaceutical formulation comprising: An anti-Cx43 antibody or an antigen-binding fragment thereof; A buffer; A surfactant, Stabilizers and Including, The pharmaceutical formulation has a pH of about 5 to about 6; The anti-Cx43 antibody or antigen-binding fragment thereof a first, second and third heavy chain complementarity determining region (CDR) sequence having the amino acid sequences of SEQ ID NOs: 1, 2 and 3, respectively; a first, second and third light chain CDR sequence having the amino acid sequences of SEQ ID NOs: 4, 5 and 6, respectively; The present invention provides a pharmaceutical formulation comprising:

[0008] In some embodiments, the anti-Cx43 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain having the amino acid sequence of SEQ ID NO:7 and a light chain variable domain having the amino acid sequence of SEQ ID NO:8.

[0009] In certain aspects, the anti-Cx43 antibody or antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-17, and a light chain having the amino acid sequence of SEQ ID NO: 18.

[0010] In certain embodiments, the anti-Cx43 antibody or antigen-binding fragment thereof binds to an epitope located within the amino acid sequence of FLSRPTEKTI (SEQ ID NO: 19). In some embodiments, the epitope can include one or more amino acids selected from the group consisting of F1, S3, R4, P5, T6, E7, K8, T9 and I10 of SEQ ID NO: 19. In one embodiment, the epitope consists of F1, S3, R4, P5, T6, E7, K8, T9 and I10 of SEQ ID NO: 19. In some embodiments, the epitope can include all ten amino acids of SEQ ID NO: 19. In certain embodiments, the epitope consists of all ten amino acids of SEQ ID NO: 19.

[0011] In some embodiments, the anti-Cx43 antibody or antigen-binding fragment thereof is present at a concentration of about 5 to about 50 mg / mL, optionally 10 to 40, or about 15 to 30 mg / mL.

[0012] In some particular embodiments, the buffer is selected from acetic acid / sodium acetate, histidine / aspartic acid, citric acid / sodium citrate, sodium hydrogen phosphate / sodium dihydrogen phosphate and histidine / histidine hydrochloride.In some particular embodiments, the buffer is histidine / aspartic acid or histidine / histidine hydrochloride.In some particular embodiments, the buffer is histidine / histidine hydrochloride.

[0013] In some embodiments, the surfactant is polysorbate 80 (PS80).

[0014] In certain embodiments, the stabilizer is selected from ethylenediaminetetraacetic acid (EDTA), sodium chloride, sorbitol, glycine, and sucrose. In certain embodiments, the stabilizer is sucrose.

[0015] In certain embodiments, the pH of the formulation is from about 5.4 to about 5.6.

[0016] In some embodiments, the formulation is an aqueous formulation. In some embodiments, the formulation is a stable aqueous formulation.

[0017] Another aspect is a pharmaceutical formulation comprising: About 10 to 50 mg / mL or about 25 mg / mL of an anti-Cx43 antibody or an antigen-binding fragment thereof (e.g., an antibody that binds to an epitope located within the amino acid sequence of FLSRPTEKTI (SEQ ID NO: 19) about 10-40 mM, or about 20 mM histidine / histidine hydrochloride buffer; about 0.005% to 0.05%, or about 0.02% w / v polysorbate 80; Approximately 1% to 20% w / v or approximately 8% w / v sucrose Including, The pharmaceutical formulation relates to a pharmaceutical formulation, wherein the formulation has a pH of about 5 to about 6, or about 5.4 to about 5.6, or about 5.5.

[0018] A further aspect is a pharmaceutical formulation comprising: About 25 mg / mL of an anti-Cx43 antibody or an antigen-binding fragment thereof comprising a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 to 17 and a light chain having the amino acid sequence of SEQ ID NO: 18; Approximately 20 mM histidine / aspartic acid buffer; Approximately 0.02% w / v polysorbate 80; Approximately 8% w / v sucrose Including, The pharmaceutical formulation relates to a pharmaceutical formulation, wherein the formulation has a pH of about 5.4 to about 5.6, or about 5.5.

[0019] Also provided herein is the use of the pharmaceutical preparation disclosed herein for promoting the opening of Cx43 hemichannel in bone cells, for example for the treatment of cancer, cancer metastasis, osteosarcoma, osteoporosis or osteopenia.Also provided is a method and kit for the treatment of diseases that are affected by the opening (or lack thereof) of Cx43 hemichannel in bone cells. [Brief description of the drawings]

[0020] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] MicroCal DSC thermogram overlay from a pH / buffer screening study of anti-Cx43 Abs.

[0021] [Diagram 2] Comparison of SEC-% main peak at 25±2° C. (left) and 40±2° C. (right) from a pH / buffer screening study.

[0022] [Diagram 3] Comparison of cIEF main peak % at 25±2°C (left) and 40±2°C (right) from a pH / buffer screening study.

[0023] [Figure 4] Comparison of non-reduced SDS-caliper % purity from a pH / buffer screening study at 25±2°C (left) and 40±2°C (right).

[0024] [Diagram 5] Comparison of reduced SDS-caliper % purity from a pH / buffer screening study at 25±2°C (left) and 40±2°C (right).

[0025] [Figure 6] Comparison of SEC-HPLC main peak % from freeze / thaw studies.

[0026] [Figure 7] Comparison of cIEF main peak % from freeze / thaw studies.

[0027] [Figure 8] Comparison of % purity from freeze / thaw studies in non-reduced SDS-caliper (left) and reduced SDS-caliper (right).

[0028] [Figure 9] Comparison of SEC-HPLC main peak % from the stirring study.

[0029] [Figure 10] Comparison of cIEF main peak % from agitation studies.

[0030] [Figure 11] Comparison of % purity from agitation studies in non-reduced SDS-caliper (left) and reduced SDS-caliper (right).

[0031] [Figure 12] Comparison of SEC-main peak % at 2-8°C (left), 25±2°C (center) and 40±2°C (right).

[0032] [Figure 13] Comparison of cIEF main peak % at 2–8°C (left), 25 ± 2°C (center) and 40 ± 2°C (right).

[0033] [Figure 14] Comparison of non-reduced SDS-caliper purity % at 2–8°C (left), 25±2°C (center), and 40±2°C (right).

[0034] [Figure 15] Comparison of reduced SDS-caliper purity % at 2–8°C (left), 25±2°C (center), and 40±2°C (right).

[0035] [Figure 16] MicroCal DSC thermogram overlay from anti-Cx43Ab formulation validation study. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] Detailed Description In some embodiments, a stable aqueous pharmaceutical formulation of an anti-Cx43 antibody is disclosed herein. Such a formulation can include an anti-Cx43 antibody or an antigen-binding fragment thereof, a buffer, a surfactant, and a stabilizer. The pharmaceutical formulation can have a pH of about 5 to about 6, or about 5.4 to 5.6, or about 5.5.

[0037] In some embodiments, the anti-Cx43 antibody or antigen-binding fragment thereof can have first, second and third heavy chain complementarity determining region (CDR) sequences having the amino acid sequences of SEQ ID NOs: 1, 2 and 3, respectively, and / or first, second and third light chain CDR sequences having the amino acid sequences of SEQ ID NOs: 4, 5 and 6, respectively.

[0038] In some embodiments, the anti-Cx43 antibody or antigen-binding fragment thereof can have a heavy chain variable domain having the amino acid sequence of SEQ ID NO:7 and a light chain variable domain having the amino acid sequence of SEQ ID NO:8.

[0039] In certain aspects, the anti-Cx43 antibody or antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-17, and a light chain having the amino acid sequence of SEQ ID NO: 18.

[0040] In certain embodiments, the anti-Cx43 antibody or antigen-binding fragment thereof binds to an epitope located within the amino acid sequence of FLSRPTEKTI (SEQ ID NO: 19).

[0041] In various embodiments, the formulations disclosed herein can have improved stability such that they do not exhibit significant changes (such as appearance, antibody concentration, pH, antibody aggregation, and antibody purity) observed at a given temperature (e.g., -20° C. or refrigerated temperatures of 2-8° C.) for a period of time, e.g., at least three months, at least six months, at least one year, or up to two years. definition

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The following references provide those of ordinary skill in the art with general definitions of many of the terms used in this disclosure: Academic Press Dictionary of Science and Technology,Morris(Ed.),Academic Press(1 st ed., 1992);Oxford Dictionary of Biochemistry and Molecular Biology, Smith et al. (Eds.), Oxford University Press (revised ed., 2000); Encyclopaedic Dictionary of Chemistry, Kumar (Ed.), Anmol Publications Pvt. Biology,Singleton et al. (Eds.),John Wiley&Sons(3 rd ed.,2002);Dictionary of Chemistry,Hunt(Ed.),Routledge(1 st ed., 1999); Dictionary of Pharmaceutical Medicine, Nahler (Ed.), Springer-Verlag Telos (1994); Dictionary of Organic Chemistry, Kumar and Anandand (Eds.), Anmol Publications Pvt. Ltd. (2002); and A Dictionary of Biology (Oxford Paperback Reference), Martin and Hine (Eds.), Oxford University Press (4 th ed., 2000). Further clarification of some of these terms as they apply specifically to the present disclosure is provided herein.

[0043] As used herein, the articles "a" and "an" refer to one or more than one, e.g., at least one, of the grammatical object of the article. The use of the terms "a" or "an" when used in conjunction with the term "comprising" herein can mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more than one."

[0044] As used herein, "about" and "approximately" generally refer to an acceptable degree of error for the quantity measured, given the nature or precision of the measurement. Exemplary degrees of error are within 20 percent (%), typically within 10%, and more typically within 5% of a given value range. The term "substantially" means more than 50%, preferably more than 80%, and most preferably more than 90% or 95%.

[0045] As used herein, the terms "comprising" or "comprises" are used in reference to compositions, methods and their respective component(s) that are present in a given embodiment but are open to including elements not specified.

[0046] As used herein, the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the disclosure.

[0047] The term "consisting of" refers to compositions, methods, and their respective components described herein, and excludes any element not recited in that description of an embodiment.

[0048] An "anti-Cx43 antibody" is an antibody that immunospecifically binds to Cx43 (e.g., its extracellular domain). The antibody can be an isolated antibody. Such binding to Cx43 can be, for example, an antibody having a K value of 1 μM or less, 100 nM or less, or 50 nM or less. D Indicates K D can be measured by any method known to those skilled in the art, such as surface plasmon resonance assay or cell binding assay.Anti-Cx43 antibody can be a monoclonal antibody or an antigen-binding fragment thereof.In some embodiments, the antibody can be one disclosed in International Application No. PCT / US2019 / 025363, the entirety of which is incorporated herein by reference.

[0049] An "antibody" as used herein is a protein that contains a binding domain that binds to a target epitope. The term antibody includes monoclonal antibodies, including immunoglobulin heavy and light chain molecules, single heavy chain variable domain antibodies, and variants and derivatives thereof, including chimeric variants of monoclonal and single heavy chain variable domain antibodies. The binding domain is substantially encoded by an immunoglobulin gene or a fragment of an immunoglobulin gene, and the protein immunospecifically binds to an antigen. Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as a myriad of immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively. For most vertebrates, including human and mouse species, a typical immunoglobulin structural unit comprises a tetramer composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD). L " and "V H " refers to the variable domains of these light and heavy chains, respectively. L " and "C H " refers to the constant domains of the light and heavy chains.L and V H The loops of the upper β chains are responsible for binding to the antigen and are called "complementarity determining regions" or "CDRs". The "Fab" (fragment, antigen binding) region consists of one constant domain and one variable domain, i.e., V, from each heavy and light chain of the antibody. L , C L , V H and C H Includes 1.

[0050] Antibodies include intact immunoglobulins as well as antigen-binding fragments thereof. The term "antigen-binding fragment" refers to a polypeptide fragment of an antibody that binds an antigen or competes with the intact antibody (i.e., with the intact antibody from which it is derived) for antigen binding (i.e., specific binding). Antigen-binding fragments can be produced by recombinant or biochemical methods well known in the art. Exemplary antigen-binding fragments include Fv, Fab, Fab', (Fab')2, CDR, paratope and V. H Chain and V L Included are single chain Fv antibodies (scFv), in which chains are linked together (directly or via a peptide linker) to form a contiguous polypeptide.

[0051] Antibodies also include variants, chimeric antibodies and humanized antibodies. The term "antibody variant" as used herein refers to an antibody with single or multiple mutations in the heavy and / or light chain. In some embodiments, the mutations are in the variable region. In some embodiments, the mutations are in the constant region. "Chimeric antibody" refers to an antibody in which one portion of each of the amino acid sequences of the heavy and light chains is homologous to the corresponding sequence in an antibody from a particular species or belonging to a particular class, while the remaining segments of these chains are homologous to the corresponding sequence of another. Typically, in these chimeric antibodies, the variable regions of both the light and heavy chains mimic the variable regions of an antibody from one species of mammal, while the constant portions are homologous to the sequences in an antibody from another species. One distinct advantage of such chimeric forms is that the variable regions can be conveniently obtained from currently known sources, for example, using readily available hybridomas or B cells from non-human host organisms, in combination with constant regions from, for example, human cell preparations. The variable region has the advantage of being easy to prepare and the specificity is not affected by its source, whereas the constant region is human and therefore less likely to elicit an immune response from a human subject when the antibody is injected than a constant region from a non-human source. However, the definition is not limited to this particular example. A "humanized" antibody refers to a molecule having an antigen-binding site that is substantially derived from an immunoglobulin from a non-human species and the remaining immunoglobulin structure of the molecule is based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site may comprise either a complete variable domain fused onto a constant domain or only the complementarity determining regions (CDRs) grafted onto appropriate framework regions in the variable domain. The antigen-binding site may be wild-type or modified by one or more amino acid substitutions, e.g., modified to more closely resemble human immunoglobulins. Some forms of humanized antibodies preserve all CDR sequences (e.g., a humanized mouse antibody that contains all six of the CDRs from mouse antibodies).Other forms of humanized antibodies have one or more CDRs (1, 2, 3, 4, 5, or 6) that are altered relative to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs.

[0052] As described herein, amino acid residues of an antibody may be numbered according to Kabat's conventional numbering system (Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th edition. Public Health Service, NIH, Bethesda, MD).

[0053] The term "binding" as used herein in relation to the binding between an antibody and a target epitope of Cx43 refers to the process of non-covalent interactions between molecules. Preferably, the binding is specific. The specificity of an antibody can be determined based on affinity. A specific antibody is one that is more specific than an antibody ... -7 Less than M, preferably 10 -8 A binding affinity or dissociation constant, K, for that epitope that is less than M D may have the following structure:

[0054] The term "antigen" refers to a molecule or portion of a molecule that can be bound by a selective binding agent, such as an antibody, and can be used in an animal to produce antibodies capable of binding to an epitope of that antigen. An antigen can have one or more epitopes.

[0055] The term "epitope" includes any determinant, preferably a polypeptide determinant, capable of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitopic determinants include chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl, and in certain embodiments may have specific three-dimensional structural characteristics and / or specific charge characteristics. In one embodiment, an epitope is a region of an antigen that is bound by an antibody. In certain embodiments, an antibody is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. Methods for epitope mapping are well known in the art, such as X-ray cocrystallography, array-based oligopeptide scanning, site-directed mutagenesis, high-throughput mutagenesis mapping, and hydrogen-deuterium exchange. Epitopes can be formed both from adjacent amino acids or non-adjacent amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually at least 5 or 8-10 amino acids in a unique spatial conformation.

[0056] The term "subject" or "patient" includes humans or other mammals receiving either prophylactic or therapeutic treatment.

[0057] The terms "treat", "treating" and "treatment" as used herein refer to therapeutic or preventive measures such as those described herein. A method of "treatment" uses the administration of a Cx43 ligand provided herein to a patient, such as a patient with cancer, to prevent, cure, delay, reduce the severity of, or improve one or more symptoms of cancer or recurrent cancer, or to extend the patient's survival beyond the expected survival in the absence of such treatment. A method of "treatment" also uses the administration of a Cx43 ligand (e.g., an antibody) provided herein to a patient to provide cancer therapy in the patient beyond that expected in the absence of such treatment.

[0058] The term "cancer" broadly refers to the uncontrolled, abnormal growth of the host's own cells, resulting in the invasion of surrounding tissues and potentially tissues distant from the initial site of abnormal cell growth in the host. Major classes include carcinomas, which are cancers of epithelial tissues (e.g., skin, squamous cells); sarcomas, which are cancers of connective tissues (e.g., bone, cartilage, fat, muscle, blood vessels, etc.); leukemias, which are cancers of blood-forming tissues (e.g., bone marrow tissue); lymphomas and myelomas, which are cancers of immune cells; and central nervous system cancers, including cancers from brain and spinal cord tissues. "Cancer(s)", "neoplasm(s)" and "tumor(s)" are used interchangeably herein. As used herein, "cancer" refers to all types of cancers or neoplasms or malignant tumors, including leukemias, carcinomas and sarcomas, whether new or recurrent. Specific examples of cancers are carcinomas, sarcomas, myelomas, leukemias, lymphomas, and mixed tumors. Non-limiting examples of cancers are new or recurrent cancers of the brain, melanoma, bladder, breast, cervix, colon, head and neck, kidney, lung, non-small cell lung, mesothelioma, ovary, prostate, sarcoma, stomach, uterus, and medulloblastoma.

[0059] The term "effective amount" as used herein refers to an amount of an agent, such as a Cx43 ligand, e.g., an anti-Cx43 antibody, that is sufficient to treat, prognose or diagnose cancer when administered to a patient. The therapeutically effective amount varies depending on the patient and disease state being treated, the patient's weight and age, the severity of the disease state, the mode of administration, and the like, which can be readily determined by one of ordinary skill in the art. Dosages for administration, as provided herein, include, for example, about 1 ng to about 10,000 mg, about 5 ng to about 9,500 mg, about 10 ng to about 9,000 mg, about 20 ng to about 8,500 mg, about 30 ng to about 7,500 mg, about 40 ng to about 7,000 mg, about 50 ng to about 6,500 mg, about 100 ng to about 6,000 mg, about 200 ng to about 5,500 mg, about 300 ng to about 5,000 mg, and the like. , about 400ng to about 4,500mg, about 500ng to about 4,000mg, about 1μg to about 3,500mg, about 5μg to about 3,000mg, about 10μg to about 2,600mg, about 20μg to about 2,575mg, Approximately 30μg to approximately 2,550mg, approximately 40μg to approximately 2,500mg, approximately 50μg to approximately 2,475mg, approximately 100μg to approximately 2,450mg, approximately 200μg to approximately 2,425mg, approximately 300μg to approximately 2,000, Approximately 400μg to approximately 1,175mg, approximately 500μg to approximately 1,150mg, approximately 0.5mg to approximately 1,125mg, approximately 1mg to approximately 1,100mg, approximately 1.25mg to approximately 1,075mg, approximately 1.5mg to approximately 1,05 0mg, about 2.0mg to about 1,025mg, about 2.5mg to about 1,000mg, about 3.0mg to about 975mg, about 3.5mg to about 950mg, about 4.0mg to about 925mg, about 4.5mg to about 900mg , about 5 mg to about 875 mg, about 10 mg to about 850 mg, about 20 mg to about 825 mg, about 30 mg to about 800 mg, about 40 mg to about 775 mg, about 50 mg to about 750 mg, about 100 mg to about 725 mg, about 200 mg to about 700 mg, about 300 mg to about 675 mg, about 400 mg to about 650 mg, about 500 mg, or about 525 mg to about 625 mg of the antibody or antigen-binding portion thereof. Administration can be, for example, weekly, every two weeks, every three weeks, every four weeks, every five weeks, or every six weeks. Dosage regimens can be adjusted to provide the optimal therapeutic response.An effective amount is also an amount in which any toxic or adverse effects (side effects) of the agent are minimized and / or are outweighed by the beneficial effects. Dosing can be intravenously administered at or about 6 mg / kg or 12 mg / kg weekly, or 12 mg / kg or 24 mg / kg every other week. Further dosing regimens are described below.

[0060] As used herein, a "formulation" is a composition of a pharma- ceutical active drug, such as a biologically active protein (e.g., an antibody), that is suitable for parenteral administration (including, but not limited to, intravenous, intramuscular, or subcutaneous) to a patient in need thereof and contains only pharma- ceutical acceptable excipients, diluents, and other additives deemed safe by the U.S. Food and Drug Administration or other foreign national authorities.

[0061] As used herein, the phrases "liquid formulation" and "aqueous formulation" are used interchangeably to refer to a liquid agent or preparation containing a biologic in combination with one or more excipients (e.g., chemical additives) dissolved in a suitable solvent.

[0062] A "stable" formulation is one in which no significant change is observed over a period of time, e.g., at least 3 months, at least 6 months, at least 1 year, or up to 2 years, at a given temperature (e.g., -20°C or refrigerated temperatures of 2-8°C). The stability of the formulations disclosed herein can be assessed using one or more of the following criteria: 1) the aqueous formulation is colorless or clear to slightly milky by visual analysis; 2) the protein content is maintained within + / - 5 mg / mL of the initial concentration; 3) the pH is maintained within + / - 0.2 pH units of the target pH; 4) the percent monomer by SEC is 95% or greater; 5) the purity is 90% or greater as measured by CE-SDS and the relative potency based on ELISA is within 50-150%.

[0063] As used herein, the term "excipient" is intended to mean a therapeutically inactive substance that is included in the formulation for a wide variety of purposes, for example, as a buffer, stabilizer, tonicity agent, surfactant, antioxidant, cryoprotectant or diluent.

[0064] Suitable excipients include, but are not limited to, polyols (also known as sugar alcohols) such as mannitol or sorbitol, sugars such as sucrose, lactose or dextrose, salts such as NaCl, KCl or calcium phosphate, amino acids such as histidine, lysine, aspartic acid or glutamic acid, surfactants, and water. The purity of the excipient should meet official standards (e.g., United States Pharmacopoeia, European Pharmacopoeia, Japanese Pharmacopoeia) and be of sufficient purity for subcutaneous, intramuscular or intravenous injection into humans.

[0065] The term "buffer" or "buffering agent" as used herein refers to a pharma- ceutically acceptable excipient that stabilizes the pH of a pharmaceutical preparation. Suitable buffers are well known in the art and can be found in the literature. For example, citrate, acetate, histidine, succinate, malate, phosphate or lactate, and / or their respective free acids or bases, as well as mixtures of various salts and / or acids and bases thereof, can be used. In certain embodiments, pharma-ceutically acceptable buffers include, but are not limited to, histidine buffers, citrate buffers, succinate buffers, acetate buffers and phosphate buffers. In certain embodiments, the buffer is an acetate buffer, such as a sodium acetate buffer. Other particular buffers are histidine buffers, i.e., buffers having histidine, typically L-histidine, as a buffering agent. A particular buffer is L-histidine / HCl buffer, which contains L-histidine or a mixture of L-histidine and L-histidine hydrochloride, where pH adjustment is achieved with hydrochloric acid. Unless otherwise indicated, the term "L-histidine" when used herein to describe a buffering agent refers to an L-histidine / HCl buffer. The L-histidine / HCl buffer can be prepared by dissolving an appropriate amount of L-histidine and L-histidine hydrochloride in water, or by dissolving an appropriate amount of L-histidine in water and adjusting the pH to the desired value by the addition of hydrochloric acid. The above buffers are generally used at concentrations of about 1 mM to about 100 mM, about 10 mM to about 50 mM, about 15 to 30 mM or 20 mM. Regardless of the buffer used, the pH can be adjusted to a value in the range of about 4.0 to about 7.0, about 5.0 to about 6.0, about 5.4 to about 5.6 or about 5.5 using acids or bases known in the art, such as hydrochloric acid, acetic acid, phosphoric acid, sulfuric acid and citric acid, sodium hydroxide and potassium hydroxide.

[0066] The term "surfactant" as used herein refers to a pharma-ceutically acceptable surface active agent. In certain embodiments, non-ionic surfactants are used. Examples of pharma-ceutically acceptable surfactants include, but are not limited to, polyoxyethylene-sorbitan fatty acid esters (Tween®), polyoxyethylene alkyl ethers (Brij), alkylphenyl polyoxyethylene ethers (TritonX), polyoxyethylene-polyoxypropylene copolymers (Poloxamer, Pluronic) and sodium dodecyl sulfate (SDS). In certain embodiments, the polyoxyethylene-sorbitan fatty acid esters are polysorbate 20 (polyoxyethylene sorbitan monolaurate sold under the trademark Tween® 20™) and polysorbate 80 (polyoxyethylene sorbitan monooleate sold under the trademark Tween® 80™). In certain embodiments, the polyethylene-polypropylene copolymer is one sold under the name Pluronic® F68 or Poloxamer 188™. In certain embodiments, the polyoxyethylene alkyl ether is one sold under the trademark Brij™. In certain embodiments, the alkylphenyl polyoxyethylene ether is one sold under the trade name TritonX, such as p-tert-octylphenoxypolyethoxyethanol (sold under the trade name TritonX-100™). When polysorbate 20 (Tween® 20™) and polysorbate 80 (Tween® 80™) are used, they are generally used in a concentration range of about 0.001 to about 1%, about 0.01 to about 0.1%, or about 0.02% to about 0.05%. In the formulations of the present disclosure, the concentrations of the surfactants are described as percentages expressed as weight / volume (w / v).

[0067] The term "stabilizer" as used herein refers to a pharma- ceutically acceptable excipient that protects active pharmaceutical ingredients and / or formulations from chemical and / or physical degradation during manufacture, storage and application.Stabilizers include, but are not limited to, sugars, amino acids, polyols such as mannitol, sorbitol, xylitol, dextran, glycerol, arabitol, propylene glycol, polyethylene glycol, cyclodextrins such as hydroxypropyl-β-cyclodextrin, sulfobutylethyl-β-cyclodextrin, β-cyclodextrin, polyethylene glycols such as PEG3000, PEG3350, PEG4000, PEG6000, albumins such as human serum albumin (HSA), bovine serum albumin (BSA), salts such as sodium chloride, magnesium chloride, calcium chloride, and chelating agents such as EDTA, as defined below. As described herein above, the stabilizer can be present in the formulation in an amount of about 1 to about 500 mM, in an amount of about 10 to about 300 mM, or in an amount of about 120 mM to about 300 mM. More than one stabilizer selected from the same or different groups can be present in the formulation.

[0068] The term "saccharide" as used herein includes monosaccharides and oligosaccharides. Monosaccharides are monomeric carbohydrates that are not hydrolyzable by acids, including simple sugars and their derivatives, such as amino sugars. Sugars are usually in their D-configuration. Examples of monosaccharides include glucose, fructose, galactose, mannose, sorbose, ribose, deoxyribose, neuraminic acid. Oligosaccharides are carbohydrates consisting of more than one monomeric sugar unit connected via glycosidic bond(s), either branched or linear. The monomeric sugar units in an oligosaccharide can be the same or different. Depending on the number of monomeric sugar units, an oligosaccharide is a disaccharide, trisaccharide, tetrasaccharide, pentasaccharide, etc. In contrast to polysaccharides, monosaccharides and oligosaccharides are water-soluble. Examples of oligosaccharides include sucrose, trehalose, lactose, maltose, and raffinose. In certain embodiments, the sugar is sucrose and trehalose (i.e., α,α-D-trehalose), e.g., sucrose. Trehalose is available as trehalose dihydrate. The sugar can be present in the formulation in an amount of about 100 to about 500 mM, in an amount of about 200 to about 300 mM, or in an amount of about 240 mM.

[0069] A subgroup within the stabilizers are the cryoprotectants. The term "cryoprotectant" refers to a pharma- ceutically acceptable excipient that protects labile active ingredients (e.g., proteins) from destabilizing conditions during the lyophilization process, subsequent storage, and reconstitution. Cryoprotectants include, but are not limited to, the group consisting of sugars, polyols (e.g., sugar alcohols, etc.), and amino acids. In certain embodiments, the cryoprotectant can be selected from the group consisting of sugars, such as sucrose, trehalose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, neuraminic acid, amino sugars, such as glucosamine, galactosamine, N-methylglucosamine ("Meglumine"), polyols, such as mannitol and sorbitol, and amino acids, such as arginine and glycine, or mixtures thereof. Cryoprotectants are generally used in amounts of about 10 to 500 mM, in amounts of about 10 to about 300 mM, or in amounts of about 100 to about 300 mM.

[0070] Another subgroup within the stabilizers are antioxidants. The term "antioxidant" refers to a pharma- ceutically acceptable excipient that prevents oxidation of an active pharmaceutical ingredient. Antioxidants include, but are not limited to, ascorbic acid, glutathione, cysteine, methionine, citric acid, and EDTA. Antioxidants can be used in amounts of about 0.01 to about 100 mM, in amounts of about 5 to about 50 mM, or in amounts of about 5 to about 25 mM.

[0071] A formulation according to the present disclosure may also include one or more isotonicity agents. The term "isotonicity agent" refers to a pharma- ceutically acceptable excipient used to modulate the isotonicity of a formulation. A formulation may be hypotonic, isotonic or hypertonic. Isotonicity generally refers to the osmolality of a solution, usually relative to the osmolality of human serum (about 250-350 mOsmol / kg). A formulation according to the present disclosure may be hypotonic, isotonic or hypertonic. In certain embodiments, a formulation is isotonic. An isotonic formulation is a liquid that has been reconstituted from a liquid or solid form, e.g., from a lyophilized form, and represents a solution that has the same tonicity as some other solution to which it is compared, such as physiological salt solutions and serum. Suitable isotonicity agents include, but are not limited to, sodium chloride, potassium chloride, glycerin, and any constituent from the group of amino acids or sugars, particularly glucose. Isotonicity agents are generally used in amounts of about 5 mM to about 500 mM.

[0072] Among the stabilizers and isotonicity agents, there is a group of compounds that can function in both ways, i.e., they can be stabilizers and isotonicity agents at the same time. Examples can be found in the group of sugars, amino acids, polyols, cyclodextrins, polyethylene glycols and salts. An example of a sugar that can be a stabilizer and isotonicity agent at the same time is trehalose.

[0073] The "isoelectric point" or "pI" of a protein is the pH at which the protein has a net total charge equal to 0, i.e., the pH at which the protein has an equal number of positive and negative charges. Determination of the pI for any given protein can be performed according to well-established techniques, such as isoelectric focusing. Isoelectric focusing is a technique for separating different molecules by differences in their isoelectric points (pI). It is a type of zone electrophoresis, usually performed on proteins in a gel, that takes advantage of the fact that the total charge on a molecule of interest is a function of the pH of its surroundings.

[0074] Various aspects of the disclosure are described in further detail below. Additional definitions are set forth throughout the specification. Pharmaceutical preparations

[0075] In some embodiments, the present disclosure provides a pharmaceutical composition comprising an anti-Cx43 antibody or antigen-binding fragment thereof described herein. The anti-Cx43 antibody or antigen-binding fragment thereof can have a first, second and third heavy chain complementarity determining region (CDR) sequence having the amino acid sequence of SEQ ID NO: 1, 2 and 3, respectively, and a first, second and third light chain CDR sequence having the amino acid sequence of SEQ ID NO: 4, 5 and 6, respectively.

[0076] In some embodiments, the anti-Cx43 antibody or antigen-binding fragment thereof can comprise a heavy chain variable domain having the amino acid sequence of SEQ ID NO:7 and a light chain variable domain having the amino acid sequence of SEQ ID NO:8.

[0077] In certain aspects, the anti-Cx43 antibody or antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-17, and a light chain having the amino acid sequence of SEQ ID NO: 18.

[0078] In certain embodiments, the anti-Cx43 antibody or antigen-binding fragment thereof binds to an epitope located within the amino acid sequence of FLSRPTEKTI (SEQ ID NO: 19).

[0079] In some embodiments, the anti-Cx43 antibody or antigen-binding fragment thereof can be present at a concentration of about 5 to about 50 mg / mL, or 10 to 40, or about 15 to 30 mg / mL.

[0080] In various embodiments, anti-Cx43 antibody or its antigen-binding fragment can be formulated in a pharma- ceutically acceptable amount and in a pharma-ceutically acceptable composition.As used herein, "pharma-ceutically acceptable" refers to a substance that is generally safe, non-toxic, and useful for preparing pharmaceutical compositions that are not biologically or otherwise undesirable, including those that are acceptable for veterinary and human pharmaceutical use.Examples of "pharma-ceutically acceptable liquid carriers" include water and organic solvents.Preferred pharma-ceutically acceptable aqueous liquids include PBS, saline, dextrose solution, and the like.

[0081] As used herein, the term "pharmaceutically acceptable salt" refers to any pharmaceutically acceptable salt of the compounds disclosed herein. For example, the "pharmaceutically acceptable salt" of any compound described herein includes salts that are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, or allergic response, within the scope of sound medical judgment, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977 and Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008. Salts can be prepared in situ during the final isolation and purification of the compounds described herein, or separately by reacting free base groups with a suitable organic acid.

[0082] To facilitate the selection of a pharma- ceutically acceptable carrier or excipient, various references are available, e.g., Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa. (1984); Hardman et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis et al., (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman et al., (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse See, Weiner, Wang, W., Int. J. Pharm. 185:129-188 (1999) and Wang, W., Int. J. Pharm. 203:1-60 (2000), and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY.

[0083] In some embodiments, the antibody formulation may include a buffer (e.g., histidine, acetate, phosphate, or citrate buffer), a detergent (e.g., polysorbate), and / or a stabilizer (e.g., sucrose), or the like.

[0084] Buffers are used to control pH in a range that optimizes therapeutic efficacy, especially when stability is pH dependent. Buffers can be present at concentrations ranging from about 50 mM to about 250 mM. Buffering agents suitable for use in the present disclosure include both organic and inorganic acids and their salts. For example, citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate. Additionally, buffers can be composed of trimethylamine salts such as histidine and tris.

[0085] In certain embodiments, the buffer can be selected from acetic acid / sodium acetate, histidine / aspartic acid, citric acid / sodium citrate, sodium hydrogen phosphate / sodium dihydrogen phosphate and histidine / histidine hydrochloride. When referring to a buffer composition "A / B", " / " as used herein means that both component A and component B (e.g., a salt of component A) are present. In certain embodiments, the buffer is histidine / aspartic acid or histidine / histidine hydrochloride. In certain embodiments, the buffer is histidine / histidine hydrochloride.

[0086] The non-ionic surfactant or detergent (also known as a "wetting agent") is present to help solubilize the therapeutic agent and to protect the therapeutic protein from aggregation caused by agitation, and also allows the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein or antibody. The non-ionic surfactant is present in the range of about 0.05 mg / ml to about 1.0 mg / ml, or about 0.07 mg / ml to about 0.2 mg / ml.

[0087] Suitable nonionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), poloxamers (184, 188, etc.), PLURONIC® polyols, TRITON®, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, sucrose fatty acid esters, methylcellulose and carboxymethylcellulose. Anionic detergents that can be used include sodium lauryl sulfate, sodium dioctylsulfosuccinate (dioctyle sodium sulfosuccinate) and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride. In some embodiments, the surfactant is polysorbate 80 (PS80).

[0088] In certain embodiments, the stabilizer is selected from ethylenediaminetetraacetic acid (EDTA), sodium chloride, sorbitol, glycine, and sucrose. In certain embodiments, the stabilizer is sucrose.

[0089] Additional excipients include agents that can act as one or more of the following: (1) bulking agents, (2) solubility enhancers, (3) stabilizers, and (4) agents that prevent denaturation or sticking to container walls. Such excipients include polyhydric sugar alcohols (listed above); amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, and the like; sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycols, and the like. These include organic sugars or sugar alcohols such as choline; sulfur-containing reducing agents such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol and sodium thiosulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin or other immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose); disaccharides (e.g., lactose, maltose, sucrose); trisaccharides such as raffinose; and polysaccharides such as dextrin or dextran.

[0090] In some embodiments, the antibody formulation may include a pharma- ceutically acceptable carrier, including, for example, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, sucrose, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, polyethylene-polyoxypropylene-block polymers and polyethylene glycol. In some embodiments, the antibody formulation further includes a surfactant. In some embodiments, the surfactant is selected from the group consisting of polysorbates, sodium dodecyl sulfate, and non-ionic surfactants.

[0091] A formulation according to the present disclosure may be in liquid form, in lyophilized form, or in liquid form reconstituted from a lyophilized form. In certain embodiments, the formulation is in liquid form. The term "liquid" as used herein in connection with a formulation according to the present disclosure refers to a formulation that is liquid at a temperature of at least about 2 to about 8°C under atmospheric pressure. The term "lyophilized" as used herein in connection with a formulation according to the present disclosure refers to a formulation produced by a lyophilization method known per se in the art. The solvent (e.g., water) is removed by freezing, followed by sublimation of the ice under vacuum and desorption of the residual water at elevated temperature. Lyophilizates usually have a residual moisture of about 0.1 to 5% (w / w) and exist as a powder or a physically stable cake. Lyophilizates are characterized by rapid dissolution after addition of a reconstitution medium.

[0092] The term "reconstituted form" as used herein in connection with a formulation according to the present disclosure refers to a formulation that has been lyophilized and reconstituted by the addition of a reconstitution medium. Suitable reconstitution media include, but are not limited to, water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), surfactant-containing solution (e.g., 0.02% polysorbate 80), pH buffer solution (e.g., phosphate buffer solution).

[0093] The formulation according to the present disclosure is physiologically well tolerated, can be easily prepared, can be accurately dispensed, and is stable to degradation products and aggregates during repeated freeze-thaw cycles and mechanical stress over the period of storage, and is stable for periods of more than one year at storage temperatures (e.g., -20°C or 2-8°C).

[0094] The antibody formulation of the present disclosure can be an aqueous solution.In some embodiments, the antibody formulation is not subjected to freezing temperature and / or is not frozen, i.e., they remain in liquid state.In some embodiments, the antibody in the antibody formulation is not subjected to lyophilization.

[0095] In some embodiments, the antibody formulations disclosed herein have improved stability compared to other formulations.As used herein, the term "stability" generally refers to maintaining the integrity or minimizing the degradation, denaturation, aggregation or unfolding of bioactive agents such as proteins, peptides or other bioactive macromolecules.As used herein, "improved stability" generally means that the protein of interest (e.g., an antibody such as anti-Cx43Ab), peptide or other bioactive macromolecules maintains greater stability compared to control proteins, peptides or other bioactive macromolecules under conditions known to cause degradation, denaturation, aggregation or unfolding.

[0096] In some embodiments, stability refers to antibody formulations with low to undetectable particle formation. The phrase "low to undetectable particle formation" as used herein refers to samples containing less than 30 particles / mL, less than 20 particles / ml, less than 20 particles / ml, less than 15 particles / ml, less than 10 particles / ml, less than 5 particles / ml, less than 2 particles / ml, or less than 1 particle / ml, as determined by HIAC analysis or visual analysis. In some embodiments, no particles are detected in the antibody formulation by either HIAC analysis or visual analysis.

[0097] In some embodiments, stability refers to reduced fragmentation of the antibody. The term "low to undetectable fragmentation" as used herein refers to a sample that contains equal to or greater than 80%, 85%, 90%, 95%, 98% or 99% of total protein in a single peak determined, for example, by HPSEC, or in two peaks (e.g., heavy and light chains) by reduced capillary gel electrophoresis (rCGE) (or as many peaks as subunits are present), and does not contain other single peaks that correspond to undegraded antibody or undegraded fragments thereof, each having more than 5%, 4%, 3%, 2%, 1% or 0.5% of total protein. The term "reduced capillary gel electrophoresis" as used herein refers to capillary gel electrophoresis under reducing conditions sufficient to reduce disulfide bonds in the antibody.

[0098] Those skilled in the art will appreciate that protein stability depends on other characteristics in addition to the composition of the formulation. For example, stability can be affected by temperature, pressure, humidity, pH, and external forms of radiation. Thus, unless otherwise stated, stability referred to herein is considered to be measured at -20°C, one atmosphere of pressure, 50% relative humidity, pH of 5.5, and normal background levels of radiation. Antibody stability in antibody formulations can be determined by various means. In some embodiments, antibody stability is determined by size exclusion chromatography (SEC). SEC separates analytes (e.g., macromolecules such as proteins and antibodies) based on a combination of hydrodynamic size, diffusion coefficient, and surface properties. Thus, for example, SEC can separate antibodies in their native three-dimensional conformation from antibodies in various denatured states and / or degraded antibodies. In SEC, the stationary phase is generally composed of inert particles packed into a dense three-dimensional matrix in a glass or steel column. The mobile phase can be pure water, aqueous buffers, organic solvents, mixtures of these, or other solvents. Stationary phase particles have small pores and / or channels that only allow species below a certain size to enter. Thus, larger particles are excluded from these pores and channels, while smaller particles are removed from the flowing mobile phase. The time a particle spends immobilizing within the pores of the stationary phase depends, in part, on how far the particle can penetrate into the pores. Removal of the particle from the mobile phase stream causes it to take longer to elute from the column, resulting in separation between the particles based on their size differences.

[0099] In some embodiments, SEC is combined with identification technology to identify or characterize protein or its fragments.Protein identification and characterization can be achieved by various techniques, including but not limited to chromatographic techniques, such as high performance liquid chromatography (HPLC), immunoassay, electrophoresis, ultraviolet / visible / infrared spectroscopy, Raman spectroscopy, surface-enhanced Raman spectroscopy, mass spectrometry, gas chromatography, static light scattering (SLS), Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), urea-induced protein unfolding technology, intrinsic tryptophan fluorescence, differential scanning calorimetry, and / or ANS protein binding.

[0100] In some embodiments, protein identification is achieved by high pressure liquid chromatography. Various instruments and devices for carrying out HPLC are known to those skilled in the art. In general, HPLC involves loading a liquid solvent containing the protein of interest into a separation column where separation takes place. The HPLC separation column is filled with solid particles (e.g., silica, polymer, or adsorbent), and the sample mixture is separated into compounds as it interacts with the column particles. HPLC separation is affected by the conditions of the liquid solvent (e.g., pressure, temperature), the chemical interactions between the sample mixture and the liquid solvent (e.g., hydrophobicity, protonation, etc.), and the chemical interactions between the sample mixture and the solid particles packed inside the separation column (e.g., ligand affinity, ion exchange, etc.).

[0101] In some embodiments, SEC and protein identification are performed in the same instrument or simultaneously. For example, SEC and HPLC can be combined, often referred to as SE-HPLC.

[0102] The stability of the antibodies described herein may be enhanced by the use of non-toxic "water-soluble polyvalent metal salts". Examples include Ca2+, Mg2+, Zn2+, Fe2+, Fe3+, Cu2+, Sn2+, Sn4+, Al2+ and Al3+. Examples of anions that can form water-soluble salts with the polyvalent metal cations include those formed from inorganic and / or organic acids. Such water-soluble salts have a solubility in water (20° C.) of at least about 20 mg / ml, alternatively at least about 100 mg / ml, alternatively at least about 200 mg / ml.

[0103] Suitable inorganic acids that can be used to form the "water-soluble polyvalent metal salt" include hydrochloric acid, acetic acid, sulfuric acid, nitric acid, thiocyanic acid, and phosphoric acid. Suitable organic acids that can be used include aliphatic carboxylic acids and aromatic acids. Aliphatic acids within this definition can be defined as saturated or unsaturated C2-9 carboxylic acids (e.g., aliphatic mono-, di-, and tri-carboxylic acids). For example, exemplary monocarboxylic acids within this definition include the saturated C2-9 monocarboxylic acids acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, and capryonic acid, and the unsaturated C2-9 monocarboxylic acids acrylic acid, propriolic acid, methacrylic acid, crotonic acid, and isocrotonic acid. Exemplary dicarboxylic acids include the saturated C2-9 dicarboxylic acids malonic acid, succinic acid, glutaric acid, adipic acid, and pimelic acid, and unsaturated C2-9 dicarboxylic acids include maleic acid, fumaric acid, citraconic acid, and mesaconic acid. Exemplary tricarboxylic acids include the saturated C2-9 tricarboxylic acids tricarballylic acid and 1,2,3-butanetricarboxylic acid. Additionally, carboxylic acids in this definition may also contain one or two hydroxyl groups to form hydroxycarboxylic acids. Exemplary hydroxycarboxylic acids include glycolic acid, lactic acid, glyceric acid, tartronic acid, malic acid, tartaric acid, and citric acid. Aromatic acids within this definition include benzoic acid and salicylic acid.

[0104] Commonly used water-soluble polyvalent metal salts that can be used to help stabilize the encapsulated polypeptides of the present disclosure include, for example, (1) inorganic acid metal salts of metal halides (e.g., zinc chloride, calcium chloride), sulfates, nitrates, phosphates, and thiocyanates; (2) aliphatic carboxylates (e.g., calcium acetate, zinc acetate, calcium propionate, zinc glycolate, calcium lactate, zinc lactate, and zinc tartrate); and (3) aromatic carboxylates (e.g., zinc benzoate) and salicylates.

[0105] In some embodiments, the aqueous formulation comprises about 2 mg / ml to about 100 mg / ml of an antibody, the antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences as defined in Kabat in SEQ ID NOs: 1-3, and the light chain variable region comprising CDR1, CDR2 and CDR3 sequences as defined in Kabat in SEQ ID NOs: 4-6, and the formulation is stable when stored at about 40° C. for at least 1 month. In some embodiments, the formulation is stable when stored at about 25° C. for at least 3 months. In some embodiments, the formulation is stable when stored at about 5° C. for at least 6 months. In some embodiments, the formulation is stable when stored at about 5° C. for at least 12 months. In some embodiments, the formulation is stable when stored at about 5° C. for at least 18 months. In some embodiments, the formulation is stable when stored at about 5° C. for at least 24 months or 36 months.

[0106] The term "stable" may be relative and not absolute. Thus, in some embodiments, an antibody is stable if less than 20%, 15%, 10%, 5% or 2% of the antibody is degraded, denatured, aggregated or unfolded as measured by SEC HPLC when stored at -20°C for 6 months. In some embodiments, an antibody is stable if less than 20%, 15%, 10%, 5% or 2% of the antibody is degraded, denatured, aggregated or unfolded as measured by SEC HPLC when stored at -20°C for 12 months. In some embodiments, an antibody is stable if less than 20%, 15%, 10%, 5% or 2% of the antibody is degraded, denatured, aggregated or unfolded as measured by SEC HPLC when stored at -20°C for 18 months in an antibody formulation. In some embodiments, an antibody is stable if, when the antibody in the antibody formulation is stored at -20°C for 24 months, less than 20%, less than 15%, less than 10%, less than 5%, or less than 2% of the antibody degrades, denatures, aggregates, or unfolds as measured by SEC HPLC.

[0107] In some embodiments, the SEC is greater than or equal to 100% when the antibody is stored at 23° C. to 27° C. for 3 months. An antibody is stable if less than 20%, 15%, 10%, 5% or 2% of the antibody is degraded, denatured, aggregated or unfolded as measured by HPLC. In some embodiments, an antibody is stable if less than 20%, 15%, 10%, 5% or 2% of the antibody is degraded, denatured, aggregated or unfolded as measured by SEC HPLC when stored at 23°C-27°C for 6 months. In some embodiments, an antibody is stable if less than 20%, 15%, 10%, 5% or 2% of the antibody is degraded, denatured, aggregated or unfolded as measured by SEC HPLC when stored at 23°C-27°C for 12 months. In some embodiments, an antibody is stable if, when stored at 23° C.-27° C. for 24 months, less than 20%, less than 15%, less than 10%, less than 5%, or less than 2% of the antibody degrades, denatures, aggregates, or unfolds as measured by SEC HPLC.

[0108] In some embodiments, an antibody is stable if less than 6%, 4%, 3%, 2%, or 1% of the antibody degrades, denatures, aggregates, or unfolds per month as measured by SEC HPLC when stored at 40° C. In some embodiments, an antibody is stable if less than 6%, 4%, 3%, 2%, or 1% of the antibody degrades, denatures, aggregates, or unfolds per month as measured by SEC HPLC when stored at 5° C.

[0109] In some embodiments, the antibody formulations of the present disclosure can be considered stable if the antibody shows little or no loss of binding activity of the antibody (including its antibody fragment) of the formulation compared to the reference antibody over a period of 8 weeks, 4 months, 6 months, 9 months, 12 months, or 24 months, as measured by, for example, an antibody binding assay known to those skilled in the art, such as ELISA. In some embodiments, the antibody stored at about 40° C. for at least one month retains at least 60%, at least 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% of its binding ability to Cx43 compared to the reference antibody that has not been stored. In some embodiments, the antibody stored at about 5° C. for at least six months retains at least 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% of its binding ability to Cx43 compared to the reference antibody that has not been stored. In some embodiments, an antibody stored at about 40° C. for at least one month retains at least 95% of its ability to bind to Cx43 compared to a reference antibody that has not been stored. In some embodiments, an antibody stored at about 5° C. for at least six months retains at least 95% of its ability to bind to Cx43 compared to a reference antibody that has not been stored.

[0110] The antibody formulation may result in low to undetectable levels of antibody aggregation. The phrase "low to undetectable levels of aggregation" as used herein refers to a sample containing less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1% and less than about 0.5% aggregates by weight of protein as measured by high performance size exclusion chromatography (HPSEC) or static light scattering (SLS) techniques. In some embodiments, less than 2% of the antibody forms aggregates when stored at about 40°C for at least 4 weeks as measured by HPSEC. In some embodiments, less than 2% of the antibody forms aggregates when stored at about 5° for at least 3 months, at least 6 months, at least 9 months, at least 12 months, at least 15 months, at least 18 months, at least 24 months, or at least 36 months as measured by HPSEC.

[0111] The antibody formulations provided herein may be characterized as having a chromatographically distinct chromatographic identity as measured by visual inspection, micro-flow imaging (MFI) or size exclusion chromatography (SEC). It has been discovered herein that when the formulation is stored at about 40° C. for at least 1 month, the formulation is substantially free of particles, as determined by visual inspection. In some embodiments, the formulation is substantially free of particles, as determined by visual inspection, when stored at about 5° C. for at least 6 months, at least 9 months, at least 12 months, at least 15 months, at least 18 months, at least 24 months, or at least 36 months.

[0112] The formulations may also contain auxiliary agents such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms can be ensured by sterilization procedures and by the inclusion of various antibacterial and antifungal agents, such as paraben, chlorobutanol, phenol, sorbic acid, and the like. Preservatives are generally used in amounts of about 0.001 to about 2% (w / v). Preservatives include, but are not limited to, ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl paraben, benzalkonium chloride.

[0113] The antibody formulations described herein can have a variety of viscosities. Methods for measuring the viscosity of antibody formulations are known to those skilled in the art and can include, for example, a rheometer (e.g., an Anton Paar MCR301 Rheometer with either a 50 mm, 40 mm, or 20 mm plate accessory). In some embodiments of the present disclosure, the viscosity is reported at the high shear limit of a shear rate of 1000 / sec. In some embodiments, the antibody formulation has a viscosity of less than 20 centipoise (cP), less than 18 cP, less than 15 cP, less than 13 cP, or less than 11 cP. In some embodiments, the antibody formulation has a viscosity of less than 13 cP. Those skilled in the art will understand that viscosity is temperature dependent, and thus, unless otherwise specified, the viscosities provided herein are measured at 25°C unless otherwise specified.

[0114] Antibody formulations can have different osmolarities. Methods for measuring the osmolarity of antibody formulations are known to those of skill in the art and can include, for example, an osmometer (e.g., a freezing point depression osmometer from Advanced Instrument Inc 2020). In some embodiments, the formulation has an osmolarity of 200-600 mosm / kg, 260-500 mosm / kg, or 300-450 mosm / kg.

[0115] The antibody formulations of the present disclosure can have a variety of pH levels. In some embodiments, the pH of the antibody formulation is 4-7, 4.5-6.5, 5-6, or 5.4-5.6. In some embodiments, the pH of the antibody formulation is 5.5. In some embodiments, the pH of the antibody formulation is 6.0. In some embodiments, the pH of the antibody formulation is 7.0 or higher. Various means can be utilized to achieve the desired pH level, including, but not limited to, the addition of an appropriate buffer.

[0116] In some embodiments, the antibody formulation can include about 10-50 mg / mL or about 25 mg / mL of an anti-Cx43 antibody or antigen-binding fragment thereof, about 10-40 mM, or about 20 mM histidine / histidine hydrochloride buffer, about 0.005%-0.05%, or about 0.02% w / v polysorbate 80, and about 1%-20% w / v or about 8% w / v sucrose, wherein the formulation has a pH of about 5.4 to about 5.6, or about 5.5.

[0117] In some embodiments, the antibody formulation can comprise about 25 mg / mL of an anti-Cx43 antibody or antigen-binding fragment thereof, comprising a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NO:9-17, and a light chain having the amino acid sequence of SEQ ID NO:18, about 20 mM histidine / aspartate buffer, about 0.02% w / v polysorbate 80, and about 8% w / v sucrose, wherein the formulation has a pH of about 5.4 to about 5.6, or about 5.5.

[0118] In one aspect, the antibody formulation can comprise about 25 mg / mL of an anti-Cx43 antibody or antigen-binding fragment thereof, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 9 and a light chain having the amino acid sequence of SEQ ID NO: 18, about 20 mM histidine / aspartate buffer, about 0.02% w / v polysorbate 80, and about 8% w / v sucrose, wherein the formulation has a pH of about 5.4 to about 5.6, or about 5.5.

[0119] In one aspect, the antibody formulation can comprise about 25 mg / mL of an anti-Cx43 antibody or antigen-binding fragment thereof, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 10 and a light chain having the amino acid sequence of SEQ ID NO: 18, about 20 mM histidine / aspartate buffer, about 0.02% w / v polysorbate 80, and about 8% w / v sucrose, wherein the formulation has a pH of about 5.4 to about 5.6, or about 5.5.

[0120] In one aspect, the antibody formulation can comprise about 25 mg / mL of an anti-Cx43 antibody or antigen-binding fragment thereof, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 11 and a light chain having the amino acid sequence of SEQ ID NO: 18, about 20 mM histidine / aspartate buffer, about 0.02% w / v polysorbate 80, and about 8% w / v sucrose, wherein the formulation has a pH of about 5.4 to about 5.6, or about 5.5.

[0121] In one aspect, the antibody formulation can comprise about 25 mg / mL of an anti-Cx43 antibody or antigen-binding fragment thereof, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 12 and a light chain having the amino acid sequence of SEQ ID NO: 18, about 20 mM histidine / aspartate buffer, about 0.02% w / v polysorbate 80, and about 8% w / v sucrose, wherein the formulation has a pH of about 5.4 to about 5.6, or about 5.5.

[0122] In one aspect, the antibody formulation can comprise about 25 mg / mL of an anti-Cx43 antibody or antigen-binding fragment thereof, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 13 and a light chain having the amino acid sequence of SEQ ID NO: 18, about 20 mM histidine / aspartate buffer, about 0.02% w / v polysorbate 80, and about 8% w / v sucrose, wherein the formulation has a pH of about 5.4 to about 5.6, or about 5.5.

[0123] In one aspect, the antibody formulation can comprise about 25 mg / mL of an anti-Cx43 antibody or antigen-binding fragment thereof, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 14 and a light chain having the amino acid sequence of SEQ ID NO: 18, about 20 mM histidine / aspartate buffer, about 0.02% w / v polysorbate 80, and about 8% w / v sucrose, wherein the formulation has a pH of about 5.4 to about 5.6, or about 5.5.

[0124] In one aspect, the antibody formulation can comprise about 25 mg / mL of an anti-Cx43 antibody or antigen-binding fragment thereof, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 14 and a light chain having the amino acid sequence of SEQ ID NO: 18, about 20 mM histidine / aspartate buffer, about 0.02% w / v polysorbate 80, and about 8% w / v sucrose, wherein the formulation has a pH of about 5.4 to about 5.6, or about 5.5.

[0125] In one aspect, the antibody formulation can comprise about 25 mg / mL of an anti-Cx43 antibody or antigen-binding fragment thereof, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 15 and a light chain having the amino acid sequence of SEQ ID NO: 18, about 20 mM histidine / aspartate buffer, about 0.02% w / v polysorbate 80, and about 8% w / v sucrose, wherein the formulation has a pH of about 5.4 to about 5.6, or about 5.5.

[0126] In one aspect, the antibody formulation can comprise about 25 mg / mL of an anti-Cx43 antibody or antigen-binding fragment thereof, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 16 and a light chain having the amino acid sequence of SEQ ID NO: 18, about 20 mM histidine / aspartate buffer, about 0.02% w / v polysorbate 80, and about 8% w / v sucrose, wherein the formulation has a pH of about 5.4 to about 5.6, or about 5.5.

[0127] In one aspect, the antibody formulation can comprise about 25 mg / mL of an anti-Cx43 antibody or antigen-binding fragment thereof, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 17 and a light chain having the amino acid sequence of SEQ ID NO: 18, about 20 mM histidine / aspartate buffer, about 0.02% w / v polysorbate 80, and about 8% w / v sucrose, wherein the formulation has a pH of about 5.4 to about 5.6, or about 5.5.

[0128] In some aspects, the disclosure provides kits comprising any of the antibody formulations described herein, the containers described herein, the unit dosage forms described herein, or the pre-filled syringes described herein. therapeutic use

[0129] In some embodiments, the antibody formulations of the present disclosure can be used for pharmaceutical purposes. Antibodies used in pharmaceutical applications must generally have a high level of purity, especially with respect to contaminants from cell culture, including cellular protein contaminants, cellular DNA contaminants, viruses and other infectious agents. See ''WHO Requirements for the use of animal cells as in vitro substrates for the production of biologicals: Requirements for Biological Substances No. 50'' No. 878. Annex 1, 1998. In response to concerns about contaminants, the World Health Organization (WHO) has set limits on the levels of various contaminants. For example, the WHO has recommended a DNA limit of less than 10 ng per dose for protein products. Similarly, the United States Food and Drug Administration (FDA) has set a DNA limit of less than or equal to 0.5 pg / mg protein. Thus, in some embodiments, the present disclosure relates to antibody formulations that meet or exceed the contaminant limits defined by one or more government agencies, such as the United States Food and Drug Administration and / or the World Health Organization.

[0130] The antibody formulation of the present disclosure can be administered to a subject through various means.In some embodiments, the antibody formulation is suitable for parenteral administration, such as inhalation (e.g., powder or aerosol spray), transmucosal, intravenous, subcutaneous or intramuscular administration.In some embodiments, the formulation is an injectable formulation.In some embodiments, the present disclosure relates to a sealed container that contains any of the antibody formulations described herein.

[0131] In some aspects, the present disclosure relates to various pharmaceutical dosage forms. Various dosage forms may be applicable to the formulations provided herein. For example, see Pharmaceutical Dosage Form: Parenteral Medications, Volume 1, 2 nd See the US Pat. No. 5,393,136, Edition. In one embodiment, the pharmaceutical unit dosage of the present disclosure comprises an antibody formulation in a suitable container, such as a vial or syringe. In one embodiment, the pharmaceutical unit dosage of the present disclosure comprises an antibody formulation delivered intravenously, subcutaneously, or intramuscularly. In another embodiment, the pharmaceutical unit dosage of the present disclosure comprises an antibody formulation delivered by aerosol. In a particular embodiment, the pharmaceutical unit dosage of the present disclosure comprises an antibody formulation delivered subcutaneously. In another embodiment, the pharmaceutical unit dosage of the present disclosure comprises an antibody formulation delivered by aerosol. In a further embodiment, the pharmaceutical unit dosage of the present disclosure comprises an antibody formulation administered intranasally.

[0132] The compositions of the present disclosure can be administered by a variety of methods known in the art. As will be appreciated by those of skill in the art, the route and / or mode of administration will vary depending on the desired results.

[0133] To administer the compositions of the present disclosure by certain routes of administration, it may be necessary to dilute the composition in a diluent. Pharmaceutically acceptable diluents include saline, glucose, Ringer's and aqueous buffer solutions.

[0134] In certain embodiments, formulations according to the present disclosure are administered intravenously (iv), subcutaneously (sc) or by any other parental administration means such as those known in the pharmaceutical arts.

[0135] The phrases "parenteral administration" and "administered parenterally" as used herein mean modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

[0136] The composition must be sterile and fluid to the extent that the composition can be delivered by syringe or injection system. Besides water, the carrier can be an isotonic buffered saline solution, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof.

[0137] Formulations according to the present disclosure can be prepared by methods known in the art, such as ultrafiltration-diafiltration, dialysis, addition and mixing, lyophilization, reconstitution, and combinations thereof. Examples of preparations of formulations according to the present disclosure can be found below.

[0138] The pharmaceutical compositions described herein may be used in the treatment of cancer, cancer metastasis, osteosarcoma, osteoporosis or osteopenia.

[0139] Cancer metastasis occurs when cancer spreads from the part of the body where it originated (e.g., breast or prostate) to other parts of the body (e.g., liver or bone) and establishes secondary tumors. Bone is one of the most common sites of cancer metastasis. Cancers that metastasize to bone include, but are not limited to, breast cancer, prostate cancer, lung cancer, and skin cancer (e.g., melanoma). Bone metastasis can be identified in up to 75% of patients with advanced breast and prostate cancer. Bone metastasis is associated with many significant clinical and quality of life consequences, including, but not limited to, intractable pain, pathological fractures, spinal cord and nerve compression, bone marrow infiltration, and movement disorders. In many cases, the systemic presence of cancer can also make the cancer incurable.

[0140] Osteosarcoma is the most common primary bone malignancy, accounting for 60% of all malignant pediatric bone tumors. Before multiagent chemotherapy, amputation only resulted in a long-term survival rate of approximately 20%. Since the 1970s, combination chemotherapy has been the main treatment for osteosarcoma, along with limb-sparing surgery. Currently, the 5-year survival rate for patients with osteosarcoma is reported to be 50%-80%. However, this survival rate has not improved in the past decade, and fully 40% of patients with osteosarcoma die from their disease.

[0141] Osteoporosis is a systemic skeletal disease characterized by reduced bone mass and deterioration of bone tissue microarchitecture, resulting in increased bone fragility and susceptibility to fracture.Hip, spine and wrist are the common bones that are broken or fractured in subjects with or at risk of osteoporosis, but any bone can be affected by osteoporosis.

[0142] Osteoporosis in postmenopausal white women is defined as a bone mineral density (BMD) value that is more than 2.5 SD below the youth mean, i.e., a T-score of 2.5 SD. Severe osteoporosis (established osteoporosis) uses the same threshold, but is accompanied by one or more previous fragility fractures. The preferred site for diagnostic purposes is the hip joint, BMD measurement performed at either the total hip or the femoral neck. For men, the same thresholds used for women are appropriate, since for any given BMD, the age-adjusted fracture risk is roughly the same.

[0143] Osteopenia is a pre-osteoporotic condition characterized by mild thinning of bone mass that is less severe than osteoporosis. Osteopenia occurs when bone formation is insufficient to offset normal bone loss. Osteopenia is generally considered the first step to osteoporosis. Decreased bone mineralization may also be referred to as osteopenia, whether or not osteoporosis is present. EXAMPLES

[0144] Working Example The following examples are put forward so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the compositions and methods, and are not intended to limit the scope of what the inventors regard as their invention. [Example 1] Materials and methods [Table 38-1] [Table 38-2]

[0145] [Table 39]

[0146] [Table 40-1] [Table 40-2]

[0147] These anti-Cx43Ab formulation development studies aimed to develop a viable and stable liquid formulation supporting long-term storage of anti-Cx43Ab drug product. Studies included pH / buffer screening, excipient and PS80 strength screening. The effect of buffer system, pH, excipients and PS80 on product stability was evaluated through freeze / thaw, agitation and accelerated stability studies.

[0148] pH / buffer screening studies showed that the anti-Cx43 Ab was more stable in 20 mM histidine / histidine hydrochloride buffer at pH 5.5 and 20 mM histidine / aspartate buffer at pH 5.5 compared to other buffer candidates examined.

[0149] Excipient and PS80 strength screening studies showed that anti-Cx43 Ab in histidine buffer containing sucrose was relatively more stable than those containing sodium chloride, sorbitol or glycine. The addition of PS80 significantly improved the stability of anti-Cx43 Ab at an optimal concentration of 0.02%, while the addition of EDTA showed a slight improvement in the stability of anti-Cx43 Ab.

[0150] Anti-Cx43 Ab at 25 mg / mL in 20 mM histidine / histidine hydrochloride at pH 5.5 with 8% sucrose and 0.02% (w / v) PS80 was selected for formulation validation studies. Sample number management rules Sample number: PPP-YYYYMMNN-X-CC-TT

[0151] PPP represents the numeric portion of the project name (this project is 2142). YYYY, MM and NN represent the year, month and serial number of the sample preparation for this month, respectively.

[0152] X represents the test conditions, e.g., FT and A represent freeze-thaw and agitation, respectively.

[0153] CC stands for test temperature, e.g. 05, 25 and 40 stand for 2-8°C, 25°C and 40°C, respectively.

[0154] TT represents the test time, for example, TO, 7D, 4W and 1M represent the start time, 7 days, 4 weeks and 1 month, respectively.

[0155] F represents the formulation number. For example, F1 and F2 represent formulation 1 and formulation 2, respectively.

[0156] For example, 2142-20180601-25-4W represented the first sample of the project anti-Cx43 Ab prepared in June 2018. Samples were stored upright at 25°C for 4 weeks. Analysis method exterior

[0157] The appearance of the samples, including transparency, color, and visible particles, was examined against black and white backgrounds using a YB-2 light box. pH

[0158] pH was measured using a Mettler Toledo S40 pH Meter. The pH meter was calibrated before use. Osmolality

[0159] Osmolality was measured using an Advanced 2020 Multi-Sample Osmometer using 20 μL of sample. Osmometer testing precision was confirmed using a 290 mOsmol / kg reference standard. MFI

[0160] For the analysis of sub-visible particles, a microflow imaging (MFI) system was used. The MFI test was performed with samples over 1.3 mL according to the user manual. The MFI data was analyzed with MVAS software. The final data was reported as total particle counts in different size ranges. particulate matter

[0161] The size and number of subvisible particles were measured under a laminar flow cabinet utilizing a HACH Particulate Analyzer. All samples were kept in the cabinet for at least 0.5 h before testing to avoid the introduction of air bubbles and interference during the examination. Each sample was tested four consecutive times, 1 mL each. Results were presented as the average number of particles ≥ 10 μm and ≥ 25 μm per mL (method according to the United States Pharmacopeia <788> (compatible with particulate matter in injectables). Protein concentration

[0162] Protein concentrations were determined by Thermo UV spectrophotometer. According to the Beer-Lambert law, the relationship between the absorbance value (A), protein concentration (c), optical path length (b) and extinction coefficient (ε) of a protein solution at a specific UV wavelength is given by the following equation: A=ε*b*c (A is the absorbance value, ε is the extinction coefficient, b is the optical path length, and c is the concentration). The extinction coefficient of anti-Cx43 Ab is 1.531AU*mL*mg -1 *cm -1 The UV absorption was measured at 280 nm using a Nanodrop2000 spectrophotometer. DSC

[0163] Differential scanning calorimetry (DSC) was utilized to measure the thermal stability of proteins by detecting the heat capacity of samples during heat flow. Specifically, DSC was used to measure the thermal transition midpoint (Tm) and onset of melting (Tmonset), which are indicators of the relative stability of proteins in solution. Samples were diluted to 1 mg / mL in reference buffer. An aliquot of 400 μL of reference buffer was added to each odd-numbered well of a 96-well plate, and an aliquot of 400 μL of each sample was added to the corresponding even-numbered well. Scanning temperatures ranged from 20 °C to 100 °C, and the scan rate was 200 °C / hr. Data analysis was performed using MicroCal VP Capillary DSC Automated data analysis software 2.0. mDSC

[0164] Modulated differential scanning calorimetry (mDSC) is performed, for example, by using a DSC-Q2000 system (TA instruments-Waters LLC). Tzero aluminum crucibles and Tzero aluminum lids, all from TA instruments, were used to contain the samples to be measured and the crucibles were sealed by a Tzero press. An empty Tzero crucible was similarly prepared and used as a reference. Approximately 10 μL of DS was added and transferred by a Tzero press to the Tzero crucible, which was pressed flat and sealed with a Tzero lid. A calibration scan program was run with equilibration at -60.00°C for 5 min and then at a constant temperature rate of 5.00°C / min up to 10.00°C. Data acquisition and processing were performed with the help of the Universal Analysis Software package. cIEF

[0165] The method of imaged capillary isoelectric focusing (iCIEF) separates proteins based on their charge differences in a pH gradient. Under an external electric field, charge variants of a monoclonal antibody migrate along a continuous pH gradient formed by an ampholyte additive. The charge variant stops where the pH is equal to its pI. The pI values ​​and relative abundances of the resolved peaks can be identified and quantified using software. A master mix was prepared in the following proportions (relative to the amount of one sample): 0.5 μL of pI 7.05 marker; 0.5 μL of pI 9.22 marker; 4 μL of Pharmalyte 3-10; 35 μL of 1% methylcellulose; 40 μL of H2O. The solution for one sample injection consisted of 20 μL of 1.0 mg / mL diluted sample and 80 μL of master mix. SDS-Caliper (reduced and non-reduced)

[0166] SDS-Caliper is a high-throughput chip-based method that separates proteins primarily by their molecular size. Before each sample was tested, it required pretreatment, such as incubation with sample buffer, SDS and N-ethylmaleimide (for non-reduced) or dithiothreitol (for reduced) at 70°C for 10 min. Then, the loading mix in a minimum volume of 42 μL (final protein concentration of 0.045 mg / mL) was tested by LabChip GXII Touch at excitation / emission wavelengths of 635 nm and 700 nm. The final results were analyzed by Empower software. Cation Exchange Chromatography (CEX)

[0167] CEX measures the charge heterogeneity of a monoclonal antibody solution by separating proteins according to differences in their net charge number in a buffer solution. Samples in low salt buffers at a pH below the isoelectric point have a net positive charge and adsorb to a negatively charged chromatography resin. A pH gradient is used to elute the different protein species based on their charge heterogeneity, with the most positively charged species binding most strongly and therefore requiring a higher pH. The different eluted charged species are detected by UV absorbance at 280 nm. The percentages of the main peak, acid peak and basic peak of the sample are calculated using a method of peak area normalization. The elution rate is determined by the following equation: . CEX was performed on an Agilent 1260 Series Infinity system and a propac WCX-10 column. The mobile phase A used here was 16 mM 2-methylpiperazine, 16 mM imidazole, 16 mM Tris, pH 5.0 ± 0.1. The mobile phase B was 16 mM 2-methylpiperazine, 16 mM imidazole, 16 mM Tris, 80 mM NaCl, pH = 10.9 ± 0.1. The flow rate was set at 1 mL / min. The sample was diluted to 1 mg / mL with mobile phase A and 100 μL of sample was eluted with a gradient of increasing amounts of mobile phase B. The detection wavelength was set at 280 nm. The run time was 60 min. CE-SDS (reduced and non-reduced)

[0168] Non-reducing capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) is a purity analysis method that separates proteins based on their electrophoretic mobility, with smaller sized proteins moving faster and larger sized proteins moving slower. In this method, a diluted protein sample is first alkylated by N-ethylmaleimide (NEM) to prevent heat-induced fragmentation, then denatured with SDS, before being injected into an uncoated capillary filled with a viscous SDS gel solution. Constituents of different molecular sizes in the protein sample are detected as they pass through the capillary equipped with a 220 nm PDA detector.

[0169] Briefly, non-reduced CE-SDS was performed using a Beckman Coulter PA800 Enhanced or PA800 Plus instrument equipped with a photodiode array detector. Samples were diluted to 4mg / mL with diluent (PB-CA) and then heated at 60°C for 10 minutes in the presence of 75μL of SDS sample buffer and 5μL of 100mM NEM for non-reduced CE-SDS. Samples were injected using -5kV for 20 seconds followed by separation at -15kV for 35 minutes. Detection was performed at 220nm.

[0170] Reduced capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) is a purity analysis method that separates proteins based on their electrophoretic mobility, with smaller sized proteins moving faster and larger sized proteins moving slower. In this method, a diluted protein sample is first denatured with SDS and then reduced with β-mercaptoethanol (BME) before being injected into an uncoated capillary filled with a viscous SDS gel solution. Constituents of different molecular sizes in the protein sample are detected as they pass through the capillary equipped with a 220 nm PDA detector.

[0171] Briefly, reduced CE-SDS was performed using a Beckman Coulter PA800 Enhanced or PA800 Plus instrument equipped with a photodiode array detector. Samples were diluted to 4 mg / mL with diluent (PB-CA) and then heated at 70° C. for 10 min in the presence of 75 μL of SDS sample buffer and 5 μL of 2-mercaptoethanol for reduced CE-SDS. Samples were injected for 20 s using −5 kV, followed by separation for 35 min at −15 kV. Detection was performed at 220 nm. SEC-HPLC

[0172] Size Exclusion Chromatography (SEC) is a purity analysis method that separates proteins based on their size. After separation, the relative percentages of HMW, monomeric and LMW species are quantified by UV detection. SEC was performed as follows: if the sample was greater than 10 mg / mL, it was diluted to 10 mg / mL with mobile phase before SEC analysis. 100 μg of sample was injected into an Agilent 1260 HPLC system equipped with a TSKgel G3000SWXL column (7.8×300 mm, particle size 5 μm) and a UV detector (detection wavelength: 280 nm). The mobile phase was 50 mM phosphate buffer containing 300 mM sodium chloride (pH 6.8±0.1). An isocratic gradient was applied for 20 min at a flow rate of 1 mL / min. [Example 2] pH / Buffer Screening

[0173] The pH / buffer screening study was to determine the optimal pH / buffer system for anti-Cx43Ab drug formulation. The ultimate goal of this study was to select one pH / buffer system with the greatest stabilizing capacity for anti-Cx43Ab drug for further formulation development studies.

[0174] Nine pH / buffer systems were designed based on the pI of the molecule and the application of the buffer system. Anti-Cx43 Ab DS (Lot: 2142S180507Y) formulated in 20 mM histidine / histidine hydrochloride buffer at pH 5.5 was generated from a 50 L pool. The DS was then exchanged into the nine prepared buffers by ultrafiltration centrifugation, respectively. The anti-Cx43 Ab concentration in this study was 25 mg / mL. Samples were stored at 25 ± 2 °C and 40 ± 2 °C for up to 4 weeks. Samples were collected at each time point in time and kept at 2-8 °C before analysis. Test items including appearance, pH, Conc_UV280, SEC-HPLC, cIEF, SDS-caliper (R&NR), and DSC were performed in this study. The sampling plan is listed in Table 1. Table 1. Study parameters from anti-Cx43 Ab pH / buffer screening. [Table 1] Note: x = appearance; SEC-HPLC; cIEF; SDS-Caliper (R&NR); y = DSC; z = pH; Conc_UV280; (opt) = as required.

[0175] Buffer exchange of anti-Cx43Ab DS was performed using an ultrafiltration centrifugation device (30,000 MWCO PES, VIVASPIN20). Nine pH / buffer systems were used to screen the optimal buffer system. Table 1 shows the detailed buffer systems. Multiple rounds of ultrafiltration were performed until the exchange rate was over 98%. The protein concentration was then adjusted to 25mg / mL using the corresponding pH / buffer system. Each sample was filtered through a 0.22μm filter (Millipore Express PES Membrane) and then distributed into 2R vials with 1mL / vial filling volume. The vials were stoppered, sealed and labeled immediately after filling. All filtration, filling and sealing operations were performed in a biosafety hood.

[0176] An appropriate number of vials for each pH / buffer system sample were placed in stability chambers at 25±2° C. and 40±2° C., respectively. Samples were taken and analyzed at predetermined time points.

[0177] Thermograms of anti-Cx43 Ab in different buffer systems are shown in Figure 1. Tm onset, the temperature at which the mAb starts to unfold, was taken as an indication of the overall thermal stability.

[0178] As shown in Table 2, the B2 and B8 samples had lower Tm onset than the other samples, which indicated that the thermal stability of anti-Cx43 Ab was not significantly affected by other pH / buffer systems except for B2 and B8. Table 2. DSC data from anti-Cx43 Ab pH / buffer screening studies. [Table 2]

[0179] NOTE: A5.0: 20 mM acetic acid / sodium acetate buffer at pH 5.0; HD-5.0: 20 mM histidine / aspartate buffer at pH 5.0; HD-5.5: 20 mM histidine / aspartate buffer at pH 5.5; C5.5: citric acid / sodium citrate buffer at pH 5.5; C6.0: citric acid / sodium citrate buffer at pH 6.0; H5.5: 20 mM histidine / histidine hydrochloride buffer at pH 5.5; H6.0: 20 mM histidine / histidine hydrochloride buffer at pH 6.0; H6.5: 20 mM histidine / histidine hydrochloride buffer at pH 6.5; P7.0: 20 mM sodium hydrogen phosphate / sodium dihydrogen phosphate buffer at pH 7.0.

[0180] The appearance, protein concentration and pH results of anti-Cx43 Ab in different buffer systems are summarized in Tables 3 and 4.

[0181] The concentration of the nine samples was approximately 25 mg / mL, and the pH values ​​were near the target pH. All samples were colorless, slightly milky, and free of visible particles at T0, but the milky level of samples B4, B5, and B9 was darker than the other samples. Due to the absence of PS80, all samples showed slightly visible particles after 2 weeks of storage at 25 ± 2 °C and 40 ± 2 °C.

[0182] This data suggested that the anti-Cx43 Ab was relatively more stable in the pH / buffer systems of B1, B2, B3, B6, B7 and B8 than the other candidates. Table 3. Protein concentration and pH results from pH / buffer screening studies. [Table 3] Table 4. Appearance results from pH / buffer screening studies. [Table 4] Note: A = colorless, slightly milky, no visible particles; B = colorless, slightly milky, slightly visible particles.

[0183] The SEC-HPLC results for all samples are shown in Table 5 and FIG.

[0184] All samples had comparable SEC purity at T0 with a main peak of approximately 97%. After 4 weeks of incubation at 25°C, the main peak purity of all samples showed no obvious decrease. After 2 weeks of storage at 40°C, a slight decrease in the main peak was observed. After 4 weeks of incubation at 40°C, the decrease in the main peak ranged from 0.4% to 2.4%. The distinction between samples was not significant except for B9. The purity decrease in the B9 sample was 2.4%.

[0185] The SEC data showed that the anti-Cx43 Ab was relatively more stable in B2, B3 and B6. Table 5. SEC-HPLC results from pH / buffer screening studies. [Table 5]

[0186] cIEF was used to determine the isoelectric point (pI) and charge variant distribution of anti-Cx43 Ab. The cIEF results for all samples are shown in Table 6 and Figure 3.

[0187] The pI values ​​of all samples were around 8.1 and did not change significantly under different conditions.

[0188] After 4 weeks of storage at 25±2° C., the main peak of all samples decreased slightly. The main peak of the B9 sample decreased by 9.7%, which was the largest among all samples.

[0189] After 4 weeks of storage at 40±2°C, the main peaks of all samples decreased significantly, along with a significant increase in the acidic peak. The main peaks of B5, B8 and B9 decreased to 42.6%, 46.6% and 18.5%, respectively. In contrast, the decrease in the main peaks of B3 and B4 was relatively less severe than that of the other samples.

[0190] The cIEF data showed that the anti-Cx43 Ab was relatively more stable in B3 and B4. Table 6. cIEF results from pH / buffer screening studies. [Table 6]

[0191] The SDS-caliper results for all samples are shown in Table 7, Figures 4 and 5.

[0192] After 4 weeks of storage at 25±2° C., there were no significant changes in non-reduced and reduced SDS-caliper purity for all samples.

[0193] After 4 weeks of storage at 40±2°C, the non-reduced SDS-caliper purity of B4, B5 and B9 decreased to 86.9%, 82.5% and 55.2%, respectively, which were greater than those of the other samples. The decrease in the main peak of B2 and B3 was relatively less than that of the other samples. The reduced SDS-caliper purity of all samples decreased slightly, except for B4, B5 and B9.

[0194] The SDS-caliper data showed that the anti-Cx43 Ab was relatively more stable in B2, B3 and B6. Table 7. SDS-caliper results from pH / buffer screening studies. [Table 7]

[0195] In this study, nine samples in various pH / buffer systems were designed and incubated at 25±2°C and 40±2°C. Based on all the results, the performance of B6 (20 mM histidine / histidine hydrochloride buffer at pH 5.5) and B2 (20 mM histidine / aspartic acid buffer at pH 5.0) was superior to the other samples. In conclusion, for further study, 20 mM histidine / histidine hydrochloride buffer at pH 5.5 (B6) is used as the leading pH / buffer system and 20 mM histidine / aspartic acid buffer at pH 5.0 (B2) is used as the backup pH / buffer system. [Example 3] Excipient and PS80 Strength Screening

[0196] The objective of the excipient and PS80 strength screening study was to identify the most stabilizing excipients and evaluate the optimal strength of PS80 for anti-Cx43 Ab in candidate buffer systems.

[0197] 20 mM histidine / histidine hydrochloride buffer at pH 5.5 (B6) was selected for combination studies of sodium chloride, sorbitol, glycine, sucrose, PS80 and EDTA addition. 20 mM histidine / aspartate buffer system at pH 5.0 (B2) was used as excipient and reserve buffer for PS80 strength screening studies. Eight formulations were designed as listed in Table 8. Table 8. List of formulation candidates from excipient and PS80 strength screening studies [Table 8-1] [Table 8-2] NOTE: H5.5: 20 mM histidine / histidine hydrochloride buffer at pH 5.5; HD-5.0: 20 mM histidine / aspartate buffer at pH 5.0.

[0198] The formulations were freeze / thawed (-40 ± 5 °C / RT) for 5 cycles, stirred at 300 rpm at 25 °C for 7 days, and stored at 2-8 °C, 25 ± 2 °C and 40 ± 2 °C for 4 weeks, respectively. Samples were collected at each time point in time and kept at 2-8 °C before analysis. Test items including appearance, pH, Conc_UV280, SEC-HPLC, cIEF, SDS-caliper (R&NR) and MFI were performed for this study. Table 9 shows the sampling conditions for excipient and PS80 strength screening study. Table 9. Sampling and testing plan from anti-Cx43 Ab excipient and PS80 strength screening [Table 9] NOTE: x = Appearance, pH, SEC-HPLC, cIEF, MFI, SDS-Caliper; z = Conc_UV280, Osmolality; () = as required.

[0199] Anti-Cx43 Ab DS (Lot: 2142S180507Y) formulated in 20 mM histidine / histidine hydrochloride buffer at pH 5.5 was produced from the 50 L pool. The components of each final target formulation were calculated and prepared as described in Table 8. Buffer exchange of anti-Cx43 Ab DS was performed using an ultrafiltration centrifugation device (30,000 MWCO PES, VIVASPIN20). Multiple ultrafiltrations were performed until the exchange rate was greater than 98%. The protein concentration was then adjusted to 25 mg / mL using the corresponding formulation buffer. Buffer exchange was performed using a 0.22 μm filter (Millipore Express PES Each formulation was filtered through a 500 mL / membrane and then dispensed into 6R vials with a 4 mL / vial fill volume. The vials were stoppered and sealed immediately after filling. All filtering, filling and sealing operations were performed in a biosafety hood.

[0200] The appropriate number of vials for each formulation were arranged and tested as described in Table 9. Samples were taken and analyzed at predetermined time points.

[0201] The appearance, protein concentration, osmolality and pH results from the freeze / thaw study are summarized in Table 10.

[0202] Protein concentrations and osmolality were all near target values ​​at T0.

[0203] The pH values ​​of the eight samples were all near the target value after five freeze / thaw cycles (-40±5°C / RT). The samples were all colorless, slightly milky, and free of visible particles at T0. Due to the absence of PS80, many visible particles were observed in the F7 sample after five freeze / thaw cycles (-40±5°C / RT). The milky level of the F1 sample became deeper after five freeze / thaw cycles (-40±5°C / RT).

[0204] This data suggested that anti-Cx43 Ab was relatively more stable in F2, F4, F5, F6 and F8. Table 10. Protein concentration, pH, osmolality and appearance results from freeze / thaw studies [Table 10] Note: A = colorless, slightly opalescent, no visible particles; C = colorless, slightly opalescent, many visible particles

[0205] The freeze / thaw MFI results are summarized in Table 11.

[0206] The particle number in F7 was much higher than the others at T0 and after five freeze / thaw cycles (-40±5°C / RT). Table 11. MFI results from freeze / thaw studies [Table 11-1] [Table 11-2]

[0207] The SEC-HPLC results for all formulations are listed in Table 12 and FIG.

[0208] At TO, all formulations had similar SEC purity with the main peak at approximately 97.5%. After five freeze / thaw cycles (-40±5°C / RT), all formulations had comparable SEC main peak purity of approximately 97.5%, except for the F3 sample, which showed a slightly higher decrease in main peak purity at 8.1%. Table 12. SEC-HPLC results from freeze / thaw studies. [Table 12]

[0209] The cIEF results for all formulations are listed in Table 13 and FIG.

[0210] The pI values ​​of all samples were around 8.1 and did not change significantly after five freeze / thaw cycles (-40±5°C / RT).

[0211] Compared to TO, the proportions of the main peak, acidic peak and basic peak also had no significant changes for all samples through five freeze / thaw cycles (-40±5°C / RT). [Table 13-1] [Table 13-2]

[0212] The SDS-caliper data for all formulations is summarized in Table 14 and FIG.

[0213] All preparations showed comparable purity after five freeze / thaw cycles (-40±5° C. / RT) by either non-reduced or reduced SDS-caliper. Table 14. SDS-caliper results from freeze / thaw studies [Table 14]

[0214] The appearance, protein concentration, osmolality and pH value results of the agitation study are summarized in Table 15.

[0215] Protein concentrations and osmolality were all near target values ​​at T0.

[0216] All formulations, except F7, remained stable in pH value and appearance after 7 days of stirring at 300 rpm and 25° C. Numerous visible particles were observed in the F7 sample after 7 days of stirring at 300 rpm and 25° C. The level of milkiness in the F1 sample became deeper after 7 days of stirring at 300 rpm and 25° C. Table 15. Protein concentration, pH value, osmolality and appearance results from agitation studies. [Table 15-1] [Table 15-2] Note: A = colorless, slightly milky, no visible particles; C = colorless, slightly milky, many visible particles.

[0217] The MFI data for all samples is listed in Table 16.

[0218] The particle count of F7 was slightly higher than the others at TO. After 7 days of stirring at 300 rpm at 25°C, the particle count in F7 increased significantly due to the absence of PS80. Except for F7, all other samples had similar particulate counts and no growth trend was observed. Table 16. MFI results from agitation studies [Table 16]

[0219] The SEC-HPLC results for all formulations are listed in Table 17 and FIG.

[0220] After 7 days of stirring at 300 rpm at 25° C., all formulations had similar SEC main peak purity of >97%. Table 17. SEC-HPLC results from the stirring study. [Table 17]

[0221] The cIEF results for all formulations are listed in Table 18 and FIG.

[0222] The pI values ​​of all samples were around 8.1 and did not change significantly after 7 days of stirring at 300 rpm and 25°C.

[0223] After 7 days of stirring at 300 rpm at 25° C., the main peak purity of all formulations remained stable. Table 18. cIEF results from agitation studies [Table 18]

[0224] The SDS-caliper results for all formulations are listed in Table 19 and FIG.

[0225] All formulations showed comparable purity in either non-reduced or reduced SDS-caliper after 7 days of stirring at 300 rpm at 25°C. Table 19. SDS-caliper results from agitation studies. [Table 19]

[0226] The appearance, protein concentration, osmolality and pH value results for the accelerated stability study are summarized in Tables 20 and 21.

[0227] Protein concentrations and osmolality were all near target values ​​at T0.

[0228] After 4 weeks of storage at 2–8°C, 25 ± 2°C or 40 ± 2°C, the pH value remained unchanged for all formulations, but slightly visible particles were found in F7 due to the absence of PS80. Table 20. Appearance results from accelerated stability studies [Table 20]

[0229] Note: A = colorless, slightly milky, no visible particles; B = colorless, slightly milky, slightly visible particles. Table 21. Protein concentration, osmolality and pH results from accelerated stability studies. [Table 21]

[0230] The MFI data for all samples is listed in Table 22.

[0231] After 4 weeks of storage at 2-8°C and 25±2°C, there was no apparent change in the number of subvisible particles in all formulations.

[0232] After 4 weeks of storage at 40±2° C., the increase in subvisible particle count (ECD≧10 μm and ECD≧25 μm) in F7 was much higher than that in the other formulations, and there was a slight trend toward increased particle count for F7. Table 22. MFI results from accelerated stability studies [Table 22-1] [Table 22-2]

[0233] The SEC-HPLC data for all samples is listed in Table 23 and FIG.

[0234] After storage at 2-8°C or 25±2°C for 4 weeks, there was no obvious change in main peak purity in all formulations.

[0235] A significant decrease in the main peak was observed after 2 weeks at 40±2°C. After 4 weeks of storage at 40±2°C, the decrease in the main peak was in the range of 0.3% to 6.6%. The decrease in the main peak purity in F1 and F6 was 6.6% and 3.0%, respectively. In contrast, the decrease in the main peak in F5 and F8 was relatively milder than that in the other formulations. Table 23. SEC-HPLC results from accelerated stability studies [Table 23-1] [Table 23-2]

[0236] The cIEF data for all samples is listed in Table 24 and FIG.

[0237] The pI values ​​of all samples were approximately 8.1 and did not change significantly after storage at 2-8°C, 25±2°C or 40±2°C.

[0238] After 4 weeks of storage at 2-8°C or 25±2°C, there was no significant change in the main peak purity for all formulations.

[0239] After 4 weeks of storage at 40 ± 2°C, the main peak purity of all samples decreased significantly, along with a significant increase in the acidic peak. There was no significant difference in the main peak percentage of all samples, and the decrease in the main peak was within the range of 15.1% to 21.1%. Table 24. cIEF results from accelerated stability studies. [Table 24-1] [Table 24-2]

[0240] The SDS-caliper data for all samples is listed in Table 25, Figure 14 and Figure 15.

[0241] After 4 weeks of storage at 2-8°C or 25±2°C, all formulations showed comparable purity in non-reduced and reduced SDS-calipers.

[0242] After 4 weeks of storage at 40±2° C., the purity of all formulations decreased significantly in non-reduced and reduced SDS-caliper. The decrease in non-reduced purity in F1 and F3 was 7.2% and 7.3%, which was the largest decrease in all formulations. The decrease in reduced purity in F1, F3 and F6 was 2.7%, 2.3% and 2.2%, respectively. The decrease in non-reduced or reduced SDS-caliper purity in F2, F5 and F7 was relatively lower than the other formulations. Table 25. SDS-Caliper results from accelerated stability studies [Table 25-1] [Table 25-2]

[0243] After five freeze / thaw cycles (-40±5°C / RT), anti-Cx43 Ab in all formulations had no significant differences in protein concentration, pH value, osmolality and purity (SDS-caliper reduced and non-reduced). The visible and subvisible particle counts (MFI) in F7 (without PS80) were much higher than the other formulations. The SEC main peak of F3 sample showed a slightly higher reduction at 8.1%. The milky color level of F1 sample became deeper after five freeze / thaw cycles (-40±5°C / RT).

[0244] After 7 days of stirring at 300 rpm at 25°C, anti-Cx43 Ab in all formulations had no significant differences in protein concentration, pH value, osmolality and purity (SEC-HPLC, cIEF, SDS-caliper reduced and non-reduced). The visible and subvisible particle counts (MFI) in F7 (without PS80) were much higher than the other formulations. The milky level of F1 sample became deeper after 7 days of stirring at 300 rpm at 25°C.

[0245] After 4 weeks of storage at 2-8°C, anti-Cx43 Ab in all formulations showed no significant differences in protein concentration, pH value, osmolality, subvisible particles and purity (SEC-HPLC, cIEF, SDS-caliper reduced and non-reduced). After 4 weeks of storage at 2-8°C, only slightly visible particles were seen in F7.

[0246] After 4 weeks of storage at 25±2°C, anti-Cx43Ab in all formulations showed no significant differences in protein concentration, pH value, osmolality, subvisible particles and purity (SEC-HPLC, SDS-caliper reduced and non-reduced). Furthermore, due to the absence of PS80, only a few visible particles were observed in formulation F7. The cIEF main peak of all samples was slightly reduced, but no significant differences were observed among the eight formulations after 4 weeks of storage at 25±2°C.

[0247] After 4 weeks of storage at 40±2°C, anti-Cx43Ab in all formulations had no significant differences in protein concentration, pH value and osmolality. Due to the absence of PS80, slightly visible particles were observed in formulation F7. The increase in the number of subvisible particles (ECD ≥ 10 μm and ECD ≥ 25 μm) in F7 was much higher than that in the other formulations. The purity (SEC-HPLC, cIEF, SDS-caliper reduced and non-reduced) of all samples decreased significantly. The decrease in the SEC main peak in F1 and F6 was much higher than that in the other formulations. The decrease in the SEC main peak in F5 and F8 was relatively milder than that in the other formulations. There was no significant difference in the percentage of cIEF main peak in all samples, and the decrease in the main peak was within the range of 15.1% to 21.1%. The decrease in SDS-caliper purity (non-reduced) in F1 and F3 was higher than that in the other formulations. F1 showed a higher decrease in SDS-caliper purity (reduced) than the others. The decrease in non-reduced or reduced SDS-caliper purity in F2, F5 and F7 was relatively lower than the other formulations.

[0248] In summary, formulation development studies including pH / buffer screening, excipient and PS80 strength screening were performed to determine the top formulation.

[0249] In the pH / buffer screen, the histidine / histidine hydrochloride buffer system showed the best ability for protein stabilization.

[0250] In the excipient and PS80 strength screening, sodium chloride, sorbitol, glycine and sucrose (F1, F2, F3 and F4) were selected to investigate their stabilizing ability for anti-Cx43Ab. The results suggested that anti-Cx43Ab was relatively more stable in histidine buffer with sucrose as an excipient. The stability data of samples containing different concentrations of PS80 (F4, F6 and F7) showed that F4 (containing 0.02% PS80) provided better stabilization for anti-Cx43Ab than F6 and F7 (containing 0% or 0.05% PS80, respectively). Based on the results of the EDTA study (F4 and F5), EDTA did not provide any further stabilization of anti-Cx43Ab.

[0251] Finally, 25 mg / mL anti-Cx43 Ab in 20 mM histidine / histidine hydrochloride, pH 5.5, containing 8% sucrose and 0.02% (w / v) PS80 was considered the lead formulation for formulation validation studies. [Example 4] Formulation verification study

[0252] A validation study of the anti-Cx43 Ab formulation was performed to confirm the stability of the selected formulation using the final process DS. Conditions evaluated in the validation study include long-term storage conditions, accelerated conditions, stress conditions, freeze / thaw and agitation. The formulation selected from the formulation screening study was 25 mg / mL anti-Cx43 Ab in 20 mM histidine / histidine hydrochloride buffer at pH 5.5 containing 8% (w / v) sucrose and 0.02% (w / v) PS80. Table 26. Study parameters from anti-Cx43Ab formulation validation study [Table 26] Note: x=Appearance, pH, Osmolarity, Conc_UV280, SEC-HPLC, CEX, CE-SDS(R&NR), HIAC;y=mDSC

[0253] The leading formulation was evaluated in a formulation validation study. The mAb material (first 15 L DS) was formulated as 20 mM histidine / histidine hydrochloride buffer at pH 5.5 containing 25 mg / ml protein, 8% sucrose, and 0.02% (w / v) PS80. In a biosafety hood, the formulated DS was filtered using a 0.22 μm PVDF filter and filled into 6 mL glass vials (6.0 mL / vial), stoppered, and sealed.

[0254] An appropriate number of vials were placed in a 2-8°C refrigerator, 25°C and 40°C stability chambers, respectively. Meanwhile, vials were frozen in a -40°C freezer for 5 cycles, thawed at room temperature or fixed in a thermostatic shaker at 100 rpm for 7 days at 25°C. Samples were collected and analyzed at the designated time points.

[0255] The thermogram of anti-Cx43Ab mAb in the final formulation is shown in Figure 16. The Tg' onset, the temperature at which the sample begins to undergo glass translation, was taken as an indication for the formation of a glassy state. The Tg' onset for anti-Cx43Ab was -31.61°C. Table 27. mDSC data from anti-Cx43Ab formulation validation study [Table 27]

[0256] The visual appearance, protein concentration, pH and osmolality results of the freeze / thaw and agitation studies are summarized in Table 28.

[0257] After 5 freeze / thaw cycles (-40±5°C / RT) and 7 days of agitation, there were no obvious changes in appearance, protein concentration, pH and osmolality. All samples appeared colorless, slightly milky and free of visible particles. No obvious changes were observed in protein concentration and all results were within the specification of 25.0±2.5mg / mL. No obvious changes were observed in pH and osmolality tests compared to T0. Table 28. Appearance, protein concentration, pH and osmolality results from freeze / thaw and agitation studies. [Table 28] Note: A = colorless, slightly milky, no visible particles.

[0258] Particulate matter results from the freeze / thaw and agitation studies are summarized in Table 29. No trend toward increased particle counts (ECD > 10 μm and ECD > 25 μm) was observed after five freeze / thaw cycles (-40 ± 5 °C / RT) and 7 days of agitation at 25 °C. Table 29. HIAC data from freeze / thaw and agitation studies. [Table 29]

[0259] The SEC-HPLC results of the freeze / thaw and agitation studies are summarized in Table 30. No obvious changes were observed after five freeze / thaw cycles. A slight decrease (1.2%) in SEC main peak purity was observed after 7 days of agitation (100 rpm) at 25° C. Table 30. SEC data from freeze / thaw and agitation studies. [Table 30]

[0260] The CE-SDS (NR and R) results of the freeze / thaw and agitation studies are summarized in Table 31. No obvious changes in CE-SDS-NR and CE-SDS-R purity were observed after five freeze / thaw cycles. After 7 days of agitation (100 rpm) at 25° C., a slight decrease in CE-SDS-NR purity (1.0%) and CE-SDS-R purity (2.1%) was observed. Table 31. CE-SDS data from freeze / thaw and agitation studies. [Table 31]

[0261] The CEX results of the freeze / thaw and agitation studies are summarized in Table 32. There was no obvious change in the percentage of the main peak, acidic peak and basic peak after five freeze / thaw cycles (-40±5°C / RT) compared to TO. A significant decrease in the main peak (up to 17.9%) was observed in samples agitated (100 rpm) at 25°C for 7 days. Table 32. CEX data from freeze / thaw and agitation studies. [Table 32-1] [Table 32-2]

[0262] The results of appearance, protein concentration, pH and osmolality for different storage conditions are summarized in Table 33. All samples were free of visible particles under different storage conditions except for one sample kept at 2-8°C for 1 month which appeared to contain some visible particles by chance. The color of the sample changed slightly to yellow at 25°C-1 month / 2 months / 3 months and 40°C-2 weeks / 4 weeks. No obvious changes were observed in protein concentration, pH and osmolality compared to T0, which were all within the specifications. Table 33. Appearance, protein concentration, pH and osmolality results from stability studies [Table 33] Note: A = colorless, slightly milky, no visible particles; B = slightly yellow, slightly milky, no visible particles; C = colorless, slightly milky, visible particles.

[0263] The HIAC results of samples under different storage conditions are summarized in Table 34. There was no obvious change in the subvisible particle count at 2-8°C, 25±2°C and 40±2°C for 2 weeks. The data generated at 2-8°C for 1 month was for reference for the generation of visible particles. Table 34. HIAC data from stability studies [Table 34]

[0264] The SEC-HPLC results of samples under different storage conditions are summarized in Table 35. For samples incubated at 2-8°C, a slight decrease in the main peak was observed after 3 months of storage (the decrease in the main peak was equal to 1.1%). For samples incubated at 25°C, a slight decrease in the main peak was observed after 3 months of storage (the decrease in the main peak was equal to 2.6%). For samples incubated at 40°C, a significant decrease in the main peak was observed after 4 weeks of storage (the decrease in the main peak was equal to 5.1%). Table 35. SEC data from stability studies [Table 35]

[0265] The CE-SDS (NR&R) results of samples under different storage conditions are shown in Table 36. In samples incubated at 2-8°C, a decrease in CE-SDS purity was observed after 3 months of storage (the decrease in the CE_NR main peak was equal to 1.3% and the decrease in the CE_R main peak was equal to 2.5%). In samples incubated at 25°C, a decrease in CE-SDS purity was observed after 3 months of storage (the decrease in the CE_NR main peak was equal to 6.1% and the decrease in the CE_R main peak was equal to 8.0%). In samples incubated at 40°C, a decrease in CE-SDS purity was observed after 4 weeks of storage (the decrease in the CE_NR main peak was equal to 12.8% and the decrease in the CE_R main peak was equal to 5.8%). Table 36. CE-SDS data from stability studies [Table 36]

[0266] The CEX results of samples under different storage conditions are shown in Table 37. For samples incubated at 2-8°C, a decrease in the main peak was observed after 3 months of storage (CEX main peak decrease was equal to 20.4%). For samples incubated at 25°C, a decrease in the main peak was observed after 3 months of storage (CEX main peak decrease was equal to 20.0%). For samples incubated at 40°C, a decrease in the main peak was observed after 4 weeks of storage (CEX main peak decrease was equal to 30.6%). Table 37. CEX data from stability studies [Table 37]

[0267] After five freeze / thaw cycles (-40±5°C / RT), anti-Cx43 Ab in selected formulations showed no significant changes in appearance, protein concentration, pH value, osmolality and purity (SEC-HPLC, CEX-HPLC, CE-SDS reduced and non-reduced).

[0268] After 7 days of stirring at 25° C., the anti-Cx43 Ab in the selected formulations showed no significant differences in appearance, protein concentration, pH value, or osmolality. The purity of the selected formulations (SEC-HPLC, CEX-HPLC, CE-SDS reduced and non-reduced) decreased slightly.

[0269] After storage for 3 months at 2-8 °C, anti-Cx43 Ab in the selected formulations did not change significantly in appearance, protein concentration, pH value, osmolality and particulate matter. The purity (SEC-HPLC, CEX-HPLC, CE-SDS reduced and non-reduced) of the selected formulations decreased slightly.

[0270] After 3 months of storage at 25±2°C, anti-Cx43 Ab in selected formulations showed no significant changes in protein concentration, pH value, osmolality and particulate matter. The color of the samples changed slightly to yellow. The purity (SEC-HPLC, CEX-HPLC, CE-SDS reduced and non-reduced) of selected formulations decreased after 3 months of storage at 25±2°C.

[0271] After 4 weeks of storage at 40±2°C, anti-Cx43 Ab in selected formulations showed no significant changes in protein concentration, pH value, osmolality and particulate matter. The color of the samples changed slightly to yellow. The purity (SEC-HPLC, CEX-HPLC, CE-SDS reduced and non-reduced) of selected formulations decreased.

[0272] Based on confirmatory study data, -20°C was recommended as the DP storage condition.

[0273] In summary, 25 mg / mL protein in 20 mM histidine / histidine hydrochloride buffer at pH 5.5 containing 8% sucrose and 0.02% (w / v) PS80 was considered as the formulation for anti-Cx43 Ab. According to the validation study data, -20°C was recommended as the DP storage condition. Modifications

[0274] Modifications and variations of the described methods and compositions of the present disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the present disclosure.Although the present disclosure has been described in connection with specific embodiments, it should be understood that the present disclosure claimed should not be unduly limited to such specific embodiments.Indeed, various modifications of the described modes for carrying out the present disclosure are contemplated and will be understood by those skilled in the relevant field to which the present disclosure pertains, within the scope of the present disclosure, as expressed by the following claims. INCORPORATION BY REFERENCE

[0275] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each individual patent and publication was specifically and individually indicated to be incorporated by reference. The present invention provides, for example, the following items. (Item 1) 1. A pharmaceutical formulation comprising: An anti-Cx43 antibody or an antigen-binding fragment thereof; A buffer; A surfactant, Stabilizers and Including, The pharmaceutical formulation has a pH of about 5 to about 6; The anti-Cx43 antibody or antigen-binding fragment thereof a first, second and third heavy chain complementarity determining region (CDR) sequence having the amino acid sequences of SEQ ID NOs: 1, 2 and 3, respectively; a first, second and third light chain CDR sequence having the amino acid sequences of SEQ ID NOs: 4, 5 and 6, respectively; 13. A pharmaceutical formulation comprising: (Item 2) The pharmaceutical formulation of item 1, wherein the anti-Cx43 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain having the amino acid sequence of SEQ ID NO: 8. (Item 3) 3. The pharmaceutical formulation according to item 2, wherein the anti-Cx43 antibody or antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 to 17, and a light chain having the amino acid sequence of SEQ ID NO: 18. (Item 4) 4. The pharmaceutical formulation according to any one of items 1 to 3, wherein the anti-Cx43 antibody or antigen-binding fragment thereof binds to an epitope located within the amino acid sequence of FLSRPTEKTI (SEQ ID NO: 19). (Item 5) 5. The pharmaceutical formulation according to item 4, wherein the epitope comprises one or more amino acids selected from the group consisting of F1, S3, R4, P5, T6, E7, K8, T9 and I10 of SEQ ID NO: 19, or consists of F1, S3, R4, P5, T6, E7, K8, T9 and I10 of SEQ ID NO: 19. (Item 6) The epitope comprises all ten amino acids of SEQ ID NO: 19, or 5. The pharmaceutical formulation according to item 4, consisting of all ten amino acids. (Item 7) 7. The pharmaceutical formulation according to any one of items 1 to 3 and 5 to 6, wherein the anti-Cx43 antibody or antigen-binding fragment thereof is present at a concentration of about 5 to about 50 mg / mL, or about 10 to about 40 mg / mL, or about 15 to about 30 mg / mL. (Item 8) 7. The pharmaceutical formulation according to any one of items 1 to 3 and 5 to 6, wherein the buffer is selected from acetic acid / sodium acetate, histidine / aspartic acid, citric acid / sodium citrate, sodium hydrogen phosphate / sodium dihydrogen phosphate and histidine / histidine hydrochloride. (Item 9) 9. The pharmaceutical formulation according to item 8, wherein the buffer is histidine / aspartic acid or histidine / histidine hydrochloride. (Item 10) 10. The pharmaceutical formulation according to item 9, wherein the buffer is histidine / histidine hydrochloride. (Item 11) 10. The pharmaceutical formulation according to any one of items 1-3, 5-6 and 8-9, wherein the surfactant is polysorbate 80 (PS80). (Item 12) 10. The pharmaceutical formulation according to any one of items 1-3, 5-6 and 8-9, wherein the stabilizer is selected from ethylenediaminetetraacetic acid (EDTA), sodium chloride, sorbitol, glycine and sucrose. (Item 13) 13. The pharmaceutical formulation according to item 12, wherein the stabilizer is sucrose. (Item 14) 14. The pharmaceutical formulation according to any one of items 1 to 3, 5 to 6, 8 to 9 and 13, wherein the pH is from about 5.4 to about 5.6. (Item 15) 14. The pharmaceutical formulation according to any one of items 1-3, 5-6, 8-9 and 13, wherein the formulation is an aqueous formulation. (Item 16) 1. A pharmaceutical formulation comprising: About 10 to 50 mg / mL or about 25 mg / mL of an anti-Cx43 antibody or antigen-binding fragment thereof that binds to an epitope located within the amino acid sequence of FLSRPTEKTI (SEQ ID NO: 19); about 10-40 mM, or about 20 mM histidine / histidine hydrochloride buffer; about 0.005% to 0.05%, or about 0.02% w / v polysorbate 80; Approximately 1% to 20% w / v or approximately 8% w / v sucrose Including, The pharmaceutical formulation, wherein the formulation has a pH of about 5.4 to about 5.6, or about 5.5. (Item 17) 1. A pharmaceutical formulation comprising: About 25 mg / mL of an anti-Cx43 antibody or an antigen-binding fragment thereof comprising a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 to 17 and a light chain having the amino acid sequence of SEQ ID NO: 18; Approximately 20 mM histidine / aspartic acid buffer; Approximately 0.02% w / v polysorbate 80; Approximately 8% w / v sucrose Including, The pharmaceutical formulation, wherein the formulation has a pH of about 5.4 to about 5.6, or about 5.5. (Item 18) A pharmaceutical formulation according to any one of items 1, 16 and 17 for use in promoting the opening of Cx43 hemichannels in bone cells and, optionally, for the treatment of cancer, cancer metastasis, osteosarcoma, osteoporosis or osteopenia. (Item 19) Use of a pharmaceutical preparation according to any one of items 1, 16 and 17 for promoting the opening of Cx43 hemichannels in bone cells, and optionally for the treatment of cancer, cancer metastasis, osteosarcoma, osteoporosis or osteopenia.

Claims

[Claim 1] The invention described in the specification.

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