Antibody pharmaceutical compositions and their preparation and use

Abstract

The present disclosure provides a stable pharmaceutical formulation comprising: an antibody comprising a polypeptide having a canine CH2, CH3, IgG Fc region variant or a canine FcRn binding region thereof; a buffer having a pH in the range of 4.5 to 7.0; an osmotic pressure regulator and / or stabilizer; a chelating agent; and a surfactant; wherein the formulation can maintain stability up to twenty-four months in solution and is suitable for oral, rectal, transmucosal, intestinal or parenteral administration.

Description

[0001] Citation of relevant applications

[0002] This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 503,659, filed May 22, 2023, which is hereby incorporated by reference in its entirety.

[0003] field The present invention generally relates to stable pharmaceutical formulations of pharmacologically active antibodies (including bispecific antibodies, trispecific antibodies, etc.) or antibody fragments or domains for administration to subjects in need, said antibodies comprising a polypeptide having a complementarity-determining region (CDR), CH2, CH3, IgG Fc region variant or its canine or cat FcRn binding region of a canine or cat variable antibody domain, or a mixture of these antibody molecules. Background Technology

[0004] Currently, to maintain stability over a product shelf life of at least two years, many antibodies are offered in lyophilized or freeze-dried formulations. Lyophilization or freeze-drying helps prevent physical and chemical reactions in the polypeptide chains by removing or reducing water in the formulation, thus avoiding instability during long-term storage. Lyophilized antibody formulations have several limitations, including a long lyophilization process leading to high manufacturing costs. Furthermore, lyophilized formulations must be aseptically and accurately reconstituted by professionals before administration to subjects.

[0005] Antibodies are also available in liquid formulations at concentrations comparable to or higher than those of the reconstituted lyophilized formulation, eliminating the need for reconstitution before administration. This allows users and practitioners to administer antibodies to subjects more quickly and easily. However, liquid antibody formulations have a shorter shelf life, and antibodies may lose biological activity due to chemical and physical instability during storage. Factors contributing to chemical instability include, but are not limited to, deamidation, racemization, hydrolysis, oxidation, β-elimination, or disulfide exchange. Factors contributing to physical instability include, but are not limited to, antibody denaturation, aggregation, particle formation, precipitation, and adsorption. Among these factors, aggregation, deamidation, and oxidation are known to be the most common causes of antibody degradation (Wang et al., 1988). J. of Parenteral Science & Technology 42 (Supplement) S4-S26; Cleland et al., 1993 Critical Reviews in Therapeutic Drug Carrier Systems 10(4): 307-377).

[0006] For proteins, and especially for antibodies, to maintain biological activity, formulations must retain the conformational integrity of at least the core amino acid sequence of the protein, while protecting multiple functional groups from degradation. Protein degradation pathways can involve chemical instability (i.e., any process involving modification of the protein through bond formation or breakage to create new chemical entities) or physical instability (i.e., changes in the protein's higher-order structure). For a general review of the stability of protein drugs and the factors affecting stability, see, for example, Manning et al. (1989). Pharmaceutical Research 6:903-918 (the contents of which are incorporated herein by reference in their entirety). Furthermore, stability must be maintained when the formulation does not include a carrier peptide.

[0007] While the possibility of protein instability is generally recognized, specific instability issues for particular proteins cannot be predicted. Any one or a combination of these instabilities can lead to the formation of peptide byproducts or derivatives, which can have adverse effects such as reduced activity or efficacy, increased toxicity, and / or increased immunogenicity. In fact, peptide precipitation can lead to thrombosis and / or dosage form heterogeneity, and particle formation can induce undesirable immune responses in subjects receiving peptides. Therefore, the safety, efficacy, and immunogenicity of any peptide drug formulation are directly related to its stability.

[0008] Therefore, there remains a need for formulations that not only maintain the stability and bioactivity of bioactive peptides during storage and delivery, but are also suitable for various therapeutic routes of administration.

[0009] Therefore, this article provides antibody formulation compositions suitable for presentation in single- or multi-dose vials or syringes, which provide acceptable product quality and stability in a liquid state and have a shelf life of more than 24 months. Summary of the Invention

[0010] According to the purposes of the present invention, as embodied and broadly described herein, in one aspect, the present invention relates to a stable pharmaceutical formulation comprising: (a) 5 to 100 mg / ml of an antibody or antibody fragment or domain comprising a polypeptide having a canine or feline variable antibody domain CDR, CH2, CH3, an IgG Fc region variant, or a canine or feline FcRn binding region thereof; (b) 5 to 50 mM of a buffer having a pH in the range of 4.0 to 7.2; (c) 0.01 to 35% (w / v) of an osmotic pressure regulator and / or stabilizer; and (d) 0.002 to 10% (w / v) of a surfactant; wherein the formulation is a single-dose or multi-dose formulation, and wherein the formulation maintains stability in solution for up to twenty-four months.

[0011] In one embodiment, the formulation further comprises a chelating agent of 0.01 to 5.0 mM. In another embodiment, the multi-dose formulation further comprises an antimicrobial preservative at a concentration of about 0.1% to 1.5% (wv), said antimicrobial preservative being selected from the group consisting of benzyl alcohol, phenol, m-cresol, benzalkonium chloride, benzalkonium chloride, phenoxyethanol, and methylparaben, or mixtures thereof. In a specific embodiment, the antimicrobial preservative is benzyl alcohol.

[0012] In one embodiment, the buffer is selected from the group consisting of succinate, histidine, histidine hydrochloride (HCl), phosphate, Tris, diethanolamine, citrate, acetate, other organic acids, and mixtures thereof. The buffer is preferably acetate, histidine, or histidine hydrochloride. In another embodiment, the buffer is present at a concentration of 5 to 50 mM and maintained at a physiologically suitable pH in the range of pH 4.0 to pH 7.2.

[0013] In another embodiment, the osmotic pressure regulator and / or stabilizer is selected from the group consisting of: CaCl2, NaCl, MgCl2, lactose, sorbitol, sucrose, mannitol, trehalose, raffinose, polyethylene glycol, hydroxyethyl starch, cyclodextrin, glycine or other stable amino acids (including proline, arginine, glutamine, etc.), and mixtures thereof. In an alternative embodiment, the osmotic pressure regulator and / or stabilizer is sucrose, trehalose, or sorbitol. In one embodiment, the osmotic pressure regulator and / or stabilizer is present at a concentration of 0.01% to 35% (w / v); while in an alternative embodiment, the osmotic pressure regulator and / or stabilizer may be present at 0.01% to 15% (w / v).

[0014] In one embodiment, the chelating agent is selected from the group consisting of: aminopolycarboxylic acids, hydroxyaminocarboxylic acids, N-substituted glycine, 2-(2-amino-2-oxoethyl)aminoethanesulfonic acid (BES), deferoxamine (DEF), citric acid, nicotinamide, deoxycholate, diethylenetriaminepentaacetic acid (DTPA), hypozinotriacetic acid (NTA), N-2-acetamido-2-iminodiacetic acid (ADA), bis(aminoethyl)ethylene glycol ether, N,N,N′,N′-tetraacetic acid (EGTA), trans-diaminocyclohexanetetraacetic acid (DCTA), glutamic acid and aspartic acid, N-hydroxyethyliminodiacetic acid (HIMDA), N,N-bishydroxyethylglycine (bicine), and N-(tris(hydroxymethyl)methyl) 10. Glycine, glycylglycine, sodium deoxycholate, ethylenediamine, propylenediamine, diethylenetriamine, triethylenetetraamine, ethylenediaminetetraacetic acid (EDTA), disodium EDTA, calcium EDTA, oxalic acid, malate, citric acid, citric acid monohydrate and trisodium citrate dihydrate, 8-hydroxyquinoline salt, amino acids, histidine, cysteine, methionine, peptides, polypeptides and proteins, and mixtures thereof. In another embodiment, the chelating agent is EDTA, disodium EDTA or calcium EDTA. The concentration of the chelating agent is from 0.01 to 5.0 mM. In an alternative embodiment of the formulation according to the invention, the chelating agent is present at a concentration of from 0.1 to about 2.0 mM.

[0015] In yet another embodiment, the surfactant is selected from the group consisting of: polysorbate, poloxamer, triaton, sodium lauryl sulfate, sodium lauryl sulfate, sodium octyl glucoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl - Betaine, cetyl-betaine, lauramidopropyl-betaine, cocamidopropyl-betaine, linoleamide-propyl-betaine, myristamidopropyl-betaine, palmamidopropyl-betaine, isostearamidopropyl-betaine, myristamidopropyl-dimethylamine, palmamidopropyl-dimethylamine, isostearamidopropyl-dimethylamine, sodium methylcocoyl taurate, disodium methyloleoyl taurate, dihydroxypropyl PEG 5 linoleamide chloride, polyethylene glycol, polypropylene glycol, and mixtures thereof. In another embodiment, the polysorbate is selected from the group consisting of: polysorbate 20, polysorbate 21, polysorbate 40, polysorbate 60, polysorbate 61, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85, and mixtures thereof. The concentration of the surfactant is from 0.002 to 1.0% (w / v).

[0016] In yet another embodiment, the pharmaceutical formulation further comprises an antioxidant selected from the group consisting of: GLA (gamma-linolenic acid)-lipoic acid; DHA (docosahexaenoic acid)-lipoic acid; GLA-tocopherol; di-GLA-3,3′-thiodipropionic acid; DGLA (dihomo-gamma-linolenic acid); AA (arachidonic acid); SA (salicylic acid); EPA (eicosapentaenoic acid) or DHA (docosahexaenoic acid); phenolic antioxidants, including polyenes; unsaturated sterols; ascorbic acid; organosulfur compounds; terpenes; and amino acid antioxidants. In one embodiment, the amino acid antioxidant is selected from the group consisting of: methionine, cysteine, carnosine, and analogues thereof. The concentration of the antioxidant is from 0.02 mM to about 100 mM.

[0017] In yet another embodiment, the pharmaceutical formulation further comprises a preservative selected from the group consisting of phenol, m-cresol, benzyl alcohol, benzalkonium chloride, benzyl chloride, phenoxyethanol, and methylparaben. The concentration of the preservative is from about 0.001% w / v to about 10% w / v.

[0018] In another embodiment, the pharmaceutical preparation is suitable for oral, rectal, mucosal, intestinal, or parenteral administration. In another embodiment, parenteral administration is selected from intravenous, intra-arterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular, intraosseous, intradermal, or subcutaneous administration.

[0019] Other advantages of the invention will be set forth in part in the description which follows, and will also be apparent in part from the description, or may be learned by practice of the invention. The advantages of the invention will be realized and achieved by means of the elements and combinations particularly pointed out in the appended claims. It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only, and do not limit the claimed invention. Attached Figure Description

[0020] This specification incorporates and forms part of the accompanying drawings, which illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.

[0021] Figure 1 The image shows a DSC thermogram overlay of the pH / buffer screening study evaluation of IVX-01101.

[0022] Figures 2A-2C The visual appearance of the samples at different time points is shown, with formulation numbers from 01 to 06 from left to right (as shown in Table 26). Figure 2A T0; ( Figure 2B 40℃-2W;Figure 2C 40℃-4W.

[0023] Figures 3A-3D Stability data for IVX-03023 monoclonal antibody produced in a 15 L bioreactor in a non-GMP stabilization tank at -70 °C ± 10 °C are presented. Figures 3A-3B The results showed that the active pharmaceutical ingredient remained stable for one year at the recommended long-term storage temperature of -70 ± -10℃, and all product quality attributes significantly exceeded the acceptance criteria, including the percentage of size heterogeneous monomers. Figure 3A Purity % Figure 3B ), protein concentration ( Figure 3C ) and charge heterogeneity ( Figure 3D ).

[0024] Figures 4A-4H Stability data for IVX-03023 monoclonal antibody produced in a 15 L bioreactor in a non-GMP stabilization tank at 5 °C ± 3 °C are presented. Figures 4A-4H The results showed that the pharmaceutical products remained stable for one year at the recommended long-term storage temperature of 2-8°C, and all product quality attributes significantly exceeded the acceptance criteria, including the percentage of size heterogeneous monomers. Figure 4A ), size heterogeneity HMW% ( Figure 4B ),purity( Figure 4C ), charge heterogeneity ( Figure 4D ), protein concentration ( Figure 4E ), ELISA efficacy ( Figure 4F Sub-visible particles >10 / mL Figure 4G ) and subvisible particles >25 / mL ( Figure 4H The effectiveness of the molecules is maintained.

[0025] Figures 5A-5E Stability data for IVX-03023 monoclonal antibody produced in a 15 L bioreactor in a non-GMP stabilization tank at 25 °C ± 2 °C are presented. Figures 5A-5E The results showed that the pharmaceutical product remained stable for 3 months at the recommended long-term storage temperature of 25°C, and all product quality attributes significantly exceeded the acceptance criteria, including the percentage of size heterogeneous monomers. Figure 5A ), size heterogeneity HMW% ( Figure 5B ),purity( Figure 5C ), charge heterogeneity ( Figure 5D ) and protein concentration ( Figure 5E ).

[0026] Figures 6A-6D Stability data for IVX-06076 monoclonal antibody produced in a 15 L bioreactor in a non-GMP stabilization tank at -70 °C ± 10 °C are presented.Figures 3A-3B The results showed that the active pharmaceutical ingredient remained stable for 6 months at the recommended long-term storage temperature of -70 ± -10℃, and all product quality attributes significantly exceeded the acceptance criteria, including the percentage of size heterogeneous monomers. Figure 6A Purity % Figure 6B ), protein concentration ( Figure 6C ) and charge heterogeneity ( Figure 6D ).

[0027] Figures 7A-7H Stability data for IVX-06076 monoclonal antibody produced in a 15 L bioreactor in a non-GMP stabilization tank at 5 °C ± 3 °C are presented. Figures 7A-7H The results showed that the pharmaceutical products remained stable for one year at the recommended long-term storage temperature of 5°C, and all product quality attributes significantly exceeded the acceptance criteria, including the percentage of size heterogeneous monomers. Figure 7A ), size heterogeneity HMW% ( Figure 7B ),purity( Figure 7C ), charge heterogeneity ( Figure 7D ), protein concentration ( Figure 7E ), ELISA efficacy ( Figure 7F Sub-visible particles >10 / mL Figure 7G ) and subvisible particles >25 / mL ( Figure 7H The effectiveness of the molecules is maintained.

[0028] Figures 8A-8H Stability data for IVX-06076 monoclonal antibody produced in a 15 L bioreactor in a non-GMP stabilization tank at 25 °C ± 2 °C are presented. Figures 8A-8H The results showed that the pharmaceutical products remained stable for 6 months at the recommended long-term storage temperature of 25°C, and all product quality attributes significantly exceeded the acceptance criteria, including the percentage of size heterogeneous monomers. Figure 8A ), size heterogeneity HMW% ( Figure 8B ),purity( Figures 8C-8D ), protein concentration ( Figure 8E ), combined with ELISA ( Figure 8F Sub-visible particles >10 / mL Figure 8G ) and subvisible particles >25 / mL ( Figure 8H ). Detailed Implementation

[0029] The invention can be more readily understood by referring to the following detailed description of preferred embodiments of the invention and the examples included therein, as well as by referring to the accompanying drawings and their preceding and following description.

[0030] I. Definition

[0031] To facilitate understanding of the principles and features of the various embodiments of this disclosure, various exemplary embodiments will be explained herein. While exemplary embodiments of this disclosure have been explained in detail, it should be understood that other embodiments are also covered. Therefore, the scope of this disclosure is not intended to be limited to the details of the construction and arrangement of the components set forth in the specification or embodiments. This disclosure can have other embodiments and can be practiced or implemented in various ways.

[0032] For clarity, specific terminology will be used in describing exemplary embodiments. Unless the context clearly requires otherwise, the singular forms “a,” “an,” and “the” as used in this specification and the appended claims include multiple indicators. For example, reference to a component is also intended to include a combination of multiple components. Reference to a composition containing “a” component is intended to include components other than those specified.

[0033] The range herein may be expressed as “about” or “approximately” or “substantially” a particular value and / or to “about” or “approximately” or “substantially” another particular value. When such a range is expressed, other embodiments include from said one particular value and / or to said other particular value.

[0034] Similarly, as used herein, expressions such as “substantially free of” or “substantially pure” can include “at least substantially free of” or “at least substantially pure” a substance, or “completely free of” or “completely pure” a substance.

[0035] "Comprising," "containing," or "including" means that the composition, article, or method contains at least the said compound, ingredient, particle, or method step, but does not exclude the presence of other compounds, materials, particles, or method steps, even if such other compounds, materials, particles, or method steps have the same function as the said compound.

[0036] All formulations of antibodies and / or antibody fragments that specifically bind to antigens of interest are collectively referred to herein as "formulations of this disclosure", "liquid formulations of this disclosure", "high-concentration stable liquid formulations of this disclosure", "antibody liquid formulations of this disclosure" or "antibody formulations of this disclosure".

[0037] As used herein, the terms "antibody" and "antibodies" (immunoglobulins) encompass monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two complete antibodies, human antibodies, humanized antibodies, camelified antibodies, chimeric antibodies, single-chain Fv (scFv), single-drain antibodies, single-domain antibodies, domain antibodies, Fab fragments, F(ab′)2 fragments, antibody fragments with desired biological activity, disulfide-linked Fv (sdFv), and anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies against the antibodies of this disclosure), intracellular antibodies, and epitope-binding fragments of any of the above antibodies. Specifically, antibodies include immunoglobulin molecules, biologically active fragments of the disclosed molecules, and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing antigen-binding sites. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0038] Natural antibodies are typically heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by a covalent disulfide bond, and the number of disulfide bonds between heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end, followed by several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, while the variable domain of the light chain is aligned with the variable domain of the heavy chain. Light chains are classified as λ chains or κ chains based on the amino acid sequence of their constant regions. The variable domain of the κ light chain may also be referred to as VK in this paper. The term "variable region" can also be used to describe the variable domains of either the heavy or light chains. It is believed that specific amino acid residues form interfaces between the variable domains of the light and heavy chains. These antibodies can be derived from any mammal, including but not limited to humans, monkeys, pigs, horses, rabbits, dogs, cats, mice, etc.

[0039] The term "antibody fragment" refers to an incomplete antibody structure, including but not limited to isolated single antibody chains, Fv constructs, Fab constructs, Fc constructs, light chain variable regions or complementarity-determining regions (CDR) sequences, etc.

[0040] The term "variable" refers to the fact that certain portions of the variable domain in an antibody exhibit highly variable sequences, resulting in the binding specificity of each specific antibody to its specific antigen. However, variability is not uniformly distributed throughout the variable domain of an antibody. It is contained within segments called complementarity-determining regions (CDRs) in both the light and heavy chain variable domains. The more conserved portions of the variable domain are called framework regions (FRs). The variable domains of the native heavy and light chains each contain four FW regions, which are mostly β-sheet conformations linked by three CDRs, forming loops connecting the β-sheet structure and, in some cases, forming part of the β-sheet structure. The CDRs in each chain are held very tightly together by the FW regions and, together with CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service. National Institutes of Health, Bethesda, Md. (1991)). Constant domains generally do not directly participate in antigen binding, but may affect antigen binding affinity and can exhibit various effector functions, such as antibody involvement in ADCC, CDC, antibody-dependent phagocytosis, and / or apoptosis.

[0041] The term "hypervariant region," as used herein, refers to the amino acid residues in an antibody involved in its binding to an antigen. The hypervariant region encompasses the amino acid residues of the "complementarity-determining region" or "CDR" of the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991) and / or residues in the "hypervariant loop" of the light chain variable domain and the heavy chain variable domain; Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987). "Framework" or "FW" residues are variable domain residues flanking the CDR. FW residues are present in chimeric antibodies, humanized antibodies, human antibodies, domain antibodies, bifunctional antibodies, vaccine antibodies, linear antibodies, and bispecific antibodies.

[0042] As used herein, the “Fc region” includes the polypeptides that constitute the constant region of an antibody, but excludes the first constant region immunoglobulin domain. Therefore, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, along with the flexible hinges at the N-termini of these domains. For IgA and IgM, Fc may include the J-chain. For IgG, Fc contains the immunoglobulin domains Cγ2 and Cγ3 (Cy2 and Cy3) and the hinge between Cγ1 (Cγ1) and Cγ2 (Cγ2). Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is generally defined as containing residues C226 or P230 to its carboxyl terminus, where the numbering is based on the EU index in Kabat et al. (1991, NIHP Publication 91-3242, National Technical Information Service, Springfield, Va.). "The EU index described in Kabat" refers to the residue number of the human IgG1 EU antibody described in Kabat et al. (see above). Fc can refer to the region alone or to the region within an antibody, antibody fragment, or Fc fusion protein. Fc variant proteins can be antibodies, Fc fusions, or any protein or protein domain containing an Fc region. Proteins containing variant Fc regions, which are non-naturally occurring variants of the Fc region, are particularly preferred. The amino acid sequence of a non-naturally occurring Fc region (also referred to herein as a "variant Fc region") contains at least one substitution, insertion, and / or deletion of an amino acid residue compared to the wild-type amino acid sequence. Any new amino acid residue appearing in the variant Fc region sequence due to insertion or substitution can be referred to as a non-naturally occurring amino acid residue. Note: Polymorphisms have been observed at multiple Fc positions, including but not limited to Kabat 270, 272, 312, 315, 356, and 358, and therefore the presented sequences may differ slightly from those in the prior art.

[0043] The light chains of antibodies (immunoglobulins) from any vertebrate species can be designated as one of two distinct types, known as κ (kappa) and λ (lambda), based on the amino acid sequence of their constant domains.

[0044] Immunoglobulins can be designated as different classes based on the amino acid sequence of their heavy chain constant domains. Currently, there are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further subdivided into "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2 (defined by mouse and human names, respectively). The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different classes of immunoglobulins in multiple species are well known. The universality of individual isotypes and the functional activities associated with these constant domains are species-specific and must be determined experimentally.

[0045] As used herein, the term "monoclonal antibody" refers to an antibody derived from a population of substantially the same antibodies, meaning that the individual antibodies constituting the population are identical except for possibly trace amounts of potentially naturally occurring mutations. Monoclonal antibodies exhibit high specificity (targeting a single antigenic site). Furthermore, unlike polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. In addition to specificity, a key advantage of monoclonal antibodies is that they can be synthesized from hybridoma cells that are not contaminated with other immunoglobulin-producing cells. Alternative production methods are known and trained in the art; for example, monoclonal antibodies can be produced by stable or transient transfection of cells encoding the heavy and light chain genes encoding the monoclonal antibody.

[0046] The modifier “monoclonal” indicates the characteristic of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring engineering of the antibody by any particular method. The term “monoclonal” is used herein to refer to an antibody derived from a clonal cell population, including any eukaryotic, prokaryotic, or phage clone, rather than obtained through antibody engineering methods. For example, monoclonal antibodies used according to this disclosure can be produced by the hybridoma method first described by Kohler et al. in Nature, 256:495 (1975), or prepared by any recombinant DNA method (see, for example, U.S. Patent No. 4,816,567), including, for example, isolation from a phage antibody library using techniques described by Clackson et al. in Nature, 352:624-628 (1991) and Marks et al. in J. Mol. Biol., 222.581-597 (1991). These methods can be used to generate monoclonal mammalian antibodies, chimeric antibodies, humanized antibodies, human antibodies, domain antibodies, bifunctional antibodies, vaccine antibodies, linear antibodies, and bispecific antibodies.

[0047] "Human antibodies" can be antibodies derived from humans or antibodies obtained from transgenic organisms "engineered" to produce specific human antibodies in response to antigen stimulation, and can be produced by any method known in the art. In some techniques, elements of human heavy and light chain loci are introduced into an organismal strain derived from embryonic stem cell lines containing targeted disruption of endogenous heavy and light chain loci. The transgenic organism can synthesize human antibodies specific to human antigens, and the organism can be used to produce hybridomas that secrete human antibodies. Human antibodies can also be antibodies whose heavy and light chains are encoded by nucleotide sequences derived from one or more human DNA sources. Fully human antibodies can also be constructed by gene transfection or chromosome transfection methods, phage display technology, or in vitro activated T cells expressing ICOS, all of which are known in the art.

[0048] Antibodies can also refer to canine antibodies or antibodies that have undergone canine derivatization. Unless otherwise indicated, the term "dog" as used herein includes all domestic dogs, i.e., domestic dogs ( Canis lupus familiaris or Canis familiaris A "canine-derived" form of non-canine (e.g., mouse) antibody is a genetically engineered antibody containing a minimal sequence derived from a non-canine immunoglobulin. A canine-derived antibody is a canine immunoglobulin sequence (recipient antibody) in which hypervariable residues of the acceptor are replaced by hypervariable residues from a non-canine species (donor antibody; e.g., mouse) with the desired specificity, affinity, and capability. In some cases, frame region (FR) residues of the canine immunoglobulin sequence are replaced by corresponding non-canine residues. Additionally, canine-derived antibodies may include residues not found in the acceptor antibody or the donor antibody. These modifications further optimize antibody performance. Generally, canine-derived antibodies will include at least one and typically substantially all of two variable domains, wherein all or substantially all of the hypervariable regions correspond to hypervariable regions of the non-canine immunoglobulin sequence, and all or substantially all of the FRs are FRs of the canine immunoglobulin sequence. Canine-derived antibodies may also optionally include all or at least a portion of the immunoglobulin constant region (Fc), typically all or at least a portion of the constant region (Fc) of the canine immunoglobulin sequence.

[0049] Antibodies can also refer to cat antibodies or antibodies that have undergone feline derivation. As used herein, the term "cat" includes all members of the Felidae family and other members of the Felidae, including the Pantherinae and Felinae subfamilies (commonly referred to as Felidae) and the domestic cat (felis catus). "Feline-derived" forms of non-cat (e.g., mouse) antibodies are genetically engineered antibodies containing minimal sequences derived from non-cat immunoglobulins. A feline-derived antibody is a cat immunoglobulin sequence (recipient antibody) in which hypervariable residues of the acceptor are replaced by hypervariable residues from a non-cat species (donor antibody; e.g., mouse) with the desired specificity, affinity, and ability. In some cases, frame region (FR) residues of the cat immunoglobulin sequence are replaced by corresponding non-cat residues. Additionally, feline-derived antibodies may include residues not found in the acceptor antibody or the donor antibody. These modifications further optimize antibody performance. Generally, feline-derived antibodies will include at least one and typically substantially all of two variable domains, wherein all or substantially all of the hypervariable regions correspond to hypervariable regions of non-feline immunoglobulin sequences, and all or substantially all of the FRs are FRs of feline immunoglobulin sequences. Optionally, feline-derived antibodies will also include all or at least a portion of the immunoglobulin constant region (Fc), typically all or at least a portion of the constant region (Fc) of the feline immunoglobulin sequence.

[0050] Antibodies can also refer to antibodies derived from production animals. As used herein, the term "production animal antibody" includes antibodies derived from livestock, including but not limited to cattle, horses, sheep, goats, chickens, ostriches, geese, etc.

[0051] The term “heterochimeric” as defined in this article refers to an antibody in which one antibody chain (heavy or light chain) is canine or feline-derived, while the other antibody chain is chimeric.

[0052] The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. In one embodiment, the FcR is a human FcR with a natural sequence. Additionally, in some embodiments, the FcR is a receptor that binds to IgG antibodies (γ receptors) and includes receptors of the FcγRI, FcγRII, FcγRIII, and FcγRIV subclasses, including allelic variants and alternative splice forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine activation motif (ITAM) in its cytoplasmic domain. The inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine inhibition motif (ITIM) in its cytoplasmic domain. (See Daëron. Annu. Rev. Immunol., 15:203-234 (1997)). For reviews of FcRs, see Ravetch and Kinet, Annu. Rev. Immunol., 9:457-92 (1991); Capel et al., Immunomethods, 4:25-34 (i 994); and de Haas et al., J. Lab. Clin. Med., 126:330-41 (1995). The term “FcR” as used herein encompasses other FcRs, including those to be identified in the future. This term also includes the neonatal receptor FcRn, which is responsible for transferring maternal IgG to the fetus (Guyer et al., Immunol., 117:587 (1976); and Kim et al., J. Immunol., 24:249 (1994)).

[0053] The term "affinity" of an antibody for an epitope used in the treatments described herein is a well-known term in the art, referring to the degree or strength of an antibody's binding to an epitope. Affinity can be measured and / or expressed in a variety of ways known in the art, including, but not limited to, equilibrium dissociation constant (KD or Kd), apparent equilibrium dissociation constant (KD′ or Kd′), and IC50 (the amount required to achieve 50% inhibition in a competitive assay). It should be understood that, for the purposes of this disclosure, affinity is the average affinity of a given antibody population for binding to an epitope. KD′ values ​​reported herein as mg IgG / mL or mg / mL represent milligrams of Ig per milliliter of serum, but plasma may also be used. When antibody affinity is used as a basis for the administration of the treatments described herein, or as a basis for the selection of the treatments described herein, antibody affinity can be measured before and / or during treatment, and clinicians can use the obtained values ​​to assess whether a human patient is a suitable candidate for treatment.

[0054] As used herein, the term "affinity" is a measure of the overall binding strength (i.e., the two arms of the antibody) of an antibody to an antigen. Antibody affinity can be determined by measuring the dissociation of the antigen-antibody bond in the presence of antigen excess using any means known in the art, such as, but not limited to, by modifying the indirect fluorescent antibody technique described by Gray et al. in J. Virol. Meth., 44:11-24 (1993).

[0055] "Epitope" is a term well understood in the art and refers to any chemical part that exhibits specific binding to an antibody. "Antigen" is a part or molecule containing an epitope and therefore also specifically binding to an antibody.

[0056] As used herein, the term "antibody half-life" refers to the pharmacokinetic properties of an antibody, which measures the average survival time of an antibody molecule after administration. Antibody half-life can be expressed as the time required to eliminate 50% of a known amount of immunoglobulin from the patient's body or a specific compartment (e.g., the circulating half-life when measured in serum or plasma; or in other tissues). Half-lives may vary for different immunoglobulins or different classes of immunoglobulins. Generally, a prolonged antibody half-life results in a prolonged mean residence time (MRT) of the administered antibody in circulation.

[0057] The term "isotype" refers to the classification of the constant regions of the antibody heavy or light chain. The constant domains of an antibody do not participate in antigen binding but exhibit various effector functions. Based on the amino acid sequence of the heavy chain constant region, a given human antibody or immunoglobulin can be designated as one of five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM. Several of these classes can be further subdivided into subclasses (isotypes), such as IgG1 (γ1), IgG2 (γ2), IgG3 (γ3), and IgG4 (γ4), as well as IgA1 and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. The structures and three-dimensional conformations of different classes of immunoglobulins are well known. Among the various classes of human immunoglobulins, only human IgG1, IgG2, IgG3, IgG4, and IgM are known to activate complement. Human IgG1 and IgG3 are known to mediate ADCC in humans. Human light chain constant regions can be divided into two main classes: κ and λ.

[0058] As used herein, the term “immunogenicity” means that a compound is able to elicit an immune response (stimulate the production of specific antibodies and / or the proliferation of specific T cells).

[0059] As used in this article, the term "antigenic" means that a compound is recognized by an antibody or can bind to an antibody and induce an immune response.

[0060] As used herein, the term "excipient" refers to an inert substance commonly used as a diluent, medium, preservative, binder, or stabilizer for pharmaceutical preparations, which imparts beneficial physical properties to the formulation, such as increased protein stability, increased protein solubility, and reduced viscosity. Examples of excipients include, but are not limited to, proteins (e.g., but not limited to serum albumin), amino acids (e.g., but not limited to aspartic acid, glutamic acid, lysine, arginine, and glycine), surfactants (e.g., but not limited to SDS, Tween 20, Tween 80, polysorbates, and nonionic surfactants), sugars (e.g., but not limited to glucose, sucrose, maltose, and trehalose), polyols (e.g., but not limited to mannitol and sorbitol), fatty acids, and phospholipids (e.g., but not limited to alkyl sulfonates and caprylates). For more information on excipients, please refer to Remington's Pharmaceutical Sciences (Joseph P. Remington, 18th edition, Mack Publishing Co., Easton. Pa.), which is incorporated herein by reference in its entirety.

[0061] As used in this article, the phrase “pharmaceutically acceptable” means approved by a federal regulatory agency or state government, or listed in the United States Pharmacopeia, the European Pharmacopeia, or other generally recognized pharmacopoeia for use in animals and more specifically for use in humans.

[0062] In the context of liquid formulations containing antibodies (including antibody fragments thereof) that specifically bind to an antigen of interest, the terms "stability" and "stable" as used herein refer to the resistance of the antibody (including antibody fragments thereof) in the formulation to aggregation, degradation, or fragmentation under given manufacturing, preparation, transport, and storage conditions. The "stable" formulations of this disclosure retain biological activity under given manufacturing, preparation, transport, and storage conditions. The stability of the antibody (including its antibody fragments) can be assessed by comparing it with a reference formulation using the following methods to measure the degree of aggregation, degradation, or fragmentation: size exclusion chromatography (SEC, including size exclusion high performance liquid chromatography (SE-HPLC) or size exclusion ultra performance liquid chromatography (SE-UPLC)), capillary electrophoresis-sodium dodecyl sulfate method (CE-SDS), reversed-phase chromatography (RP-HPLC), static light scattering (SLS), dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR), circular dichroism spectroscopy (CD), urea denaturation technique, intrinsic tryptophan fluorescence, differential scanning calorimetry, and / or ANS binding technique. For example, a reference formulation may be a reference standard frozen at -70°C, consisting of an antibody (including its antibody fragments) at a concentration of 10 mg / ml in 10 mM histidine (pH 6.0–6.5) containing 80 mM NaCl, 4% trehalose, and 0.02% polysorbate 80. This reference formulation typically yields a single monomer peak (e.g., area ≥97%) via SEC. The overall stability of formulations containing antibodies (including their antibody fragments) can be assessed by various immunoassays, including, for example, ELISA and radioimmunoassays using isolated antigen molecules.

[0063] As used herein, the phrase “low to undetectable aggregation levels” refers to aggregates in a sample that, as measured by SEC or static light scattering (SLS) techniques, do not exceed about 5%, about 4%, about 3%, about 2%, about 1%, and about 0.5% by weight of protein.

[0064] As used herein, the term “fragmentation level so low as to be undetectable” means that a sample contains equal to or greater than about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% of total protein, determined, for example, by SEC or reversed-phase chromatography in a single peak, or by reducing capillary gel electrophoresis (rCGE) in two peaks (e.g., heavy chain and light chain) (or peaks equal to the number of subunits), representing undegraded antibody or its undegraded fragments, and contains no other single peaks, each of which contains more than about 5%, more than about 4%, more than about 3%, more than about 2%, more than about 1%, or more than about 0.5% of total protein. As used herein, the term “reducing capillary gel electrophoresis” means capillary gel electrophoresis performed under reducing conditions sufficient to reduce disulfide bonds in the antibody.

[0065] Concentrations, amounts, cell counts, percentages, and other values ​​in this document may be expressed in range format. It should also be understood that this range format is used solely for convenience and brevity and should be flexibly interpreted to include not only the values ​​explicitly listed as range limits but also all individual values ​​or subranges covered within the range, as if each value and subrange were explicitly listed.

[0066] II. Antibody preparations

[0067] The present invention provides formulations for stabilizing antibodies, particularly portions and / or fragments thereof.

[0068] A. IVX-01101 monoclonal antibody preparation

[0069] In one embodiment, the present invention provides an optimized formulation for IVX-01101, a canine monoclonal antibody comprising a polypeptide with increased binding to canine nerve growth factor (NGF) and FcRn compared to a control polypeptide (e.g., the wild-type counterpart of IgG canine Fc region). In some aspects, the present invention provides stable liquid polypeptide formulations for therapeutic use. Specifically, the present invention provides methods for stabilizing antibodies, as well as their antigen-binding fragments and variants, for the treatment of diseases and / or conditions. Specifically, the formulations provided by the present invention are stabilized such that the active therapeutic polypeptide remains stable over a prolonged period and can be administered via a variety of routes of administration. Specifically, these formulations are suitable for parenteral administration. In other aspects, the present invention provides a unique stable antibody formulation, for example, that remains stable under various stresses, such as freezing, lyophilization, heat, reconstitution, degradation caused by residual metals, and other stresses that the product may encounter during antibody storage and administration. Furthermore, exemplary formulations of the present invention are capable of maintaining the stability, bioactivity, purity, and quality of antibodies or antibody fragments over a prolonged period (e.g., storage of the formulation for one year or more), even at adverse temperatures. In addition, the exemplary formulations of the present invention are suitable for administration to subjects or patients, for example, by parenteral administration to subjects or patients.

[0070] B. IVX-01286 monoclonal antibody preparation

[0071] In another embodiment, this disclosure assesses the feasibility of multi-dose formulations of the molecule by monitoring the product quality and stability of different concentrations of IVX-01286 (a canine antibody with increased binding to canine NGF against this series of molecules) in different formulations with different preservatives (antimicrobial agents). The IVX-01286 protein is an IgG monoclonal antibody (mAb) molecular model for this series of canine and feline monoclonal antibodies against healthy animals and exhibits enhanced binding to canine FcRn.

[0072] In this study, the effects of different antibody concentrations, pH / buffer systems, and various widely accepted parenteral preservatives (antimicrobial agents) on protein quality and stability were evaluated at ICH-recommended stability temperatures. Based on the results, IVX-01286, formulated in 20 mM histidine buffer, 5% (w / v) sorbitol, 0.02% (w / v) PS80, 0.05 mM EDTA, and 0.9% (w / v) benzyl alcohol (pH 6.6), exhibited relatively good thermal stability, and its stability characteristics were similar to those of the preservative-free formulation (i.e., 20 mM histidine buffer, 5% (w / v) sorbitol, 0.02% (w / v) PS80, 0.05 mM EDTA, pH 6.6).

[0073] C. IVX-03023 monoclonal antibody preparation

[0074] In yet another embodiment, this disclosure provides an assessment of the feasibility of a single-dose formulation of IVX-03023, a developed feline monoclonal antibody that exhibits higher affinity for feline interleukin-5 (IL-5) and FcRn compared to control peptides (e.g., wild-type corresponding IgG canine or feline Fc regions). This disclosure also provides proof-of-concept studies of formulations produced using a clonal pool generated from a stable expression system. Product stability was monitored using multiple analytical methods, and the product was found to be stable at -70°C, 5°C, 25°C, and 40°C, significantly exceeding acceptance criteria for all product quality attributes.

[0075] D. IVX-06076 monoclonal antibody preparation

[0076] In another embodiment, this disclosure provides a feasibility assessment of a single-dose formulation of IVX-06076, a feline monoclonal antibody containing a polypeptide with increased binding affinity to feline nerve growth factor (NGF) and FcRn compared to a control polypeptide (e.g., the wild-type counterpart IgG feline Fc region). This disclosure provides a proof-of-concept study of formulations produced using a clonal pool generated from a stable expression system. Product stability was monitored using multiple analytical methods, and the product was found to be stable at -70°C, 5°C, 25°C, and 40°C, significantly exceeding acceptance criteria for all product quality attributes.

[0077] III. Preparation Method

[0078] A. Antibody

[0079] The formulations according to the invention stably support high concentrations of bioactive antibodies in solution and offer the advantage of being suitable for parenteral administration, including intravenous, intramuscular, intraperitoneal, intradermal, or subcutaneous injection. The formulations also minimize the risk of bubble formation and allergic-like side effects.

[0080] The antibody is preferably a monoclonal antibody, polyclonal antibody, antibody fragment (e.g., Fab, Fab′, F(ab′)2, Fv, Fc, ScFv, etc.), chimeric antibody, bispecific antibody, heteroconjugated single-chain antibody (ScFv), mutants thereof, fusion protein comprising multiple antibody motifs (e.g., domain antibodies), humanized antibody, human antibody, and glycosylated variants of the antibody, amino acid sequence variants of the antibody, and covalent antibodies selected from any other modified conformation of an immunoglobulin molecule containing an antigen recognition site having the desired specificity. The antibody may be derived from rodents, rats, humans, dogs, production animals, or any other source (including chimeric or humanized antibodies). In some embodiments, the antibody may be a human antibody or a humanized antibody. In other embodiments, the antibody may be a canine antibody or a canine-derived antibody. Preferably, the antibody is isolated, and more preferably substantially pure. If the antibody is an antibody fragment, it preferably retains the functional characteristics of the original antibody, such as ligand binding and / or antagonistic activity or functional activity.

[0081] 1. Polyclonal antibodies

[0082] Polyclonal antibodies can be prepared by immunizing suitable subjects with an immunogen. Changes in antibody titers over time in immunized subjects can be monitored using standard techniques, such as enzyme-linked immunosorbent assay (ELISA) using immobilized target antigens. If necessary, antibody molecules against the target antigen can be isolated from mammals (e.g., from blood) and further purified using well-known techniques, such as protein A agarose chromatography, to obtain the antibody (e.g., IgG) fraction. At an appropriate time after immunization, for example when the anti-antigen antibody titer is highest, antibody-producing cells can be obtained from the subject, and these cells can be used to immunize the target antigen using standard techniques, such as those described by Kohler and Milstein (1975). Nature The hybridoma technique was first described in 256:495-497 (see also Brown et al. (1981)). J. Immunol. 127:539-46; Brown et al. (1980) J. Biol. Chem. 255:4980-83; Yeh et al. (1976) Proc. Natl. Acad. Sci. USA 76:2927-31; and Yeh et al. (1982) Int. J. Cancer (29:269-75) Preparation of monoclonal antibodies. For information on the preparation of chimeric polyclonal antibodies, see U.S. Patent No. 6,420,113 to Buechler et al.

[0083] 2. Monoclonal antibodies

[0084] Any of the many well-known protocols for fusing lymphocytes and immortalized cell lines can be used to produce monoclonal antibodies (see, for example, G. Galfre et al. (1977)). Nature 266:55052; Gefter et al., Somatic Cell Genet As quoted above; Lerner, Yale J. Biol. Med (See above; Kenneth, Monoclonal Antibodies, as cited above). Furthermore, those skilled in the art will understand that many variations of these methods exist, and these variations are also useful. Typically, immortalized cell lines (e.g., myeloma cell lines) are derived from the same mammalian species as the lymphocytes. For example, mouse hybridomas can be created by fusing lymphocytes from mice immunized with the immunogen formulation of the present invention with an immortalized mouse cell line. Preferred immortalized cell lines are mouse myeloma cell lines sensitive to a medium containing hypoxanthine, aminopterin, and thymidine (“HAT medium”). According to standard techniques, any of many myeloma cell lines can be used as fusion conjugates, such as the P3-NS1 / 1-Ag4-1, P3-x63-Ag8.653, or Sp2 / O-Ag14 myeloma cell lines. These myeloma cell lines are available from the ATCC. Typically, HAT-sensitive mouse myeloma cells are fused with mouse spleen cells using polyethylene glycol (“PEG”). Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused myeloma cells that do not produce fusion (unfused spleen cells die after a few days because they have not been transformed). Hybridoma cells producing the monoclonal antibodies of the present invention are detected using a standard ELISA assay by screening for antibodies binding to the target antigen in the hybridoma culture supernatant.

[0085] 3. Recombinant antibodies

[0086] As an alternative to hybridomas for preparing secretory monoclonal antibodies, monoclonal antibodies can also be identified and isolated by screening recombinant combined immunoglobulin libraries (e.g., antibody phage display libraries) with target antigens, thereby isolating immunoglobulin library members that bind to the target antigens. Kits for generating and screening phage display libraries are commercially available (e.g., Pharmacia). recombinant phage antibody system Product catalog number 27-9400-01; and Stratagene SurfZAP TM phage display kit(Product Catalog No. 240612). Furthermore, examples of methods and reagents particularly suitable for generating and screening antibody display libraries can be found, for example, in US Patent No. 5,223,409 by Ladner et al.; PCT International Publication No. WO 92 / 18619 by Kang et al.; PCT International Publication No. WO 91 / 17271 by Dower et al.; PCT International Publication No. WO 92 / 20791 by Winter et al.; PCT International Publication No. WO 92 / 15679 by Markland et al.; PCT International Publication No. WO 93 / 01288 by Breitling et al.; PCT International Publication No. WO 92 / 01047 by McCafferty et al.; PCT International Publication No. WO 92 / 09690 by Garrard et al.; PCT International Publication No. WO 90 / 02809 by Ladner et al.; and Fuchs et al. (1991). Bio / Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J. Mol. Biol. 226:889-896; Clarkson et al. (1991) Nature 352:624-628; Gram et al. (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrad et al. (1991) Bio / Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; Barbas et al. (1991) Proc. Natl. Acad. Sci. USA 88:7978-7982; and McCafferty et al., Nature (1990) 348:552-554.

[0087] 4. Chimeric antibodies and humanized antibodies

[0088] In addition, chimeric antibodies, humanized antibodies, feline antibodies, canine antibodies, and other recombinant antibodies, as well as monoclonal antibodies containing human and non-human portions that can be made using standard recombinant DNA technology, are all within the scope of this invention.

[0089] The terms "humanized immunoglobulin" or "humanized antibody" refer to an immunoglobulin or antibody comprising at least one humanized immunoglobulin or antibody chain (i.e., at least one humanized light or heavy chain). The terms "humanized immunoglobulin chain" or "humanized antibody chain" (i.e., "humanized immunoglobulin light chain" or "humanized immunoglobulin heavy chain") refer to an immunoglobulin or antibody chain having variable regions (i.e., light or heavy chains, respectively), said variable regions comprising a variable framework region substantially derived from human immunoglobulins or antibodies and a complementarity-determining region (CDR) substantially derived from non-human immunoglobulins or antibodies (e.g., at least one CDR, preferably two CDRs, more preferably three CDRs), and also including constant regions (e.g., at least one constant region or a portion thereof in the case of a light chain, and three constant regions in the case of a heavy chain). The term “humanized variable region” (e.g., “humanized light chain variable region” or “humanized heavy chain variable region”) refers to a variable region that includes a variable framework region that is substantially derived from human immunoglobulins or antibodies and a complementarity-determining region (CDR) that is substantially derived from non-human immunoglobulins or antibodies.

[0090] The phrase "substantially derived from human immunoglobulins or antibodies" or "substantially human" means that, when compared with the amino acid sequence of human immunoglobulins or antibodies for comparative purposes, the region shares at least 80-90%, 90-95%, or 95-99% identity (i.e., local sequence identity) with the human framework or constant region sequence, such as allowing conserved substitutions, shared sequence substitutions, germline substitutions, reversion mutations, etc. Introducing conserved substitutions, shared sequence substitutions, germline substitutions, reversion mutations, etc., is often referred to as "optimization" of humanized antibodies or chains. The phrase "substantially derived from non-human immunoglobulins or antibodies" or "substantially non-human" means that the immunoglobulin or antibody sequence has at least 80-95%, preferably at least 90-95%, more preferably 96%, 97%, 98%, or 99% identity with the sequence of a non-human organism (e.g., a non-human mammal).

[0091] Therefore, except for the CDR, all regions or residues of the humanized immunoglobulin or antibody, or the humanized immunoglobulin or antibody chain, are substantially identical to the corresponding regions or residues of one or more natural human immunoglobulin sequences. The term "corresponding region" or "corresponding residue" refers to a region or residue on the second amino acid or nucleotide sequence that occupies the same (i.e., equivalent) position as the region or residue on the first amino acid or nucleotide sequence when the first and second sequences are optimally aligned for comparative purposes.

[0092] The term "significant identity" means that two polypeptide sequences, when optimally aligned, such as via GAP or BESTFIT using the default vacancy weighting, share at least 50-60% sequence identity, preferably at least 60-70%, more preferably at least 70-80%, more preferably at least 80-90%, even more preferably at least 90-95%, and even more preferably at least 95% or higher (e.g., 99% or higher). For sequence comparison, one sequence typically serves as a reference sequence for comparison with the test sequence. When using a sequence comparison algorithm, the test sequence and a reference sequence are input into the computer. If necessary, the coordinates of the subsequences and the sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the percentage of sequence identity between the test sequence and the reference sequence based on the specified program parameters.

[0093] The best alignment of sequences for comparison can be performed, for example, by the methods of Smith and Waterman. Adv. Appl. Math. Local homology algorithm of 2:482 (1981); Needleman and Wunsch J. Mol. Biol. The homology alignment algorithm of 48:443 (1970); Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA Similarity search methods as described in 85:2444 (1988); computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics software package (GeneticsComputer Group, 575 Science Dr., Madison, Wis.); or visual inspection (generally see Ausubel et al., Current Protocols in Molecular Biology). The BLAST algorithm is an example of an algorithm suitable for determining sequence identity and the percentage of sequence similarity, as described in Altschul et al. J. Mol. Biol.The description is found in 215:403 (1990). The software used to perform BLAST analysis is publicly available from the National Center for Biotechnology Information (NCBI) (accessible via the NCBI Internet server of the National Institutes of Health). Typically, default program parameters are used for sequence comparisons, but custom parameters can also be used. For amino acid sequences, the BLASTP program defaults to a word length of 3 (W), an expected value of 10 (E), and a BLOSUM62 scoring matrix (see Henikoff and Henikoff, ...). Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0094] Preferably, the different residue positions differ due to conserved amino acid substitutions. To classify amino acid substitutions as conserved or non-conservative, the amino acids are grouped as follows: Group I (hydrophobic side chains): leu, met, ala, val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser, thr; Group III (acidic side chains): asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V (residues affecting chain orientation): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitution involves substitution between amino acids within the same category. Non-conservative substitution requires replacing a member of one category with a member of another.

[0095] Preferably, the affinity of the humanized immunoglobulin or antibody for the antigen is within three, four, or five times that of the corresponding non-humanized antibody. For example, if the binding affinity of the non-humanized antibody is 10... -9 M, then the binding affinity of the humanized antibody will be at least 3 × 10⁻⁶. -8 M, 4×10 -8 M, 5×10 -8 M or 10 -9M. When describing the binding properties of an immunoglobulin or antibody chain, the chain can be described based on its ability to "guide antigen binding." A chain is considered to "directly bind antigens" when it confers specific binding properties or binding affinity to an intact immunoglobulin or antibody (or its antigen-binding fragment). If a mutation (e.g., a reversion mutation) affects (e.g., reduces) the binding affinity of an intact immunoglobulin or antibody (or its antigen-binding fragment) containing a heavy or light chain, such that it differs by at least one order of magnitude from the binding affinity of an antibody (or its antigen-binding fragment) containing an equivalent chain without the mutation, then the mutation is considered to substantially affect the chain's ability to guide antigen binding. If a mutation (e.g., a reversion mutation) affects (e.g., reduces) the binding affinity of an intact immunoglobulin or antibody (or its antigen-binding fragment) containing a single chain, such that it differs by only two, three, or four times from the binding affinity of an antibody (or its antigen-binding fragment) containing an equivalent chain lacking the mutation, then the mutation is considered to substantially not affect (e.g., reduce) the chain's ability to guide antigen binding.

[0096] The terms “chimeric immunoglobulin” or “chimeric antibody” refer to an immunoglobulin or antibody whose variable region is derived from one species and whose constant region is derived from another species. Chimeric immunoglobulins or antibodies can be constructed, for example, through genetic engineering, from immunoglobulin gene segments belonging to different species. The terms “humanized immunoglobulin” or “humanized antibody” are not intended to encompass chimeric immunoglobulins or antibodies as defined below. While humanized immunoglobulins or antibodies are chimeric in their construction (i.e., containing regions of proteins from multiple species), they include other features not present in chimeric immunoglobulins or antibodies (i.e., variable regions containing donor CDR residues and receptor framework residues), as defined herein.

[0097] Such chimeric and humanized monoclonal antibodies can be produced using recombinant DNA techniques known in the art, for example, using methods described in the following documents: Robinson et al. International Application No. PCT / US86 / 02269, Akira et al. European Patent Application No. 184,187, Taniguchi, M. European Patent Application No. 171,496, Morrison et al. European Patent Application No. 173,494, Neuberger et al. PCT International Publication No. WO 86 / 01533, Cabilly et al. US Patent No. 4,816,567, Cabilly et al. European Patent Application No. 125,023, Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J.Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559; Morrison, SL (1985) Science 229:1202-1207; Oi et al. (1986) BioTechniques 4:214; Winter's U.S. Patent No. 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; and Seidler et al. (1988) J. Immunol. 141:4053-4060.

[0098] 5. Human antibodies obtained from transgenic animals and phage display technology

[0099] Alternatively, it is now possible to produce transgenic animals (e.g., mice) that, upon immunization, can generate a full set of human antibodies without the production of endogenous immunoglobulins. For example, the antibody reconnection region (J) has been described in chimeric and germline mutant mice. H Homozygous deletion of the gene leads to complete suppression of endogenous antibody production. Transferring human germline immunoglobulin gene arrays into such germline mutant mice results in the production of human antibodies upon antigen challenge. See, for example, U.S. Patent Nos. 6,150,584, 6,114,598, and 5,770,429.

[0100] Fully human antibodies can also be obtained from phage display libraries (Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581-597 (1991)). Chimeric polyclonal antibodies can also be obtained from phage display libraries (Buechler et al., US Patent No. 6,420,113).

[0101] 6. Bispecific antibodies, antibody fusion peptides, and single-chain antibodies

[0102] Bispecific antibodies (BsAbs) are antibodies that bind specifically to at least two different epitopes. Such antibodies can be derived from full-length antibodies or antibody fragments (e.g., F(ab)′2 bispecific antibodies). Methods for preparing bispecific antibodies are known in the art. A conventional method for producing full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where these two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). Due to the random combination of immunoglobulin heavy and light chains, these hybridomas (tetragenic hybridomas) can produce a potential mixture of different antibody molecules (see WO 93 / 08829; and Traunecker et al., EMBO J., 10:3655-3659 (1991)).

[0103] Bispecific antibodies also include cross-linked antibodies or "heteroconjugated" antibodies. For example, one antibody in a heteroconjugated antibody may be conjugated to avidin, while the other antibody may be conjugated to biotin or other payloads. Heteroconjugated antibodies can be prepared using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Patent No. 4,676,980, along with numerous cross-linking techniques.

[0104] In yet another embodiment, the antibody can be fused chemically or genetically with a payload, such as a reactive portion, a detectable portion, or a functional portion (e.g., an immunotoxin), to produce an antibody fusion peptide. Examples of such payloads include immunotoxins, chemotherapeutic agents, and radioisotopes, all of which are well known in the art.

[0105] According to the invention, single-chain antibodies are also suitable for stabilization. These fragments contain a heavy chain variable domain (VH) linked to a light chain variable domain (VL) via a linker, which causes each variable region to bind to each other and regenerates antigen-binding pockets for the parent antibody as the source of the VL and VH regions. See Gruber et al., J. Immunol., 152:5368 (1994).

[0106] 7. Canine antibodies

[0107] With the increasing use of peptides (such as antibodies, ligand-receptor binding domains, enzymes, ligands, and peptides) as therapeutic agents in the prevention and treatment of a variety of canine diseases, it is particularly important to develop peptides with extended half-lives, especially for the prevention or treatment of chronic diseases that require repeated administration of the peptides.

[0108] In dogs, there are four IgG heavy chains, designated A, B, C, and D. These heavy chains represent four distinct subclasses of canine IgG, designated IgGA, IgGB, IgGC, and IgGD. The DNA and amino acid sequences of these four heavy chains were first described by Tang et al. Vet. Immunol. Immunopathol. [80: 259-270 (2001)] Identification. The amino acid and DNA sequences of these heavy chains can also be obtained from the GenBank database. For example, the accession number for the IgGA heavy chain amino acid sequence is AAL35301.1, the accession number for the IgGB amino acid sequence is AAL35302.1, the accession number for the IgGC amino acid sequence is AAL35303.1, and the accession number for the IgGD amino acid sequence is (AAL35304.1). Canine antibodies also contain two types of light chains, namely κ light chains and λ light chains. The DNA and amino acid sequences of these light chains can be obtained from the GenBank database. For example, the accession number for the κ light chain amino acid sequence is ABY57289.1, and the accession number for the λ light chain amino acid sequence is ABY55569.1.

[0109] a. Fc region of canine IgGB antibody

[0110] This document provides stabilizing formulations for antibodies comprising canine CH2, CH3, Fc (e.g., canine IgG Fc region variants) or canine FcRn binding fragments thereof. The disclosure is characterized by formulations for stabilizing antibodies comprising peptides that exhibit increased binding to canine FcRn compared to control peptides (e.g., wild-type corresponding IgG canine Fc region). In some cases, these peptides exhibit increased binding to canine FcRn compared to control peptides at any pH (e.g., any pH between 5.0 and 8.0). In other cases, these peptides exhibit increased binding to canine FcRn compared to control peptides at pH 5.5, pH 6.0, and / or pH 6.5. In some cases, these peptides may bind to canine FcRn at acidic pH (e.g., pH 5.5, pH 6.0, or pH 6.5) at a higher level than at neutral pH (e.g., pH 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5). In other cases, these peptides bind to canine FcRn at pH 5.5, pH 6.0, or pH 6.5 at a higher level than at pH 7.4. This disclosure relates in part to formulations for stabilizing antibodies, said antibodies comprising peptides in dogs with a longer half-life than their wild-type counterparts. For example, the provided binding molecule (e.g., a ligand-binding portion of an antibody or receptor) has an increased half-life relative to the binding molecule not linked to an Fc region (e.g., a CH2, CH3, or CH2+CH3 region) or the canine FcRn binding region disclosed herein. Stable formulations of enzyme-Fc region fusions, ligand-Fc region fusions, nanobody-Fc fusions, and peptide-Fc region fusions are also provided, wherein these fusions have an increased half-life compared to the wild-type counterpart. The Fc region may include, in addition to one or more substitutions (wild-type canine Fc region) that increase half-life, other substitutions such as those that increase effector function, decrease effector function, increase binding to protein A, and / or reduce peptide heterogeneity (e.g., by removing one or more post-translational modifications in the Fc region). The canine CH2, CH3, and Fc region sequences can be derived from any canine antibody. In some cases, the canine CH2, CH3, and Fc region sequences are derived from canine IgG (e.g., IgG.A, IgG.B, IgG.C, or IgG.D). In other cases, the canine antibody is IVX-01101.

[0111] b. Feline antibodies

[0112] Cats typically have three IgG heavy chains, called IgG1a, IgG1b, and IgG2. These heavy chains represent three distinct subclasses of feline IgG. The amino acid and DNA sequences of these heavy chains can be found in Strietzel et al., 2014. , Vet. Immunol. Immunopathol.,The amino acid sequences of the feline IgG1a heavy chain are obtained from GENBANK databases 158:214-223 and 158:214-223. For example, the GENBANK accession number for the feline IgG1a heavy chain amino acid sequence is BAA32229.1, the GENBANK accession number for the feline IgG1b heavy chain amino acid sequence is BAA32230.1, and the GENBANK accession number for the feline IgG2 heavy chain amino acid sequence is KF811175.1. The feline antibody also includes two types of light chains: κ light chains and λ light chains. The DNA and amino acid sequences of these light chains are also available from the GENBANK database. For example, the accession number for the feline κ light chain amino acid sequence is AF198257.1, and the accession number for the feline λ light chain amino acid sequence is E07339.1. The feline antibody is described in more detail in U.S. Patent No. 11,498,953 to Brondyk et al.; the contents of which are incorporated herein by reference in their entirety.

[0113] It should be understood that any of the above-mentioned polypeptide molecules, alone or in combination, are suitable for preparation as a stabilizing agent according to the present invention.

[0114] B. Excipients

[0115] In various embodiments, the present invention provides a formulation that may include various excipients, including but not limited to buffers, antioxidants, osmolarity regulators, and stabilizers. Furthermore, the formulation may contain additional agents (e.g., HCl) for pH adjustment and diluents (e.g., water). In other embodiments, different forms of histidine may be used for pH adjustment. To some extent, the role of excipients is to maintain the stability and biological activity of antibodies (e.g., by maintaining the correct conformation of the protein), and / or to maintain pH.

[0116] 1. Buffer

[0117] In various aspects of this invention, formulations according to the invention include a buffering agent (buffer). The buffer serves to maintain a physiologically suitable pH, but it can also be used to enhance the isotonicity and chemical stability of the formulation. According to the invention, the buffer brings the pH of the liquid composition closer to the physiological pH to reduce the risk of pain or allergic-like side effects during injection, and also enhances the stability of the antibody and its resistance to aggregation, oxidation, and fragmentation. In various embodiments of the invention, the pH of the formulation is from about 4.0 to about 7.2, from about 5.5 to about 6.5, preferably from about 6.0 to about 6.5. In one particular embodiment, the pH of the formulation is about 6. Ranges between the above-mentioned pH values ​​(e.g., from about pH 4.0 to about pH 7.2, preferably pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, or pH 6.8) are also intended to be part of the invention. For example, any combination of the above values ​​used as upper and / or lower limits of the value range is intended to be included. If necessary, pH can be adjusted using techniques known in the art. For example, if necessary, HCl can be added to adjust the pH to the desired level, or different forms of histidine can be used to adjust the pH to the desired level.

[0118] Buffers may include, but are not limited to, acetates (sodium or potassium acetate), succinates (sodium or potassium succinate), histidine, phosphates (sodium or potassium phosphate), Tris (tris(hydroxymethyl)aminomethane), diethanolamine, citrates, other organic acids, amino acids (such as proline, arginine) and their salts, and mixtures thereof. In one embodiment, the buffer is histidine (e.g., L-histidine). In another specific embodiment, the buffer is a succinate. In yet another embodiment, the formulation comprises an amino acid, including but not limited to histidine, present in an amount sufficient to maintain the formulation at a physiologically suitable pH. Histidine is an exemplary amino acid that has buffering capacity within a physiological pH range. The buffering capacity of histidine derives from its imidazole group. In one exemplary embodiment, the buffer is L-histidine (base) (e.g., C6H9N3O2, FW: 155.15). In another embodiment, the buffer is L-histidine monochloride monohydrate (e.g., C6H9N3O2.HCl.H2O, FW: 209.63). In another embodiment, the buffer is L-histidine hydrochloride (e.g., C6H42-Hydroxychloride). 10 ClN3O2 (FW: 191.61). In another exemplary embodiment, the buffer is a mixture of L-histidine (base) and L-histidine monochloride monohydrate.

[0119] In one embodiment, the concentration of the buffer (e.g., L-histidine, acetate, or succinate) is from about 0.1 mM to about 50 mM, from about 0.1 mM to about 40 mM, from about 0.1 mM to about 30 mM, from about 0.1 mM to about 25 mM, from about 0.1 mM to about 20 mM, or from about 5 mM to about 15 mM, preferably 15 mM or 25 mM. In various embodiments, the concentration of the buffer can be from about 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, or 25 mM. In a particular embodiment, the buffer is present at about 18 mM, 19 mM, 20 mM, 21 mM, or 22 mM. Ranges between the above concentrations, for example from about 16 mM to about 24 mM, are also intended to be part of the invention. For example, any combination of the above values ​​as upper and / or lower limits is intended to be included. In some embodiments, the amount of buffer present is sufficient to maintain a physiologically suitable pH.

[0120] 2. Osmotic pressure regulators and / or stabilizers

[0121] In various aspects of the invention, the formulation includes a stabilizer, which may also act as an osmolarity regulator. To some extent, the osmolarity regulator helps maintain the isotonicity of the formulation and maintains protein levels. To some extent, the stabilizer helps maintain the level, ratio, or proportion of therapeutically active peptides present in the formulation. As used herein, the term "osmolarity" refers to the behavior of a biological component in a fluid environment or solution. An isotonic solution has the same osmolarity as plasma and can therefore be administered intravenously to a subject without altering the osmolarity of the subject's plasma. Indeed, in one embodiment of the invention, the amount of osmolarity regulator and / or stabilizer present is sufficient to make the formulation suitable for intravenous infusion. Typically, the osmolarity regulator also acts as a stabilizer. Thus, the agent enables the protein to overcome various pressures, such as freezing, thawing, transport, high temperatures, and shear forces.

[0122] Osmotic regulators and / or stabilizers may include, but are not limited to, CaCl2, NaCl, MgCl2, lactose, sorbitol, sucrose, mannitol, trehalose, raffinose, polyethylene glycol, hydroxyethyl starch, glycine, and mixtures thereof. In a preferred embodiment, the osmotic regulator and / or stabilizer is sucrose, trehalose, or sorbitol. According to the invention, the stabilizer imparts enhanced antibody stability and resistance to aggregation, oxidation, and fragmentation to the liquid composition during refrigerated storage (e.g., 0 to 10°C, particularly 5 to 8°C, more specifically 5°C) or frozen storage, and during multiple freeze-thaw cycles.

[0123] In one embodiment, the osmotic pressure regulator and / or stabilizer is present in an amount of about 0.1% to about 35% w / v, or about 3% to about 25% w / v. In another embodiment, the osmotic pressure regulator is present in an amount of about 5% to about 15% w / v. In another embodiment, the osmotic pressure regulator is present in an amount of about 8% to about 10% w / v. In another embodiment, the osmotic pressure regulator is present in an amount of about 20 mg / ml to about 60 mg / ml, about 30 mg / ml to about 50 mg / ml, or about 35 mg / ml to about 45 mg / ml. Preferably, the osmotic pressure regulator is present in an amount of about 4% w / v. In another embodiment, the osmotic pressure regulator is present in an amount of about 5% w / v. In another specific embodiment, the osmotic pressure regulator is present in an amount of about 6% w / v. In another specific embodiment, the osmotic pressure regulator is present in an amount of about 7% w / v. In yet another specific embodiment, the osmotic pressure regulator is present in an amount of about 8% w / v. In another specific embodiment, the osmotic pressure regulator is present in an amount of about 9% w / v. In yet another specific embodiment, the amount of the osmotic pressure regulator present is approximately 10% w / v.

[0124] The concentration range between the above-mentioned concentrations, for example, from about 3% to about 12% w / v, is also intended to be part of this invention. For example, any combination of the above values ​​as upper and / or lower limit value ranges is intended to be included. The osmotic pressure regulator and / or stabilizer should be present in sufficient quantities to maintain the osmotic pressure of the formulation.

[0125] 3. Chelating agents

[0126] In various aspects of the invention, the formulation includes a chelating agent that imparts enhanced antibody stability and / or resistance to aggregation to the liquid composition. The chelating agent in the compositions of the present invention can reduce the formation of reducing oxygen species, reduce the formation of acidic species (e.g., deamidation), reduce antibody aggregation, and / or reduce antibody fragmentation, and / or reduce antibody oxidation. Compared to antibodies not protected with a chelating agent, such a chelating agent can reduce or prevent the degradation of the formulated antibody.

[0127] According to one embodiment of the invention, the chelating agent may be selected from the group consisting of: aminopolycarboxylic acids, hydroxyaminocarboxylic acids, N-substituted glycine, 2-(2-amino-2-oxoethyl)aminoethanesulfonic acid (BES), deferoxamine (DEF), citric acid, nicotinamide, and deoxycholate, and mixtures thereof. Another embodiment covers the chelating agent selected from the group consisting of: ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), nitrotriacetic acid (NTA), N-2-acetamido-2-iminodiacetic acid (ADA), bis(aminoethyl)ethylene glycol ether, N,N,N′,N′-tetraacetic acid (EGTA), trans-diaminocyclohexanetetraacetic acid (DCTA), glutamic acid and aspartic acid, N-hydroxyethyliminodiacetic acid (HIMDA), N,N-bishydroxyethylglycine (bicine), and N-(tris(hydroxymethyl)methyl) 10. Glycine, glycylglycine, sodium deoxycholate, ethylenediamine, propylenediamine, diethylenetriamine, triethylenetetramine, ethylenediaminetetraacetic acid (EDTA), disodium EDTA, calcium EDTA, oxalic acid, malate, citric acid, citric acid monohydrate and trisodium citrate dihydrate, 8-hydroxyquinoline salts, amino acids, histidine, cysteine, methionine, peptides, polypeptides and proteins, and mixtures thereof. In another aspect of the invention, the chelating agent is selected from the group consisting of: EDTA salts, including disodium ethylenediaminetetraacetate, disodium ethylenediaminetetraacetate, calcium sodium ethylenediaminetetraacetate, sodium ethylenediaminetetraacetate, trisodium ethylenediaminetetraacetate and potassium ethylenediaminetetraacetate; and a suitable salt of deferoxamine (DEF) is deferoxamine methanesulfonate (DFM), or a mixture thereof. Where possible, the chelating agents used in this invention may exist in the form of free acid or free base or salt of the compound, or in anhydrous, solvated or hydrated form of the compound or the corresponding salt.

[0128] In yet another embodiment of the invention, the chelating agent is disodium EDTA or calcium EDTA.

[0129] The concentration of the chelating agent is generally in the following ranges: about 0.01 mM to about 50 mM, about 1 mM to about 10.0 mM, about 15 mM to about 5.0 mM, about 0.01 mM to about 1.0 mM, or about 0.03 mM to about 0.5 mM. In one embodiment, the concentration of the chelating agent is generally in the following ranges: about 0.01 mM to about 2.0 mM, about 0.01 mM to about 1.5 mM, about 0.01 mM to about 0.5 mM, about 0.01 mM to about 0.4 mM, about 0.01 mM to about 0.3 mM, about 0.01 mM to about 0.2 mM, about 0.01 mM to about 0.15 mM, about 0.01 mM to about 0.1 mM, about 0.01 mM to about 0.09 mM, about 0.01 mM to about 0.08 mM, about 0.01 mM to about 0.07 mM, about 0.01 mM to about 0.06 mM, about 0.01 mM to about 0.05 mM, about 0.01 mM to about 0.04 mM, about 0.01 mM to about 0.03 mM, about 0.01 mM to about 0.02 mM. The concentration of the chelating agent may be from about 0.01 mM or about 0.05 mM. In another embodiment, the concentration of the chelating agent may be about 0.01 mg / ml, 0.02 mg / ml, 0.03 mg / ml, about 0.04 mg / ml, about 0.05 mg / ml, about 0.06 mg / ml, about 0.07 mg / ml, about 0.10 mg / ml, or about 0.20 mg / ml. Other embodiments of the chelating agent concentration consider using about 0.045 mM, about 0.046 mM, about 0.047 mM, about 0.048 mM, about 0.049 mM, about 0.05 mM, about 0.051 mM, about 0.052 mM, about 0.053 mM, about 0.054 mM, or about 0.055 mM. The range between the above concentrations, for example from about 0.04 mM to about 0.06 mM, is also intended to be part of this invention.

[0130] Unless otherwise stated, the concentrations listed herein are concentrations under ambient conditions [i.e., 25°C and atmospheric pressure].

[0131] 4. Antioxidants

[0132] In various aspects of the invention, the formulation includes an antioxidant to partially preserve the formulation (e.g., by preventing oxidation). Preferably, the antioxidant is selected from the group consisting of methionine, sodium thiosulfate, catalase, and platinum.

[0133] Antioxidants may include, but are not limited to, GLA (gamma-linolenic acid)-lipoic acid, DHA (docosahexaenoic acid)-lipoic acid, GLA-tocopherol, di-GLA-3,3′-thiodipropionic acid, and typically, any of the following: GLA, DGLA (dihomo-gamma-linolenic acid), AA (arachidonic acid), SA (salicylic acid), EPA (eicosapentaenoic acid), or DHA (docosahexaenoic acid), and any natural or synthetic antioxidants that can be chemically linked to them. These antioxidants include phenolic antioxidants (e.g., eugenol, sage, caffeic acid, BHT (butylated hydroxyanisole), gallic acid, tocopherols, tocotrienols, and flavonoid antioxidants (e.g., myricetin and fisetin)), polyenes (e.g., retinic acid), and unsaturated sterols (e.g., Δ... 5 - Acids containing oat sterols, organosulfur compounds (e.g., allicin), terpenoids (e.g., geraniol, abietic acid), and amino acid antioxidants (e.g., methionine, cysteine, carnosine). In one embodiment, the antioxidant is ascorbic acid. In another embodiment, the antioxidant is methionine or an analogue thereof, such as selenomethionine, hydroxymethylbutyric acid, ethionine, or trifluoromethionine.

[0134] In one embodiment, the antioxidant is present at concentrations of about 0.1 mM to about 50 mM, about 0.1 mM to about 40 mM, about 0.1 mM to about 30 mM, about 0.1 mM to about 20 mM, or about 5 mM to about 15 mM. In various embodiments, the antioxidant may be present at concentrations of about 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, or 15 mM. In one particular embodiment, the antioxidant is present at about 5 mM. In another particular embodiment, the antioxidant is present at about 10 mM. In yet another particular embodiment, the antioxidant is present at about 15 mM. Ranges between the above concentrations, for example, about 3 mM to about 17 mM, are also intended to be part of the invention. For example, any combination of the above values ​​as upper and / or lower limits is intended to be included. In some implementations, the antioxidant should be present in sufficient quantities to preserve the formulation in part by preventing oxidation.

[0135] 5. Surfactants

[0136] In various aspects of the invention, the formulation includes a surfactant that, according to the invention, imparts enhanced antibody stability and / or resistance to aggregation and fragmentation to the liquid composition.

[0137] According to a preferred embodiment of the present invention, the surfactant is preferably selected from the group consisting of: polysorbate, poloxamer, triaton, sodium lauryl sulfate, sodium lauryl sulfate, sodium octyl glucoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, and so on. Myristyl-betaine, cetyl-betaine, lauramidopropyl-betaine, cocamidopropyl-betaine, linoleamide-propyl-betaine, myristamidopropyl-betaine, palmitoamide-propyl-betaine, isostearamidopropyl-betaine, myristamidopropyl-dimethylamine, palmitoamide-propyl-dimethylamine, isostearamidopropyl-dimethylamine, sodium methylcocoyl taurate, disodium methyloleoyl taurate, dihydroxypropyl PEG 5 linoleamide chloride, polyethylene glycol, polypropylene glycol, and mixtures thereof. Furthermore, preferably, the surfactant is selected from the group consisting of polysorbate 20, polysorbate 21, polysorbate 40, polysorbate 60, polysorbate 61, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85, and mixtures thereof. More preferably, the surfactant is selected from polysorbate 20, polysorbate 80, PEG3350, or mixtures thereof.

[0138] The concentration of the surfactant is generally in the following ranges: about 0.01% to about 10% (w / v), about 0.01% to about 5.0%, about 0.01% to about 2.0 mg / ml, about 0.01% to about 1.5%, about 0.01% to about 1.0%, about 0.01% to about 0.5%, about 0.01% to about 0.4%, about 0.01% to about 0.3%, about 0.01% to about 0.2%, about 0.01% to about 0.15%, about 0.01% to about 0.1%, or about 0.01% to about 0.05%. More preferably, the concentration of the surfactant is about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.10%, about 0.11%, about 0.12%, about 0.13%, about 0.14%, about 0.15%, about 0.16%, or about 0.17%. Most preferably, the concentration of the surfactant is about 0.02%. Embodiments with surfactant concentrations of 0.01%, about 0.02%, about 0.03%, about 0.04%, or about 0.05% are particularly preferred because such concentrations allow the stability of the antibody in the formulation in solution to be maintained, while also reducing the tendency of the formulation to form bubbles due to stresses associated with shaking and stirring during formulation preparation, formulation handling, and preparation for parenteral administration, especially during preparation and during transport.

[0139] 5. Preservatives

[0140] In various aspects of the invention, the formulation includes an antimicrobial preservative. In one embodiment, the preservative is selected from the group consisting of phenol, m-cresol, benzyl alcohol, benzalkonium chloride, benzyl chloride, phenoxyethanol, and methylparaben, or mixtures thereof.

[0141] The concentration of preservatives is generally in the following ranges: about 0.001% w / v to 10% w / v, about 0.005% w / v to 5% w / v, about 0.008% w / v to 2.0% w / v, or about 0.01% w / v to 1.5% w / v. Preferred preservative concentrations may be approximately 0.1% w / v, 0.2% w / v, 0.3% w / v, approximately 0.4% w / v, approximately 0.5% w / v, approximately 0.6% w / v, approximately 0.7% w / v, 0.8% w / v, 0.9% w / v, approximately 1.0% w / v, 2.0% w / v, 3.0% w / v, approximately 4.0% w / v, approximately 5.0% w / v, approximately 6.0% w / v, approximately 7.0% w / v, 8.0% w / v, 9.0% w / v, approximately 9.1% w / v, approximately 9.2% w / v, 9.3% w / v, 9.4% w / v, 9.5% w / v, 9.6% w / v, 9.7% w / v, 9.8% w / v, 9.9% w / v, and 10.0% w / v. In a particular implementation, the concentration of the preservative is from about 0.01% w / v to 9.0% w / v.

[0142] C. Pharmaceutical Composition

[0143] To prepare pharmaceutical or sterile compositions of antibodies or their antigen-binding fragments, the formulation may be mixed with a pharmaceutically acceptable carrier or excipient. For further information, please refer to various references and manuals in the art. For example, see [link to relevant documentation]. Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary , Mack Publishing Company, Easton, Pa. (1984) (The contents of which are hereby incorporated in their entirety by reference).

[0144] Formulations of therapeutic and diagnostic agents can also be prepared by mixing with acceptable carriers, excipients, or stabilizers, for example, in the form of lyophilized powders, slurries, aqueous solutions, or suspensions (see, for example, 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: Marcel Dekker, NY; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems Marcel Dekker, NY; and Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY). In one embodiment, the antibody of the present invention is diluted to an appropriate concentration in a sodium acetate solution at pH 4.5-7.0, and sorbitol or sucrose is added to adjust osmotic pressure and / or stabilize the protein. Other agents such as polysorbate 20 or polysorbate 80 may be added to enhance stability.

[0145] The toxicity and therapeutic activity or efficacy of antibody compositions, whether administered alone or in combination with another agent, can be determined in cell cultures or laboratory animals using standard pharmaceutical procedures, such as determining the LD50. 50 (Dose causing 50% mortality in the population) and ED 50 (The dose effective for 50% of the population). The dose ratio between toxic effects and therapeutic effects is the therapeutic index (LD50). 50 / ED 50 In certain respects, antibodies exhibiting a high therapeutic index are ideal. Data obtained from these cell culture assays and animal studies can be used to determine dosage ranges suitable for individuals, including humans, dogs, cats, and other mammals. The dosages of these compounds are preferably within the range of circulating concentrations, including ED... 50 It has very low or no toxicity. The dosage may vary within this range depending on the dosage form and route of administration.

[0146] D. Application

[0147] The administration methods of the formulations according to the invention may vary. Suitable routes of administration include oral, rectal, mucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intravenous, intravenous, intranasal, intraocular, inhalation, inhalation, local, skin, transdermal, or intra-arterial administration. In certain embodiments, the antibody formulations of the invention or their antigen-binding fragments may be administered via a parenteral route, such as by injection. In other embodiments of the invention, the antibodies or their antigen-binding fragments, or pharmaceutical compositions thereof, may be administered via intravenous, subcutaneous, intramuscular, intra-arterial, intratumoral administration, or by inhalation (including delivery via aerosol). Administration via non-invasive routes (e.g., oral, including but not limited to alkyl, capsule, or tablet) is also within the scope of the invention.

[0148] According to one embodiment of this invention, the formulation can be directly administered into the bloodstream, muscle, tissue, fat, or visceral organs. Suitable parenteral administration methods include intravenous, intra-arterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular, intraosseous, intradermal, and subcutaneous administration. Suitable devices for parenteral administration include needle injectors (including microneedles, micro-projection, soluble needles, and other micropore-forming technologies), needle-free injectors, and infusion techniques.

[0149] The pharmaceutical compositions disclosed herein can also be administered by infusion. Well-known examples of implants and modules for administering pharmaceutical compositions include: U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing drugs at a controlled rate; U.S. Patent No. 4,447,233, which discloses a drug infusion pump for delivering drugs at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion device for continuous drug delivery; and U.S. Patent No. 4,439,196, which discloses a permeation drug delivery system with a multi-compartmental structure. Many other such implants, delivery systems, and modules are well known to those skilled in the art.

[0150] Alternatively, antibodies can be administered locally rather than systemically, for example, by injecting them directly into arthritic joints or lesions with immunopathological features induced by pathogens, typically in a stockpiled or sustained-release formulation. Furthermore, targeted drug delivery systems, such as liposomes coated with tissue-specific antibodies, can be used to administer antibodies, for example, those targeting arthritic joints or lesions with immunopathological features induced by pathogens. The liposomes will target the affected tissue and be selectively absorbed by it.

[0151] Administration regimens depend on several factors, including serum or tissue turnover of the therapeutic antibody, symptom level, immunogenicity of the therapeutic antibody, and accessibility of target cells in the biological matrix. Preferably, the administration regimen delivers sufficient therapeutic antibody to improve the target disease state while minimizing undesirable side effects. Therefore, the delivery amount of the biologic depends in part on the specific therapeutic antibody and the severity of the disease being treated. Guidance on selecting an appropriate dose of therapeutic antibody is available [see, for example, Wawrzynczak]. Antibody Therapy , Bios Scientific Pub.Ltd, Oxfordshire, UK (1996); Kresina (ed.) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY (1991); Bach (ed.) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases , Marcel Dekker, New York, NY (1993); Baert et al., New Engl. J. Med. 348:601-608 (2003); Milgrom et al., New Engl. J. Med. 341:1966-1973 (1999); Slamon et al., New Engl. J. Med. 344:783-792 (2001); Beniaminovitz et al., New Engl. J. Med. 342:613-619 (2000); Ghosh et al., New Engl. J. Med. 348:24-32 (2003); Lipsky et al., New Engl. J. Med. 343:1594-1602 (2000)].

[0152] Those skilled in the art will readily understand how to determine an appropriate dosage and can use well-known parameters or factors that are suspected of affecting treatment to determine the appropriate dosage. Dosage typically begins at a slightly below-optimal level and is then increased in small increments until the desired or optimal effect is achieved relative to any negative side effects. Important diagnostic measures include monitoring factors associated with symptoms. For example, the levels of inflammation or inflammatory cytokines produced after treatment can be determined and / or monitored.

[0153] The antibodies (including their antigen-binding fragments) disclosed herein may be administered via continuous infusion or in multiple doses at various time intervals, including daily, 1–7 times per week, once per week, once every two weeks, once per month, once every two months, once per quarter, once every six months, and once per year. Dosage may be administered via various routes, including but not limited to intravenous, subcutaneous, topical, oral, nasal, rectal, intramuscular, intracerebral, intraspinal, or inhalation. The total weekly dose is typically at least 0.05 μg / kg body weight, and more typically at least 0.2 μg / kg, 0.5 μg / kg, 1 μg / kg, 10 μg / kg, 100 μg / kg, 0.25 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 5.0 mg / ml, 10 mg / kg, 25 mg / kg, 50 mg / kg, or higher doses [see, for example, Yang et al., New Engl. J. Med. 349:427-434 (2003); Herold et al., New Engl. J. Med. 346:1692-1698 (2002); Liu et al., J. Neurol. Neurosurg. Psych. 67:451-456 (1999); Portielji et al., Cancer Immunol. Immunother. 52:133-144 (2003)). The provided dosage can also achieve a predetermined target concentration of antibody in the subject's serum, such as 0.1, 0.3, 1, 3, 10, 30, 100, 300 μg / ml or higher. In other embodiments, the antibody of the present invention is administered subcutaneously or intravenously at doses of 10, 20, 50, 80, 100, 200, 500, 1000 or 2500 mg per subject, once weekly, every two weeks, "every four weeks", monthly, every two months or quarterly.

[0154] In some implementations, the drug is administered in a dose once a week, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, every eight weeks, every nine weeks, every ten weeks, every fifteen weeks, every twenty weeks, every twenty-five weeks, or every twenty-six weeks. In some implementations, the anti-NGF antagonist antibody is administered monthly, every two months, every three months, every four months, every five months, or every six months. Most preferably, the drug is administered in a dose every eight weeks.

[0155] In some embodiments, the volume of the dose is less than or equal to about 20 ml, about 15 ml, about 10 ml, about 5 ml, about 2.5 ml, about 1.5 ml, about 1.0 ml, about 0.75 ml, about 0.5 ml, about 0.25 ml, or about 0.01 ml.

[0156] In some embodiments, the dosage is a volume of about 20 ml, about 19 ml, about 18 ml, about 17 ml, about 16 ml, about 15 ml, about 14 ml, about 13 ml, about 12 ml, about 11 ml, about 10 ml, about 9 ml, about 8 ml, about 7 ml, about 6 ml, about 5 ml, about 4 ml, about 3 ml, about 2 ml, or about 1 ml. Or about 20.5 ml, about 19.5 ml, about 18.5 ml, about 17.5 ml, about 16.5 ml, about 15.5 ml, about 14.5 ml, about 13.5 ml, about 12.5 ml, about 11.5 ml, about 10.5 ml, about 9.5 ml, about 8.5 ml, about 7.5 ml, about 6.5 ml, about 5.5 ml, about 4.5 ml, about 3.5 ml, about 2.5 ml, about 1.5 ml, or about 0.5 ml. Or approximately 900 μL, approximately 800 μL, approximately 700 μL, approximately 600 μL, approximately 500 μL, approximately 400 μL, approximately 300 μL, approximately 200 μL, or approximately 100 μL; or approximately 950 μL, approximately 850 μL, approximately 750 μL, approximately 650 μL, approximately 550 μL, approximately 450 μL, approximately 350 μL, approximately 250 μL, approximately 150 μL, or approximately 50 μL. Most preferably, the volume of the dose is less than or equal to approximately 2.5 ml.

[0157] According to one embodiment of the present invention, the concentration range of the antibody can be from about 0.1 to about 200 mg / ml. Preferably, the antibody concentration is approximately 0.5 mg / ml, approximately 1 mg / ml, approximately 2 mg / ml, approximately 2.5 mg / ml, approximately 3 mg / ml, approximately 3.5 mg / ml, approximately 4 mg / ml, approximately 4.5 mg / ml, approximately 5 mg / ml, approximately 5.5 mg / ml, approximately 6 mg / ml, approximately 6.5 mg / ml, approximately 7 mg / ml, approximately 7.5 mg / ml, approximately 8 mg / ml, approximately 8.5 mg / ml, approximately 9 mg / ml, approximately 9.5 mg / ml, approximately 10 mg / ml, approximately 11 mg / ml, approximately 12 mg / ml, approximately 13 mg / ml, approximately 14 mg / ml, approximately 15 mg / ml, approximately 16 mg / ml, approximately 17 mg / ml, approximately 18 mg / ml, approximately 19 mg / ml, approximately 20 mg / ml, approximately 21 mg / ml, approximately 22 mg / ml, approximately 23 mg / ml, approximately 24 ... mg / ml, approximately 25 mg / ml, approximately 26 mg / ml, approximately 27 mg / ml, approximately 28 mg / ml, approximately 29 mg / ml, approximately 30 mg / ml, approximately 31 mg / ml, approximately 32 mg / ml, approximately 33 mg / ml, approximately 34 mg / ml, approximately 35 mg / ml, approximately 36 mg / ml, approximately 37 mg / ml, approximately 38 mg / ml, approximately 39 mg / ml, approximately 40 mg / ml, approximately 41 mg / ml, approximately 42 mg / ml, approximately 43 mg / ml, approximately 44 mg / ml, approximately 45 mg / ml, approximately 46 mg / ml, approximately 47 mg / ml, approximately 48 mg / ml, approximately 49 mg / ml, approximately 50 mg / ml, approximately 51 mg / ml, approximately 52 mg / ml, approximately 53 mg / ml, approximately 54 mg / ml, approximately 55 mg / ml, approximately 56 mg / ml, approximately 57 mg / ml, approximately 58 mg / ml, approximately 59 mg / ml The concentrations of the antibody may be selected from the group consisting of approximately 2 mg / ml, approximately 2.5 mg / ml, approximately 5 mg / ml, approximately 10 mg / ml, approximately 20 mg / ml, approximately 30 mg / ml, approximately 40 mg / ml, approximately 50 mg / ml, 60 mg / ml, approximately 70 mg / ml, approximately 80 mg / ml, approximately 90 mg / ml, or approximately 100 mg / ml.

[0158] According to a contemplated embodiment of the present invention, the dosage contains less than or equal to about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 26 mg, about 27 mg, about 28 mg, about 29 mg, about 30 mg, about 31 mg, about 32 mg, about 33 mg, about 34 mg, about 35 mg, about 36 mg, about 37 mg, about 38 mg, about 39 mg, about 40 mg, about 41 mg, about 42 mg, about 43 mg, about 44 mg, about 45 mg, about 46 mg, about 47 mg, about 48 mg, etc. Antibodies in mg, approximately 49 mg, approximately 50 mg, approximately 51 mg, approximately 52 mg, approximately 53 mg, approximately 54 mg, approximately 55 mg, approximately 56 mg, approximately 57 mg, approximately 58 mg, approximately 59 mg, approximately 60 mg, approximately 70 mg, approximately 80 mg, approximately 90 mg, approximately 100 mg, or approximately 110 mg.

[0159] According to another embodiment, the dose comprises an antibody amount of about 1 μg / kg, about 10 μg / kg, about 20 μg / kg, about 25 μg / kg, about 50 μg / kg, about 100 μg / kg, about 200 μg / kg, about 250 μg / kg, about 500 μg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, or about 11 mg / kg (based on the mass of the mammal to which the dose is administered). In yet another embodiment, the dose contains about 20 μg / kg, about 25 μg / kg, about 50 μg / kg, about 100 μg / kg, about 200 μg / kg, about 250 μg / kg, 1 mg / kg, or about 2 mg / kg.

[0160] Dosing regimens can be varied over time according to the pharmacokinetic decay pattern desired by the healthcare professional. For example, in some embodiments, even less frequent dosing may be used, although one to four times a week is considered. In some embodiments, the dose is administered every 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, or longer. In some embodiments, the dose is administered every 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or longer. In yet another embodiment, the dose is administered every eight weeks. The progress of this therapy is easily monitored using routine techniques and assays.

[0161] For the purposes of this invention, the appropriate dosage of the drug will depend on the antibody used. For example, administration of a formulation containing a peptide comprising a canine IgG Fc region variant or its canine FcRn binding region can manage pain. The dosage will depend on a variety of factors, including but not limited to the type and severity of the pain to be treated; whether the administration is for prevention or treatment; prior treatment; the patient's clinical history and response to the agent; the physician's judgment, etc. Typically, clinicians will administer the drug until a dosage is reached to achieve the desired outcome. The dosage can be determined empirically. For example, incremental doses can be given to animals or individuals to assess the efficacy of the drug, followed by tracking of pain indicators, such as changes in the pain numerical rating scale (NRS).

[0162] The dosage and / or frequency of administration of the formulations according to the invention may vary during treatment. Empirical considerations such as antibody half-life often help in determining the dosage. The frequency of administration may be determined and adjusted during treatment and is generally (but not necessarily) based on the treatment and / or suppression and / or improvement and / or delay of pain. Some animals or individuals may require more than one dose. The frequency of administration may be determined and adjusted during treatment. For repeated administration over several days or longer, treatment may continue until the desired suppression of symptoms is achieved, or until a level of treatment is reached sufficient to alleviate the pain, depending on the pain and its severity.

[0163] The formulation may be provided in single-dose or multi-dose form in vials or syringes. Administration of formulations containing liquid compositions may be continuous or intermittent, depending on several factors, including but not limited to the recipient's physiological condition, whether the purpose of administration is treatment or prevention, and other factors known to a skilled practitioner. Administration of a drug containing a liquid composition may be substantially continuous over a pre-selected period of time, or may be divided into a series of interval doses, such as administration before, during, or after the onset of pain.

[0164] In one embodiment, the dosage administration is considered to be parenteral, preferably selected from intravenous, intra-arterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular, intraosseous, intradermal, and subcutaneous routes. One embodiment considers providing the formulation in a unit-dose sterile liquid form suitable for parenteral administration.

[0165] Therapeutic efficacy can be assessed by monitoring the disease or condition. For example, pain relief may be characterized by the duration of relief. Thus, in some embodiments, relief is observed within approximately 24 hours after administration. In other embodiments, relief is observed within approximately 36 hours, 48 ​​hours, 60 hours, 72 hours, or 4 days after administration. In some embodiments, the frequency and / or intensity of pain is reduced, and / or the quality of life of the patient with pain is improved. In some embodiments, the duration of pain relief provided after a single dose is at least approximately 7 days, at least approximately 14 days, at least approximately 21 days, at least approximately 28 days, at least approximately 35 days, at least approximately 42 days, at least approximately 49 days, at least approximately 56 days, at least approximately 63 days, at least approximately 70 days, at least approximately 77 days, at least approximately 84 days, at least approximately 180 days, or longer.

[0166] The term “and / or” refers to one or more of the listed elements, or a combination of any two or more of the listed elements.

[0167] The terms "preferred" and "preferably" refer to embodiments of the invention that provide certain benefits in certain circumstances. However, other embodiments may also be preferred in the same or other circumstances. Furthermore, the listing of one or more preferred embodiments does not imply that other embodiments are useless, nor is it intended to exclude other embodiments from the scope of the invention.

[0168] The term "comprising" and its variations, when used in the specification and claims, are not intended to be restrictive.

[0169] Unless otherwise stated, “a”, “an”, “the” and “at least one” are used interchangeably and mean one or more.

[0170] Unless otherwise indicated, all figures used in the specification and claims to represent the quantity, molecular weight, etc., of components should be understood to be modified by the term "about" in all cases. Therefore, unless otherwise indicated, the numerical parameters set forth in the specification and claims are approximations and may vary according to the desired characteristics sought to be obtained according to the invention. To a minimum, and without attempting to limit the doctrine of equivalence to the scope of the claims, each numerical parameter should be interpreted at least according to the number of significant figures reported and by applying ordinary rounding techniques.

[0171] Although the numerical ranges and parameters stating the broad scope of the invention are approximations, the values ​​stated in particular examples are reported as accurately as possible. However, all values ​​inherently contain ranges that are necessarily generated by the standard deviations found in their respective test measurements.

[0172] For any method disclosed herein that includes discontinuous steps, these steps can be performed in any feasible order. Furthermore, any combination of two or more steps can be performed simultaneously, where appropriate.

[0173] The description illustrates exemplary embodiments. Guidance is provided throughout this application by way of a list of embodiments that can be used in various combinations. In each case, the list is intended as a representative group only and should not be construed as an exclusive list.

[0174] All headings are for the reader's convenience and should not limit the meaning of the text following them, unless otherwise stated.

[0175] The present invention is illustrated by the following embodiments. It should be understood that the specific embodiments, materials, quantities, and procedures described herein, within the scope and spirit of the invention, should be interpreted broadly.

[0176] Example

[0177] Example 1: pH / buffer screening study of IVX-01101 monoclonal antibody

[0178] This article presents a two-part design for the development of antibody stable formulations. The antibody used in this study is the IVX-01101 monoclonal antibody.

[0179] First, a pH / buffer screening study was conducted under pressure conditions (40°C incubation) to select the optimal pH / buffer system that would maximize antibody stability. Next, a screening study was conducted to evaluate and select excipient and surfactant strengths based on their stabilizing effects on IVX-01101 under pressure conditions (40°C incubation, multiple freeze-thaw cycles, and stirring).

[0180] Biophysical and analytical methods included appearance, pH, protein concentration (conc.), osmolality, subvisible particles (microfluidic imaging (MFI)), size exclusion-ultra-high performance liquid chromatography (SE-UPLC), imaging capillary isoelectric focusing (iCIEF), reduced and non-reduced CE-sodium dodecyl sulfate (CE-SDS-R & NR), and differential scanning calorimetry (DSC). Potency testing was performed on selected formulations.

[0181] Table 1 lists the data summary (DS) information used in formulation development studies.

[0182] Table 1. DS Information

[0183]

[0184] Materials and methods

[0185] Appearance Use a YB-2 clarity meter to inspect the appearance of all samples against a black and white background, including clarity, color, and visible particles.

[0186] protein concentration Protein concentration (conc.) was determined using a Thermo Nano Drop One UV spectrophotometer. The extinction coefficient used in all evaluation studies was 1.52 AU*mL*mg. -1 *cm -1 The measurements were repeated twice, with 2.5 μL of sample taken from each sample, and the average result was reported. Measurements for each sample were performed after using water as a blank control.

[0187] pH: pH was measured using a pH meter with a glass electrode. Before use, the pH meter was calibrated using three different standards (pH 4.01, 7.00, and 9.21). The calibration slope was between 95.0% and 105.0%. Each sample was measured twice; the average result was reported.

[0188] osmolar concentration Osmolality was measured using an Advanced 2020 multi-sample osmoremeter. The accuracy of the osmoremeter was verified before and after testing using a Clinitrol 290 mOsm / kg reference solution. The sample volume was 20 μL, and each sample was tested only once.

[0189] MFI:Subvisible particles were monitored using a microflow imaging (MFI) system. In a biosafety cabinet, 1.5 mL of each sample was transferred to an MFI 96-well plate for analysis. Results were analyzed using the vendor's MVAS software. The number of subvisible particles with equivalent circle diameters greater than 2 μm, 5 μm, 10 μm, and 25 μm was reported.

[0190] Differential Scanning Calorimeter (DSC) DSC is a thermal analysis technique that measures the difference in heat required to raise the temperature of a sample or reference material relative to temperature. The sample was diluted to 1 mg / mL with reference buffer. 400 μL of the corresponding reference buffer was added to the odd-numbered wells of a 96-well plate, and 400 μL of the sample was added to the even-numbered wells of the same plate. Experimental parameters were set to a scan temperature that increased from 10℃ to 95℃ at a scan rate of 200℃ / h. Data analysis was performed using Malvern VP DSC automated data analysis software.

[0191] SE-UPLC Size exclusion ultra-high performance liquid chromatography (SE-UPLC) is a purity analysis method that separates proteins based on their size. SE-UPLC was performed using an Agilent UPLC system and an SEC column (Waters Acquity BEH150 × 4.6 mm, 1.7 μm). The sampler temperature was set to 5 ± 3 °C, and the column oven temperature was set to 25 ± 3 °C. The mobile phase was 50 mM PB, 300 mM NaCl, pH 6.8 ± 0.1, and the flow rate was set to 0.4 mL / min. The injection volume for each sample was 10 μg. The detection wavelength was set to 280 nm, and the run time was 8 minutes. Data were analyzed using Agilent CDS software.

[0192] iCIEF:Imaging capillary isoelectric focusing (iCIEF) is a high-resolution technique that segments proteins into arrays based on their isoelectric point (pI). Compared to conventional CIEF systems, iCIEF offers advantages such as faster method development, higher detection sensitivity, greater reliability, and higher analytical throughput. 20 μL of a reference standard or sample (diluted to 1.0 mg / mL) was mixed with 80 μL of a master mixture consisting of: 0.5 μL of a high-pI label (7.05), 0.5 μL of a low-pI label (4.22), 1.0 μL of Pharmalyte 3-10, 3.0 μL of Pharmalyte 5-8, 35 μL of 1% methylcellulose (MC), 37.5 μL of 8 M urea solution, 2.0 μL of 200 mM iminodiacetic acid solution, and 0.5 μL of ultrapure water. The final protein concentration of the sample in the loading mixture was 0.2 mg / mL. The sample mixture was then analyzed using an iCE3 capillary isoelectric focusing analyzer equipped with an FC-COATED full-column detection capillary. Focusing was performed in two steps: (1) 1.5 kV for 1 minute; (2) 3 kV for 8 minutes, with the autosampler tray maintained at 10°C. Absorbance was measured at 280 nm. After analysis, the raw data were processed using Empower 3.

[0193] CE-SDS-NR: Non-reducing capillary electrophoresis with sodium dodecyl sulfate (CE-SDS-NR) was performed using the PerkinElmer Caliper automated electrophoresis system, which separates proteins by size and helps determine purity. In the non-reducing microchip CE-SDS analysis, samples were diluted to 1 mg / mL with pure water. 2 μL of the diluted sample was mixed with 7 μL of sample denaturation solution (a mixture of sample buffer, 10% SDS solution, and 100 mM N-ethylcis-butenedimide (NEM), volume ratio 100:10:4). The thoroughly mixed samples were incubated at 70°C for 10 min. 35 μL of water was added to each sample, vortexed for 10 sec, and centrifuged at 14000 rpm for 1 min. 42 μL of the prepared sample was transferred to a 96-well plate, centrifuged at 4000 rpm for 15 minutes, and then placed on a Labchip GXII plate holder for separation and detection in HT Protein Express LabChip containing decolorizing gel, gel dye, and a lower marker. The results were analyzed using LabChip GX Reviewer software.

[0194] CE - SDS-R:Reduced capillary electrophoresis with sodium dodecyl sulfate (CE-SDS-R) was performed using the PerkinElmerCaliper automated electrophoresis system. This system separates proteins by size after sample reduction and helps determine the purity of antibody heavy and light chains. In the reduced microarray CE-SDS analysis, the sample was diluted to 1 mg / mL with pure water. 2 μL of the diluted sample was mixed with 7 μL of sample denaturation solution (a mixture of sample buffer, 10% SDS solution, and 1 M dithiothreitol as a reducing agent, in a volume ratio of 100:10:4). The thoroughly mixed sample was incubated at 70°C for 10 minutes. 35 μL of water was added to each sample, vortexed for 10 seconds, and centrifuged at 14000 rpm for 1 minute. 42 μL of the prepared sample was transferred to a 96-well plate, centrifuged at 4000 rpm for 15 minutes, and then placed on a Labchip GXII plate holder for separation and detection in an HT Protein Express LabChip plate filled with decolorizing gel, gel dye, and low molecular weight label. The results were analyzed using LabChip GX Reviewer software.

[0195] efficacy Nerve growth factor (NGF) is a member of the neurotrophic factor family and is essential for the development and phenotypic maintenance of neurons in the peripheral nervous system and the functional integrity of cholinergic neurons in the central nervous system. This assay is an ELISA method used to analyze the binding of IVX-01101 monoclonal antibody (mAb) to canine NGF. Samples, assay controls (AC), and standards (STD) were loaded at appropriate dilutions onto 96-well high-binding plates coated with canine NGF antigen protein. After washing, peroxidase-AffiniPure rabbit anti-dog IgG (H+L) antibody was added to each well to allow it to interact with the IVX-01101 mAb captured in the previous step. After a final wash, TMB substrate solution was loaded into each well. TMB reacts specifically with peroxidase in the presence of peroxidase, producing a colorimetric signal proportional to the amount of IVX-01101 bound to the NGF-coated well.

[0196] The first step of the ELISA method is to coat a 96-well plate with 100 µL of 3 µg / mL canine NGF protein solution per well. Seal the plate with sealing film and incubate at 2–8 °C for 16–86 hours. Wash the plate 6 times with approximately 300 µL of 0.05% PBST per well, and add 200 µL of blocking buffer (1% BSA / PBS) per well. Seal the plate with plate sealant and shake it on a plate shaker at 220 RPM and 25 °C for 1–3 hours. Then, transfer 100 µL of diluted IVX-01101 antibody to the wells of the 96-well plate. Standard curves are applied from 20,000 ng / mL to 0 ng / mL. Incubate the plate on a plate shaker at 25 °C and approximately 220 RPM for 70 ± 20 minutes. Add 100 µL of peroxidase-AffiniPure rabbit anti-dog IgG (H+L) antibody solution per well. Seal the plate with plate sealant. Incubate the plate on a plate shaker at 25°C and approximately 220 RPM for 70 ± 20 minutes. Finally, add 100 µL of TMB to each well (for the KPL-5120-0047 kit, mix an equal volume of TMB peroxidase substrate with peroxidase substrate solution B). After 10–20 minutes, stop the reaction by adding 100 µL of 1 M HCl to each well.

[0197] The TMB used in the ELISA method reacts specifically with peroxides in the presence of peroxidase, generating a colorimetric signal that is proportional to the amount of IVX-01101 bound to each well. Dose-response curves for the IVX-01101 data were generated using SoftMax Pro software based on a 4-parameter logistic (automatically estimated) regression model.

[0198] Research Design

[0199] In this formulation development report, the pH / buffer system, excipients, and surfactant strength were screened. Detailed study designs are shown in Table 2.

[0200] Table 2. Study Design for IVX-01101 Formulation Development

[0201]

[0202] pH / buffer solution screening study

[0203] Target The pH / buffer screening aims to select the optimal pH / buffer system that can best stabilize proteins, and is carried out under pressure conditions (40°C incubation).

[0204] Sample preparationFirst, the IVX-01101 active pharmaceutical ingredient (DS, batch number: SH23362110) buffer was exchanged into nine different buffers, including 20 mM L-glutamic acid / NaOH (pH 4.5, pH 5.0), 20 mM citrate monohydrate / sodium citrate dihydrate buffer (pH 4.5, pH 5.0), 20 mM succinate / sodium succinate hexahydrate buffer (pH 5.0), 20 mM L-histidine / L-histidine monohydrochloride buffer (pH 6.5), and 20 mM phosphate buffer (pH 6.5, pH 7.0, pH 7.5). These buffers were then concentrated to the target protein concentration (30.0 mg / mL) (Table 3). The prepared samples were filtered through a 0.22 μm polyvinylidene fluoride (PVDF) membrane and filled into 2 mL glass vials (1.0 mL each). The vials were then immediately stoppered, capped, and labeled. All procedures were performed in a biosafety cabinet.

[0205] Table 3. List of formulation candidates for pH / buffer screening studies

[0206]

[0207] Research parameters: Table 4 shows the sampling and testing plan for the pH / buffer screening study. Samples were stored at 40°C for up to 4 weeks and retrieved at each time point. Tests performed in this study included appearance, pH, protein concentration, SE-UPLC, iCIEF, CE-SDS-NR & R, and DSC.

[0208] Table 4. Sampling and testing plan for pH / buffer solution screening studies

[0209]

[0210] X = Appearance, protein concentration, SE-UPLC, iCIEF, CE-SDS-NR & R

[0211] result

[0212] Table 5 shows a summary of the DSC results obtained from the pH / buffer screening study. T values ​​for all formulations... 起始 The values ​​are all higher than 54.9℃, indicating that the thermal conditions at 40℃ are suitable for pressure studies.

[0213] Table 5. pH / buffer solution screening study: DSC

[0214]

[0215] Appearance , pH and protein concentration:Table 6 provides data obtained from the appearance tests of the pH / buffer screening study.

[0216] Based on the T0 appearance results, all formulations were colorless, slightly opalescent liquids, and not entirely free of visible particles, except for Fb1 (20 mM L-glutamate / NaOH, pH 4.5) and Fb7 (20 mM PB buffer, pH 6.5), which showed no visible particles. Visible particles were observed in all formulations after incubation at 40°C for 1, 2, and 4 weeks. For the pH 5.0 buffers, namely Fb2 (20 mM L-glutamate / NaOH), Fb4 (20 mM citrate monohydrate / sodium citrate dihydrate), and Fb5 (20 mM succinic acid / sodium succinate hexahydrate buffer), the amount of visible particles increased significantly.

[0217] Table 6. pH / Buffer Screening Study: Appearance

[0218]

[0219] Table 7 provides data obtained from pH and concentration tests in the pH / buffer screening study.

[0220] Regarding pH results, the pH of all formulations was near the target value, but after buffer exchange, the pH of the measured Fb2 (20 mML-glutamic acid / NaOH) changed significantly from 5.0 to 5.4. Regarding concentration results, all formulations were near the target value (30.0 mg / mL). After incubation at 40°C for 4 weeks, no significant changes were observed in any of the formulations.

[0221] Table 7. pH / buffer screening study: pH and protein concentration

[0222]

[0223] SE-UPLC: Table 8 provides data obtained from the SE-UPLC test of the pH / buffer screening study.

[0224] A reduction in SEC monomers was observed in all formulations. Fb1 (20 mM L-glutamic acid / NaOH, pH 4.5, 12.3% reduction) and Fb3 (20 mM citrate monohydrate / sodium citrate dihydrate, pH 4.5, 11.3% reduction) showed the largest reductions after incubation at 40°C for 4 weeks. Fb6 (20 mM L-histidine / L-histidine monohydrochloride buffer, pH 6.5, 3.8% reduction), Fb7 (20 mM PB buffer, pH 6.5, 3.6% reduction), and Fb8 (20 mM PB buffer, pH 7.0, 3.9% reduction) showed the smallest reductions after incubation at 40°C for 4 weeks.

[0225] Table 8. pH / buffer solution screening study: SE-UPLC

[0226]

[0227] Abbreviations: HMW, High Molecular Weight; LMW, Low Molecular Weight; ND, Not Detected.

[0228] iCIEF: Table 9 provides the data obtained from the iCIEF test in the pH / buffer screening study.

[0229] After incubation at 40°C for 4 weeks, changes in the iCIEF peak were observed in all formulations. Fb6 (20 mM L-histidine / L-histidine monohydrochloride buffer, pH 6.5) showed the smallest decrease in iCIEF after 4 weeks of incubation at 40°C.

[0230]

[0231] CE-SDS-NR & R: Table 10 provides data obtained from the CE-SDS-NR and CE-SDS-R tests in the pH / buffer screening study.

[0232] For the CE-SDS-NR results, after incubation at 40°C for 4 weeks, a decrease in CE-SDS-NR purity of 7.0%–14.7% was observed in all formulations. Among all formulations, Fb1 (20 mM L-glutamic acid / NaOH, pH 4.5, decrease of 14.7%) and Fb3 (20 mM citric acid monohydrate / sodium citrate dihydrate, pH 4.5, decrease of 14.2%) showed the largest purity decreases. After incubation at 40°C for 1 or 2 weeks, among all formulations, Fb5 (20 mM succinic acid / sodium succinate hexahydrate buffer, pH 6.5, decrease of 7.6%) and Fb7 (20 mM PB buffer, pH 6.5, decrease of 7.0%) showed the smallest purity decreases.

[0233] For the CE-SDS-R results, after incubation at 40°C for 4 weeks, a decrease in CE-SDS-R purity of 1.2%–6.0% was observed in the nine formulations. Among all formulations, Fb9 (20 mM PB buffer, pH 6.5, decrease of 6.0%) showed the largest decrease in purity, while Fb7 (20 mM PB buffer, pH 6.5, decrease of 1.2%) showed the smallest decrease in purity.

[0234] Table 10. pH / buffer screening studies: CE-SDS-NR & R

[0235]

[0236] A pH / buffer screening study investigated nine pH / buffer types under pressure conditions (40°C for up to 4 weeks), and the results showed that the stability of IVX-01101 was closely related to pH and buffer type. Based on all tests performed in this study, the protein exhibited poor stability at pH 4.5 and pH 5.0, while showing better stability at pH 6.5.

[0237] Visually, no visible particles were observed in Fb1 (20 mM L-glutamate / NaOH buffer, pH 4.5) and Fb7 (20 mM PB buffer, pH 6.5) at T0. The number of visible particles was significantly increased in Fb2 (20 mM L-glutamate / NaOH, pH 5.0), Fb4 (20 mM citrate monohydrate / sodium citrate dihydrate, pH 5.0), and Fb5 (20 mM succinate / sodium succinate hexahydrate buffer, pH 5.0), accompanied by a higher level of opalescence. No significant changes in protein concentration were observed using any of the test buffers.

[0238] SE-UPLC results showed that Fb6 (20 mM L-histidine / L-histidine monohydrochloride buffer, pH 6.5), Fb7 (20 mM PB buffer, pH 6.5), and Fb8 (20 mM PB buffer, pH 6.5) produced the highest purity when the proteins were stored at 40°C for 4 weeks.

[0239] The iCIEF results showed that Fb6 (20 mM L-histidine / L-histidine monohydrochloride buffer, pH 6.5) exhibited the least variation on iCIEF.

[0240] The CE-SDS-NR&R results showed that there was little difference in purity when IVX-01101 was prepared in Fb6 (20 mM L-histidine / L-histidine monohydrochloride buffer, pH 6.5) and Fb7 (20 mM PB buffer, pH 6.5).

[0241] In summary, 20 mM L-histidine / L-histidine monohydrochloride buffer was selected for IVX-01101 protein, and its optimal pH was determined to be 6.5.

[0242] Example 2: Screening study of excipient and surfactant strength of IVX-01101

[0243] Target: The excipient and surfactant strength screening study aimed to identify the optimal excipient and surfactant strengths that would best stabilize the exemplary protein. This study was conducted under pressure conditions (40°C incubation, multiple freeze-thaw cycles, and stirring).

[0244] Sample preparation: For Fe1 to Fe11, the DS (batch number: SH23362110) buffer was first exchanged for a pre-prepared 20 mM L-histidine / L-histidine monohydrochloride buffer (pH 6.5). Then, stock solutions were prepared containing 40% (w / w) sucrose, 44% (w / w) trehalose·2H2O dihydrate, 30% (w / w) sorbitol, 700 mM sodium chloride, 700 mM L-arginine hydrochloride, 100 mM L-methionine, 10 mM EDTA, and 5% (w / w) polysorbate 80 (PS80). Based on the formulation of Fe1-Fe11, the amounts of DS, excipient stock solution, and surfactant stock solution were calculated, weighed, and thoroughly mixed. All formulation samples listed in Table 11 were ultimately filtered through a 0.22 μm PVDF membrane, filled into 6 mL glass vials (3.0 mL per vial), immediately stoppered, capped, and labeled.

[0245] Table 11. Screening study of formulation candidates and the strength lists of excipients and surfactants.

[0246]

[0247] Note: All percentages in Table 11 are expressed as % (w / v).

[0248] Research parameters:Table 12 shows the sampling and testing plan for the excipient and surfactant strength screening study. For freeze-thaw (FT), samples were completely frozen at -70°C and thawed at room temperature (RT). For thermal stress, samples were stored at 40°C for up to 4 weeks. For stirring stress, samples were stirred at 300 rpm and 25°C for up to 3 days. Tests in this study included appearance, pH, protein concentration, osmolality, SE-UPLC, CE-SDS-NR&R, iCIEF, MFI, and potency tests.

[0249] Table 12. Sampling and testing plan for the screening study of excipients and surfactant strength

[0250]

[0251] X = Appearance, protein concentration, SE-UPLC

[0252] Y = CE-SDS-NR&R, iCIEF

[0253] Z = osmolar concentration, pH

[0254] S = Subvisible particles (MFI)

[0255] P = Potential (for the selected formulation only)

[0256] result

[0257] Appearance, pH and protein concentration Table 13 provides data obtained from appearance tests on excipients in the surfactant strength screening study.

[0258] Except for Fe1, Fe4 through Fe9, all formulations in this study maintained a good appearance: clear, slightly opalescent, and free of visible particles. For sucrose-containing formulations, Fe1, Fe6 through Fe9 samples turned slightly yellow after incubation at 40°C for 4 weeks. For Fe4, Fe5, and Fe6, a deeper opalescence was observed after stirring. For Fe6, visible particles were observed after stirring, freezing / thawing, and thermal stress.

[0259] Table 13. Screening study of excipient and surfactant strength: appearance

[0260]

[0261] Table 14 provides data obtained from pH and protein concentration tests for excipients in the surfactant strength screening study.

[0262] All formulations had pH values ​​and protein concentrations near their target values, with a pH of 6.5 and a protein concentration of 30.0 mg / mL, and no significant changes were observed after stirring, freezing / thawing, and thermal stress.

[0263] Table 14. Screening study of excipients and surfactants: pH and protein concentration

[0264]

[0265] Subvisible particles (MFI): Table 15 provides data on subvisible particle (MFI) testing in the excipient and surfactant strength screening study. After 5 freeze / thaw cycles, a significant increase in MFI was observed in Fe4 (containing Arg-HCl). An increase in MFI was also observed in Fe6 (containing only sucrose but no surfactant) after 3 days of stirring, 5 freeze / thaw cycles, and 4 weeks of incubation at 40°C. No significant changes in MFI were observed in the other formulations (Fe1 to Fe3, Fe5, Fe7 to Fe11) under all pressure conditions.

[0266] Table 15. Screening study of excipient and surfactant strength: Subvisible particles (MFI)

[0267]

[0268] SE-UPLC: Table 16 provides data obtained from SE-UPLC testing in the excipient and surfactant strength screening study.

[0269] Compared with the T0 sample, no significant changes in SEC monomers were observed in the formulation after stirring for 3 days and 5 freeze / thaw stress cycles, except for the Fe6 formulation (containing only sucrose but no surfactant), in which a 0.6% reduction in SEC monomers was observed after 5 freeze / thaw stress cycles.

[0270] After incubation at 40°C for 4 weeks, a reduction of SEC monomers of 1.9-7.3% was observed in all formulations. Comparison of the excipients used confirmed that formulations containing Fe1-Fe5, Fe2 (containing trehalose), and Fe3 (containing sorbitol) outperformed other formulations containing 0.02% (w / v) PS80, as the reduction in SEC monomers was less than 3.0%. The amount of surfactant was assessed by comparing Fe1 and Fe6 with Fe9; samples with PS80 content below 0.02% (w / v) showed higher SEC monomer concentrations than other samples, with the SEC monomer concentration decreasing as the PS80 concentration increased. It was found that the addition of 10 mM L-methionine and 0.05 mM EDTA to the Fe11 formulation effectively prevented the formation of high molecular weight (HMW) species. Overall, Fe2, Fe3, and Fe11 performed best among the 11 formulations tested.

[0271]

[0272] iCIEF: Table 17 provides data obtained from iCIEF tests in the excipient and surfactant strength screening study.

[0273] Compared with the T0 sample, no significant change was observed in the main peak of iCIEF in the 11 formulations after stirring for 3 days and 5 freeze / thaw stress cycles.

[0274] After incubation at 40°C for 4 weeks, the iCIEF peak was observed to decrease in all formulations, but Fe2, Fe3 and F11 showed the best performance among the 11 formulations.

[0275]

[0276] CE-SDS-NR&R: Table 18 provides data obtained from the CE-SDS-NR&R test in the excipient and surfactant strength screening study.

[0277] Compared with the T0 sample, no significant change in purity was observed in the 11 formulations under both non-reducing and reducing conditions after stirring for 3 days and 5 freezing / thawing stress cycles.

[0278] After incubation at 40℃ for 4 weeks, the purity of CE-SDS-NR decreased by 6.0-10.7%, with Fe6 (containing sucrose) showing the largest decrease (10.7%), while the Fe2, Fe3, Fe10 and Fe11 formulations performed better.

[0279] After incubation at 40°C for 4 weeks, the purity of CE-SDS-R decreased by 1.7-8.1%, with Fe6 (containing sucrose but without added surfactant) showing the largest decrease (8.1%), while the Fe2, Fe3, Fe10 and Fe11 formulations performed better.

[0280] Table 18. Screening studies of excipient and surfactant strength: CE-SDS-NR&R

[0281]

[0282] Effect: Based on the above test results, the Fe3 formulation (20 mM L-histidine / L-histidine monohydrochloride buffer, 4.5% (w / v) sorbitol, 0.02% (w / v) PS80, pH 6.5) was selected for efficacy testing. As shown in Table 19, compared with T0, IVX-01101 showed a significant decrease in efficacy after being stored at 40°C for 4 weeks.

[0283] Table 19. Screening study of excipient and surfactant strength: efficacy

[0284]

[0285] In the excipient and surfactant strength screening study, appearance and sub-visible particle results indicated that IVX-01101 protein exhibited poor stability in formulations containing sucrose, sodium chloride, or L-arginine hydrochloride. Appearance and sub-visible particle results also showed that PS80 effectively prevented the formation of protein aggregates. For sucrose-containing formulations, Fe1, Fe6, and Fe9 samples turned slightly yellow after incubation at 40°C for 4 weeks.

[0286] SE-UPLC results showed that formulations containing trehalose and sorbitol outperformed other formulations containing 0.02% (w / v) PS80. Among the PS80-containing formulations, samples with PS80 levels below 0.02% (w / v) showed higher SEC monomer content than samples without PS80. Furthermore, when the PS80 concentration increased by more than 0.04% (w / v), the SEC monomer content decreased. The Fe2, Fe3, and Fe11 formulations showed less reduction in SEC monomer content than other formulations.

[0287] iCIEF results showed that formulations containing sodium chloride or L-arginine hydrochloride exhibited poor stability, with Fe2, Fe3, and Fe11 showing a smaller decrease in the iCIEF main peak compared to other formulations.

[0288] For the CE-SDS-NR&R results, Fe2, Fe3, and Fe11 showed smaller reductions in CE-SDS purity compared to other formulations.

[0289] Furthermore, the results showed that PS80 can effectively prevent the formation of sub-visible or visible particles, while higher concentrations (close to 0.06%) of PS80 also had some impact on the SEC main peak. To balance the protective ability of PS80, a medium concentration (0.02%) was selected.

[0290] In summary, the Fe2, Fe3, and Fe11 formulations performed best, with comparable results. Even though Fe3 (trehalose) is a suitable excipient, its use is not recommended due to its relatively high material cost compared to sucrose or sorbitol. Finally, at pH 6.5, the optimal formulation for IVX-01101 was selected as 20 mM L-histidine / L-histidine monohydrochloride buffer, 4.5% (w / v) sorbitol, 0.02% (w / v) PS80, and 0.05 mM disodium EDTA.

[0291] Formula confirmation study: Studies were conducted using bioreactor materials produced on days 14 and 16, purified using anion exchange chromatography (AEX) with and without additional salt. The production process was identical to that used for GMP manufacturing of critical research and commercial materials, except for slight variations in the production of bioreactor materials on days 14 and 16, and slight variations in the AEX salt conditions. Studies were conducted at a concentration of 60 mg / mL, as higher concentrations were considered the worst-case scenario for product quality and stability. Study details are provided below:

[0292] Objective: To confirm the formulation using the final process materials.

[0293] Methods: To evaluate the stability of proteins under the following thermal stress conditions.

[0294] Target filling volume: 1.15 mL / 2 R vials (label volume: 1 mL).

[0295] Research period: 1 month.

[0296] Table 20: Short-term formulation validation studies

[0297]

[0298] X = Appearance, SE-UPLC, CE-SDS (NR & R);

[0299] Y = potency, PS80 concentration

[0300] (X,Y): Samples have been reserved and will be tested if necessary.

[0301] The results obtained by different analytical methods are shown below:

[0302]

[0303] When comparing the results of formulations F3, F4, and F5, the EDTA-containing formulation helped to slightly reduce aggregation levels. After 4 weeks of incubation at 40°C / 25°C / 5°C, 20 mM acetate performed slightly worse than 20 mM histidine; however, the product quality remained acceptable.

[0304]

[0305] Compared to formulations F3, F4, and F5, formulations containing EDTA help reduce purity loss.

[0306]

[0307] The formulation development of IVX-01101 consisted of multiple steps: pH / buffer screening studies and excipient and surfactant strength assessment studies, followed by concentration adjustments during the formulation validation study. A variety of physicochemical and biochemical methods were employed in selecting the lead formulation, including appearance, pH, protein concentration, osmolality, DSC, SE-UPLC, sub-visible particle (MFI), iCIEF, CE-SDS (NR&R), and potency.

[0308] Based on the pH / buffer screening results, a 20 mM L-histidine / L-histidine monohydrochloride buffer at pH 6.5 was selected as the lead pH / buffer system. Excipient screening and surfactant strength assessment studies showed that IVX-01101 protein exhibited higher stability in 20 mM L-histidine / L-histidine monohydrochloride buffer containing sorbitol than other formulations. Formulation validation studies confirmed the good stability of the lead formulation. Overall, IVX-01101 protein at a concentration of 5 to 60 mg / mL in 20 mM L-histidine / L-histidine monohydrochloride buffer, 4.5% (w / v) sorbitol, 0.02% (w / v) PS80, and 0.05 mM disodium EDTA (pH 6.5 ± 0.4) was considered a lead formulation for the canine antibody IVX-01101.

[0309] Example 3: Feasibility Study of Multi-Dosage Formulation of IVX-01286

[0310] Materials and methods

[0311] The purpose of this feasibility study was to assess the impact of different preservative / antimicrobial agent types and strengths in formulations on the quality and stability of the IVX-01286 antibody product in order to obtain multi-dose formulations. This study aimed to identify preservative-containing formulations with long-term stability comparable to preservative-free formulations and to evaluate preservative concentrations proven to prevent microbial growth in the formulation. It was shown that effective antimicrobial concentrations of preservatives can cause protein aggregation and degradation of certain classes of proteins; therefore, this study employed multiple analytical techniques to monitor antibody instability at these antimicrobial concentrations.

[0312] Stress studies were conducted, including short-term incubation at 40°C and 25°C and 6 months of control recommended storage at 2–8°C, to characterize the molecule’s appearance, pH, protein concentration (conc.), osmolality, subvisible particles (analyzed by high-precision liquid particle counting (HIAC), size exclusion-high performance liquid chromatography (SE-HPLC), imaging capillary isoelectric focusing (iCIEF), caliper-sodium dodecyl sulfate (non-reduced and reduced) (caliper-SDS-NR &R)) and potency (determined by enzyme-linked immunosorbent assay (ELISA)).

[0313] Table 24 lists the information on IVX-01286 active pharmaceutical ingredient (DS) used in formulation development studies.

[0314] Table 24. Molecular Information of IVX-01286

[0315]

[0316] Appearance The appearance of all samples, including color, clarity, and visible particles, was inspected using a YB-2 clarity meter against a black and white background. The light intensity of the lightbox was set to 2000~3750 lux.

[0317] protein concentration Protein concentration (conc.) was measured using a Lunatic® UV absorbance meter (Unchained Labs). According to the Lambert-Beer law, the concentration of the protein solution can be calculated from the absorbance at a given wavelength, the optical path length of the cuvette, and the extinction coefficient. The absorbance at 280 nm depends on the absorption characteristics of the aromatic amino acid residues in the protein. After loading a 2.5 μL sample volume, the Lunatic measured the absorbance in two cuvettes in parallel and calculated the concentration. The extinction coefficient used in this evaluation was 1.52 mL*mg. -1 *cm -1 .

[0318] pH Sample pH was measured using a pH meter with a glass electrode at 25°C. Before use, the pH meter was calibrated with three different standard buffer solutions (pH 4.01, 7.00, and 9.21). The calibration slope was between 95.0% and 105.0%, and the zero-point drift was between -60.0 mV and +60.0 mV. Each sample was tested twice, and the average result was reported.

[0319] osmolar concentration Osmolar concentration was measured using an Osmotech Pro. Samples were tested directly without dilution. The accuracy of the osmoremeter was verified before and after testing using a Clinitrol 290 mOsm / kg reference solution. The sample volume was 30 μL, and each sample was tested only once.

[0320] HIAC The size and count of subvisible particles in the biosafety cabinet were measured using a liquid particle counting system (HIAC 9703+). To avoid introducing air bubbles and interference during the inspection, all samples were stored in the biosafety cabinet for at least 0.5 hours before testing. Each sample was tested four times consecutively, with 0.4 ml per test. Particles ≥ 2 μm, ≥ 10 μm, and ≥ 25 μm per ml were tested according to pharmacopoeia methods (methods conforming to the United States Pharmacopeia (USP)). <788> For particulate matter in injectables, the data from the first test are discarded, and the results are reported as the average of the remaining three tests.

[0321] SE-UPLC Size exclusion high-performance liquid chromatography (SE-UPLC) is a purity analysis method that separates proteins based on their size. Measurements were performed using an Agilent HPLC system equipped with a SEC column (300 × 7.8 mm, 5 μm). The sampler temperature was set to 5 ± 3 °C, and the column oven temperature was set to 25 ± 3 °C. The mobile phase was 50 mM PB, 300 mM NaCl, pH 6.8 ± 0.1, and the flow rate was set to 1.0 mL / min. The sample was diluted to 10 mg / mL with the mobile phase, and the injection volume was 100 μg. The detection wavelength was set to 280 nm, and the run time was 20 min. Data were analyzed using Agilent CDS software.

[0322] iCIEFImaging capillary isoelectric focusing (iCIEF) is a purity analysis method used to monitor the percentage of charged variant species or charge heterogeneity of antibodies. The isoelectric point (pI) is an inherent property of a specific protein and is the pH at which the protein molecule carries no net charge. Under the influence of an external electric field, charged variants move along a continuous pH gradient formed by amphoteric electrolytes and stop when the pH equals the protein's pI.

[0323] 20 μg of sample was mixed with 80 μL of a master mixture consisting of a carrier ampholyte, a pI label, methylcellulose, and urea. The mixture was then analyzed using a ProteinSimple iCE3 equipped with a full-column detection capillary coated with fluorocarbons (FCs). The detection wavelength was set to 280 nm to assess the charge variant distribution across different pI ranges. The raw data were processed using Chrom Perfect Analysis, and the relative percentages of charge variants are reported.

[0324] Caliper-SDS-NR The Caliper sodium dodecyl sulfate (Caliper-SDS-NR) assay was performed using the PerkinElmer Caliper automated electrophoresis system, which separates proteins by size. In the non-reducing microarray CE-SDS analysis, the sample was diluted to 1 mg / mL with pure water. 2 μL of the diluted sample was mixed with 7 μL of sample denaturation solution (a mixture of sample buffer, 10% SDS solution, and 100 mM N-ethylcis-butenedimide (NEM), volume ratio 100:10:4). The thoroughly mixed sample was incubated at 70°C for 10 min, followed by centrifugation at 14000 rpm for 1 min at room temperature. 35 μL of water was added to each sample, vortexed for 10 sec, and then centrifuged at 14000 rpm for 1 min. 42 μL of the prepared sample was transferred to a 96-well plate, centrifuged at 4000 rpm for 15 minutes, and placed on a Labchip GXII plate holder for separation and detection in an HT Protein Express LabChip plate filled with decolorizing gel, gel dye, and low molecular weight label. The results were analyzed using LabChip GXReviewer software.

[0325] Caliper-SDS-RThe Caliper sodium dodecyl sulfate (Caliper-SDS-R) assay was performed using the PerkinElmerCaliper automated electrophoresis system, which separates proteins by size after sample reduction. In the reduced microchip CE-SDS analysis, the sample was diluted to 1 mg / mL with pure water. 2 μL of the diluted sample was mixed with 7 μL of sample denaturation solution (a mixture of sample buffer, 10% SDS solution, and 1 M dithiothreitol as a reducing agent, in a volume ratio of 100:10:4). The thoroughly mixed sample was incubated at 70°C for 10 min, followed by centrifugation at 14000 rpm for 1 min at room temperature. 35 μL of water was added to each sample, vortexed for 10 sec, and centrifuged at 14000 rpm for 1 min. 42 μL of the prepared sample was transferred to a 96-well plate, centrifuged at 4000 rpm for 15 min, and placed on a Labchip GXII plate holder for separation and detection in an HT Protein Express LabChip filled with destaining gel, gel dye, and low molecular weight markers. The results were analyzed using LabChip GXReviewer software.

[0326] efficacy This assay is an ELISA method for analyzing the binding of IVX-01286 monoclonal antibody to canine NGF (nerve growth factor). Samples, assay controls (AC), and standards (STD) are loaded at appropriate dilutions onto a 96-well high-binding plate coated with canine NGF antigen. After washing, peroxidase-AffiniPure rabbit anti-dog IgG (H+L) antibody is added to each well to allow it to interact with the IVX-01286 captured in the previous step. After the final wash, TMB substrate solution is loaded into each well. TMB reacts specifically with peroxidase in the presence of peroxidase, producing a colorimetric signal proportional to the amount of IVX-01286 bound to each well.

[0327] Using SoftMax Pro software, dose-response curves for the sample, AC, and STD were plotted based on a 4-parameter logistic regression model (automatic estimation). The EC50 values ​​for each of the sample, AC, and STD were obtained on the same plate. The relative binding activity of the sample and AC was calculated using the following formula:

[0328]

[0329] Remove the canine NGF protein stock solution from a freezer at ≤ -60°C and allow it to equilibrate to room temperature, avoiding repeated freezing and thawing. To prepare the working antigen solution, dilute the antigen to 3 μg / mL with coating buffer. Coat each well of a 96-well plate with 100 μL of the 3 µg / mL canine NGF protein solution. Seal the plate with sealing film and incubate at 2–8°C for 16–86 hours. Remove the 96-well plate from the freezer at 2–8°C. Using a multichannel pipette or plate washer, wash the plate 6 times with approximately 300 µL of 0.05% PBST per well. If using a multichannel pipette, gently tap the plate on a paper towel to remove any residue. If using a plate washer, wash the plate 3 times with the front side facing forward, then flip the plate over and wash it 3 times with the back side facing backward. Gently tap the plate on a paper towel to remove any residue. When using a plate washer, add 200 µL of blocking buffer (1% BSA / PBS) to each well of the plate. Seal the plate with plate sealant and shake it on a plate shaker at 220 RPM and 25°C for 1–3 hours. Aliquot each dilution point to set up replicate wells. Transfer 100 µL of diluted STD, AC, and sample to the different wells of the 96-well plate. Seal the plate. Incubate the plate on a plate shaker at 25°C and approximately 220 RPM for 70 ± 20 minutes. Add 100 µL of peroxidase-AffiniPure rabbit anti-dog IgG (H+L) antibody working solution (1:50k) to each well. Seal the plate with plate sealant. Incubate the plate on a plate shaker at approximately 220 RPM and 25°C for 70 ± 20 minutes. Remove the 96-well plate and wash it. Add 100 µL of TMB to each well. Stop the reaction after approximately 10–20 minutes. Then, add 100 µL of 1 M HCl to each well to stop the reaction. Within 45 minutes (≤45 minutes) after the reaction stops, place the plate in the plate reader (read the absorbance at 450 nm).

[0330] Research Design

[0331] In this feasibility study report, thermal stress studies were examined to characterize the IVX-01286 molecule. Table 25 shows a brief description of the study.

[0332] Table 25. Study Design for the Feasibility Study of IVX-01286 Formulation

[0333]

[0334] Formulation screening studies

[0335] TargetThis formulation feasibility study aims to evaluate the stability of the IVX-01286 molecule and select the optimal pH / buffer and excipient system containing 0.02% (w / v) PS80 and 0.05 mM EDTA as well as the preservative, in order to identify a preservative formulation with good stability characteristics.

[0336] Sample preparation This study used IVX-01286 DS (batch number: 7671-1W230323). First, its buffer was exchanged to prepare 20 mM histidine buffer (pH 6.6) and 20 mM acetate buffer (pH 5.0). Stock solutions containing 5% (w / w) PS80, 10 mM EDTA, 20% (w / w) sorbitol, and 40% (w / w) sucrose were prepared. Based on the formulation listed in Table 26, the required amounts of DS, excipient stock solution, and antimicrobial agent were calculated, weighed, and thoroughly mixed. The formulation solutions were filtered through 0.22 μm PES filters and filled into 2 mL glass vials (1 mL each), immediately stoppered, capped, and labeled. All sample filtration and aliquoting were performed aseptically in a biosafety cabinet.

[0337] Table 26. List of formulation candidates in the formulation feasibility study

[0338]

[0339] Research parameters: The sampling and testing plan for the formulation feasibility study is shown in Table 27. For thermal stress, samples were stored at 40°C for up to 4 weeks, at 25°C for up to 3 months, and at 5°C for up to 6 months. Tests performed in this study included appearance, protein concentration, pH, osmolality, HIAC, SE-UPLC, iCIEF, caliper-SDS-NR&R, and potency testing.

[0340] Table 27. Sampling and Testing Plan in the Formulation Feasibility Study

[0341]

[0342] result

[0343] Appearance: The summary of appearance test data from formulation screening studies is provided in Table 28 and Figure 2A-2CAll formulations remained colorless, slightly opalescent, and free of visible particles at T0. After incubation at 40°C for 4 weeks, samples F01 / F02 / F05 remained colorless, slightly opalescent, and free of visible particles, while the opalescence of F03 and F04 was significantly more intense than at T0. Notably, sample F06 became gel-like and could not be further tested.

[0344] The samples remained colorless, slightly opalescent, and without visible particles after being incubated at 25°C for 3 months and at 5°C for 6 months.

[0345] Table 28. Results of the Formulation Feasibility Study: Appearance

[0346]

[0347] Abbreviations: C = colorless; SOL = slightly opalescent liquid; W = white; OG = opalescent gel; OL = opalescent liquid FP = No visible particles; EFP = Essentially free of visible particles (≤ 3); NEFP = Not entirely free of visible particles (> 3). (3); M = month; W = week.

[0348] Protein concentration: The protein concentrations of the formulations (F01-F06) maintained for several months under different thermal conditions were evaluated, as shown in Table 29 below. Compared to T0, the protein concentrations of all candidates (approximately 60 mg / mL and 30 mg / mL) remained relatively consistent after incubation at 40°C, 25°C, and 5°C, indicating that the protein concentrations were stable under the three thermal stress conditions.

[0349] Table 29. Results of the formulation feasibility study: Protein concentration

[0350]

[0351] Abbreviations: W = week; M = month; NT = not tested. Note: [1] The addition of m-cresol affected the determination of the F04 formulation concentration. The F04 concentrations all deducted the amount of m-cresol absorbed, and the concentrations were within a reasonable range.

[0352] pH and osmolar concentration As shown in Table 30, except for F06 which was not tested at 40°C, the pH of all test formulations remained relatively stable during the study period. The osmolality (non-stability indicator) of all samples ranged from 363 to 536 mOsm / kg. An increase in osmolality was observed with increasing concentrations of antimicrobial preservatives and active pharmaceutical ingredients. These samples are considered slightly hyperosmolar and are not expected to cause any patient problems during parenteral administration.

[0353] Table 30. Results of the formulation feasibility study: pH and osmolar concentration.

[0354]

[0355] Abbreviations: W = week; M = month; NT = untested.

[0356] SE-UPLC:Table 31 summarizes the SE-HPLC test results from the formulation feasibility study. After incubation at 40°C for 4 weeks, the monomer percentage of all candidates decreased (up to 12.5% ​​when m-cresol was added as an antibacterial agent). This change was most pronounced with the addition of an antibacterial agent, while F01, without any antibacterial agent, showed the smallest change in monomer purity. F04 (12.5% ​​↓) and F05 (7.1% ↓) showed the largest decreases in monomer percentage, while F06 became a gel-like substance at 40°C. When stored at 25°C for 3 months, the monomer percentage in all formulations decreased slightly compared to T0, with F06 showing the lowest monomer percentage at 97.0%.

[0357] In summary, SE-HPLC analysis showed that IVX-01286 protein tends to form high molecular weight polymers under thermal stress conditions, especially at 40°C, and exhibits the worst stability compared to the control under the interaction of acetate buffer, 9% (w / v) sucrose, and to some extent, at a slightly higher level of preservative (i.e., 1.2% benzyl alcohol). Furthermore, based on data from 4W at 40°C, at the same protein concentration, 0.4% (w / v) m-cresol has a greater effect on the percentage of SEC monomers than 1.2% (w / v) benzyl alcohol.

[0358] Based on monomer percentage, all formulations remained stable for up to 6 months under the control recommended storage conditions at 5°C.

[0359] iCIEF Table 32 presents a summary of the iCIEF test results from the formulation feasibility study. The isoelectric point of all samples was measured to be 5.4. After 4 weeks of storage at 40°C, the percentage of the main peak in all formulation systems decreased significantly (up to 29.2% with the addition of m-cresol and up to 27.2% with the addition of benzyl alcohol). Within the expected variability range of the analytical method, the performance of formulations containing 0.9% benzyl alcohol was comparable to that of their counterparts without benzyl alcohol. After incubation at 25°C and 5°C, a decrease in the percentage of the main peak was observed in all formulations, with F04 showing the largest decrease (22.9%) at 25°C–3M and the largest decrease (8.4%) at 5°C–6M. However, all formulations exhibited a similar decreasing trend and were considered relatively stable under the recommended control storage conditions of up to 6 months at 5°C.

[0360]

[0361] Abbreviations: HMW = High molecular weight; LMW = Low molecular weight; NT = Untested; W = Week; M = Month.

[0362]

[0363] Caliper-SDS-NR & RTable 33 reports the Caliper-SDS-NR & R test results for the formulation feasibility study. For Caliper-SDS-NR, the purity of all samples decreased with increasing incubation time at 40°C and 25°C, with reductions of up to 4.2% and 3.2% at 40°C for 4 weeks and 25°C for 3 months, respectively. Within the expected variability range of the analytical method, formulations containing 0.9% benzyl alcohol performed comparably to their counterparts without benzyl alcohol. Compared to the Caliper-SDS-NR results, smaller changes were observed in all samples in the Caliper-SDS-R results, suggesting that interchain disulfide bond breakage may occur during incubation at 40°C. Slight decreases in purity were observed at 40°C for up to 4 weeks and at 25°C for up to 3 months; no significant decrease was observed in samples at 5°C. All formulations were stable under the recommended control storage conditions at 5°C for up to 6 months.

[0364]

[0365] Highly visible particles (HIAC) The results of the sub-visible particle (HIAC) test in the formulation feasibility study are shown in Table 34. No significant changes in the number of sub-visible particles ≥2 μm, ≥10 μm, and ≥25 μm were observed after incubation under different heat conditions. All formulations were stable under the recommended control storage conditions at 5°C for up to 6 months.

[0366]

[0367] Abbreviations: W = week; M = month; NT = untested.

[0368] Effect: Based on the above test results, the following proteins were selected for potency testing: F02 (60 mg / mL protein, 20 mM histidine, 5% (w / v) sorbitol, 0.9% (w / v) benzyl alcohol, pH 6.6), F03 (60 mg / mL protein, 20 mM histidine, 5% (w / v) sorbitol, 1.2% (w / v) benzyl alcohol, pH 6.6), and F05 (30 mg / mL protein, 20 mM histidine, 9% (w / v) sucrose, 1.2% (w / v) benzyl alcohol, pH 6.6). As shown in Table 35, within the acceptance criteria of 50-150% based on analytical method variability, the potency of IVX-01286 protein after storage at 5°C for 6 months was not different from that at T0.

[0369] Table 35. Results of the formulation feasibility study: ELISA

[0370]

[0371] Abbreviations: W = week; M = month; NT = untested; His = histidine; Ace = acetate; Sorb = sorbitol; BA = benzyl alcohol; CR = m-cresol; Suc = sucrose; NT = not tested .

[0372] Summary of formulation feasibility study This feasibility study investigated the stability of IVX-01286 molecules formulated with different concentrations of preservatives (antimicrobial agents) under pressure conditions (up to 4 weeks at 40°C and up to 3 months at 25°C) and long-term storage conditions (up to 6 months at 5°C). The results showed that the stability of IVX-01286 molecules is closely related to the type and strength of the antimicrobial agent. At the same protein concentration, 0.4% (w / v) m-cresol had a greater effect on protein quality than 1.2% (w / v) benzyl alcohol.

[0373] According to the results, the IVX-01286 molecule formulated in 20 mM histidine buffer, 5% (w / v) sorbitol, 0.02% (w / v) PS80, 0.05 mM EDTA, and 0.9% (w / v) benzyl alcohol (pH 6.6) exhibited relatively good thermal stability, and its stability characteristics were similar to those of the molecule formulated in a preservative-free formulation (i.e., 20 mM histidine buffer, 5% (w / v) sorbitol, 0.02% (w / v) PS80, 0.05 mM EDTA, pH 6.6).

[0374] Example 4: Feasibility Study of IVX-03023 Formulation

[0375] Materials and methods

[0376] A development batch of IVX-03023 feline monoclonal antibody (mAb) (also known as IVX-03) was produced in a 15 L bioreactor for proof-of-concept studies using a clonal pool generated by a stable expression system. A 10 mg / mL IVX-03023 mAb was formulated in 20 mM acetate and sodium acetate buffer, 9% (w / w) sucrose, and 0.02% (w / w) polysorbate 80 (pH 5.0) for the preparation of the active pharmaceutical ingredient (DS) and the drug product (DP). Product stability was monitored using a variety of analytical methods, including SE-HPLC, reducing and non-reducing capillary electrophoresis-SDS (CE-SDS), and imaging capillary isoelectric focusing (iCIEF) concentration and potency, as described above. DP values ​​for subvisible particles were also monitored. All methodological information is similar to that described above. Only the ELISA method differs and is described below.

[0377] ELISA power assay:IL5 antigen was coated onto the surface of each well in a 96-well plate to capture IVX-03023 antibody. After IVX-03023 antibody binding, goat anti-cat IgG (H+L) secondary antibody fused with the HRP enzyme for detection was added to bind to IVX-03023 antibody. Color development occurred in the wells after the addition of TMB substrate. After the addition of stop solution, the plate was read at 450 nm and 630 nm. The difference in OD values ​​between 450 nm and 630 nm was proportional to the amount of IVX-03023 bound. Standard curves for samples and reference standards were plotted and fitted using a 4-parameter logistic regression model using SoftMax Pro software, and the EC50 values ​​of each curve were calculated. The conversion rate of the EC50 value of the samples to the binding power was calculated by comparing it with the reference standard measured on the same plate.

[0378] result

[0379] The following are long-term stability data for the IVX-03023 monoclonal antibody and a drug formulation containing 20 mM NaAc-HAc, 9% (w / w) sucrose, 0.02% (w / w) PS80 (pH 5.0), and 10 mg / mL mAb. Non-GMP stable pool production stability studies were conducted in a 15L bioreactor.

[0380] Table 36 lists the data summary (DS) information used in the formulation development study of IVX-03023 at 70℃±10℃.

[0381] Research parameters: The sampling and testing plan for the formulation feasibility study is shown below. For thermal stress, samples were stored at -70°C ± 10°C for up to 12 months (Table 36), at 5°C for up to 12 months (Table 37), at 25°C for up to 3 months (Table 38), and at 40°C ± 2°C for up to 1 month (Table 39). Tests included appearance, protein concentration, ELISA binding, pH, CE-SDS (reduced and non-reduced), SE-UPLC, and iCIEF.

[0382]

[0383]

[0384]

[0385] Table 38. Study parameters of IVX-03023 (25℃ ± 2℃)

[0386]

[0387] Table 39. Research parameters of IVX-03023 (40℃ ± 2℃)

[0388]

[0389] IVX-03023 active pharmaceutical ingredient remained stable for one year at the recommended long-term storage temperature of -70 ± -10℃, and the quality attributes of all products significantly exceeded the acceptance criteria (Table 36). Figures 3A-3D The pharmaceutical product remains stable for one year at the recommended long-term storage temperature of 2-8°C (Table 37). Figures 4A-4H Furthermore, it remained stable for 3 months at an accelerating temperature of 25°C (Table 48). Figures 5A-5E All product quality attributes far exceeded acceptance criteria. Molecular efficacy was maintained. Results for pH, visible particles, and osmolality all showed no significant changes over time.

[0390] Example 5: Formulation Feasibility Study of IVX-06076

[0391] Materials and methods

[0392] A development batch of IVX-06076 cat mAb (also known as IVX-06) was produced in a 15 L-scale bioreactor for a proof-of-concept study using a clone pool generated from a stable expression system. 10 mg / mL IVX-06076 mAb was formulated in 20 mM acetate and sodium acetate buffer, 9% (w / w) sucrose, and 0.02% (w / w) polysorbate 80 (pH 5.0) to prepare the active pharmaceutical ingredient (DS) and pharmaceutical product (DP) materials. Product stability was monitored using a variety of analytical methods, including SE-HPLC, reduced and non-reduced capillary electrophoresis-SDS (CE-SDS), and imaging capillary isoelectric focusing (iCIEF) concentration and potency. DP values ​​of subvisible particles were also monitored. All methods are as described above. The ELISA was the same as that presented for IVX-01101.

[0393] result

[0394] The following are long-term stability data for the IVX-00676 monoclonal antibody and a pharmaceutical formulation containing 20 mM NaAc-HAc, 9% (w / w) sucrose, 0.02% (w / w) PS80 (pH 5.0), and 10 mg / mL mAb. Production stability studies were conducted in a non-GMP stabilization tank of a 15L bioreactor.

[0395] Table 44 lists the data summary (DS) information used in the formulation development study of IVX-06076 at 70℃±10℃.

[0396] Research parameters: The sampling and testing plan for the IVX-06076 formulation feasibility study is shown below. For thermal stress, samples were stored at -70°C ± 10°C for up to 12 months (Table 40). Figures 6A-6D It can be stored at 5°C for up to 18 months (Table 41). Figures 7A-7H ), can be stored at 25°C for up to 3 months (Table 42, Figures 8A-8H The samples were stored at 40℃ ± 2℃ for up to one month (Table 43). Tests included appearance, protein concentration, ELISA binding, pH, CE-SDS (reduced and non-reduced), SE-UPLC, and iCIEF.

[0397] Table 40. Research parameters of IVX-03023 (-70℃ ± 10℃)

[0398]

[0399] Table 41. Research parameters of IVX-03023 (5℃ ± 3℃)

[0400]

[0401] Table 42. Research parameters of IVX-06076 (25℃ ± 2℃)

[0402]

[0403] Table 43. Research parameters of IVX-06076 (40℃ ± 2℃)

[0404]

[0405] IVX-06076 active pharmaceutical ingredient remains stable for 6 months at the recommended long-term storage temperature of -70 ± -10°C (Table 40). Figures 6A-6D Furthermore, the quality attributes of all products far exceed the acceptance criteria. The pharmaceutical products remain stable for one year at the recommended long-term storage temperature of 2-8°C (Table 41). Figures 7A-7H Furthermore, it remained stable for 6 months at an accelerating temperature of 25°C (Table 42). Figures 8A-8H All product quality attributes far exceeded acceptance criteria. Molecular efficacy was maintained. Results for pH, visible particles, and osmolality all showed no significant differences over time (data not shown in the figure).

[0406] All patents, patent applications, publications, and electronically available materials cited herein (including, for example, nucleotide sequences submitted to databases such as GenBank and RefSeq; amino acid sequences submitted to databases such as SwissProt, PIR, PRF, and PDB; and sequences translated from coding regions annotated in GenBank and RefSeq) are incorporated herein by reference in their entirety. In the event of any inconsistency between the disclosure of this application and the disclosure of any document incorporated herein by reference, the disclosure of this application shall prevail. The detailed descriptions and embodiments above are provided for illustrative purposes only and should not be construed as imposing any unnecessary limitations. The invention is not limited to the specific details shown and described, and various variations that will be apparent to those skilled in the art are included within the scope of the invention as defined in the claims.

Claims

1. A stable pharmaceutical preparation comprising: (a) An antibody of 5 to 100 mg / ml, said antibody comprising a variant of the CDR, CH2, CH3, IgG Fc region having a canine or cat variable antibody domain, or a polypeptide thereof having a canine or cat FcRn binding region. (b) A buffer of 4.0 to 50 mM with a pH in the range of 4.5 to 7.0; (c) 0.01 to 35% (w / v) of osmotic pressure regulators and / or stabilizers; and (d) 0.01 to 10% (w / v) surfactant; The formulation is either a single-dose formulation or a multi-dose formulation, and The formulation described herein maintains stability in solution for up to twenty-four months.

2. The pharmaceutical formulation of claim 1 further comprises 0.01 to 5.0 mM of a chelating agent.

3. The pharmaceutical formulation of claim 1, wherein the multi-dose formulation further comprises an antimicrobial preservative at a concentration of about 0.1 to 1.5% (wv), said antimicrobial preservative being selected from the group consisting of benzyl alcohol, phenol, m-cresol, benzalkonium chloride, benzyl chloride, phenoxyethanol and methylparaben, or mixtures thereof.

4. The pharmaceutical formulation of claim 1, wherein the buffer is selected from the group consisting of: sodium acetate, histidine, histidine hydrochloride (HCl), succinate, phosphate, Tris, diethanolamine, citrate, acetate, other organic acids, and mixtures thereof.

5. The pharmaceutical formulation of claim 1, wherein the buffer is present at a concentration of 5 to 30 mM and maintained at a physiologically suitable pH in the range of pH 5 to pH 6.

5.

6. The pharmaceutical formulation of claim 1, wherein the osmotic pressure regulator and / or stabilizer is selected from the group consisting of: CaCl2, NaCl, MgCl2, lactose, sorbitol, sucrose, mannitol, trehalose, raffinose, polyethylene glycol, hydroxyethyl starch, glycine, and mixtures thereof.

7. The pharmaceutical formulation of claim 6, wherein the osmotic pressure regulator and / or stabilizer is preferably sucrose, trehalose, or sorbitol.

8. The pharmaceutical formulation of claim 1, wherein the osmotic pressure regulator and / or stabilizer and / or stabilizer is present at a concentration of 0.02% to 10% (wv).

9. The pharmaceutical formulation of claim 1, wherein the chelating agent is selected from the group consisting of: aminopolycarboxylic acids, hydroxyaminocarboxylic acids, N-substituted glycine, 2-(2-amino-2-oxoethyl)aminoethanesulfonic acid (BES), deferoxamine (DEF), citric acid, nicotinamide, deoxycholate, diethylenetriaminepentaacetic acid (DTPA), nitrotriacetic acid (NTA), N-2-acetamido-2-iminodiacetic acid (ADA), bis(aminoethyl)ethylene glycol ether, N,N,N′,N′-tetraacetic acid (EGTA), trans-diaminocyclohexanetetraacetic acid (DCTA), glutamic acid and aspartic acid, N-hydroxyethyliminodiacetic acid (HIMDA), N,N-bishydroxyethylglycine (bicine) and N-(tris(hydroxymethyl)methyl) 10. Glycine, glycylglycine, sodium deoxycholate, ethylenediamine, propylenediamine, diethylenetriamine, triethylenetetramine, ethylenediaminetetraacetic acid (EDTA), disodium EDTA, calcium EDTA, oxalic acid, malate, citric acid, citric acid monohydrate and trisodium citrate dihydrate, 8-hydroxyquinoline salts, amino acids, histidine, cysteine, methionine, peptides, polypeptides and proteins, and mixtures thereof.

10. The pharmaceutical preparation of claim 9, wherein the chelating agent is preferably EDTA, disodium EDTA, or calcium EDTA.

11. The pharmaceutical formulation of claim 9, wherein the chelating agent is present at a concentration of 0.01 to 5 mM.

12. The pharmaceutical formulation of claim 1, wherein the surfactant is selected from the group consisting of: polysorbate, poloxamer, triaton, sodium lauryl sulfate, sodium lauryl sulfate, sodium octyl glucoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, Myristyl-betaine, cetyl-betaine, lauramidopropyl-betaine, cocamidopropyl-betaine, linoleamideopropyl-betaine, myristamidopropyl-betaine, palmitoamideopropyl-betaine, isostearamidopropyl-betaine, myristamidopropyl-dimethylamine, palmitoamideopropyl-dimethylamine, isostearamidopropyl-dimethylamine, sodium methylcocoyl taurate, disodium methyloleoyl taurate, dihydroxypropyl PEG 5 linoleoyl ammonium chloride, polyethylene glycol, polypropylene glycol, and mixtures thereof.

13. The pharmaceutical formulation of claim 12, wherein the polysorbate is selected from the group consisting of: polysorbate 20, polysorbate 21, polysorbate 40, polysorbate 60, polysorbate 61, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85, and mixtures thereof.

14. The pharmaceutical formulation of claim 1, wherein the surfactant is present at a concentration of 0.002 to 0.5% (w / v).

15. The pharmaceutical formulation of claim 1, further comprising an antioxidant selected from the group consisting of: GLA (gamma-linolenic acid)-lipoic acid; DHA (docosahexaenoic acid)-lipoic acid; GLA-tocopherol; di-GLA-3,3′-thiodipropionic acid; DGLA (di-homo-gamma-linolenic acid); AA (arachidonic acid); SA (salicylic acid); EPA (eicosapentaenoic acid) or DHA (docosahexaenoic acid); phenolic antioxidants, including polyenes; unsaturated sterols; ascorbic acid; organosulfur compounds; terpenes; and amino acid antioxidants.

16. The pharmaceutical formulation of claim 15, wherein the amino acid antioxidant is selected from the group consisting of methionine, cysteine, carnosine, and analogues thereof.

17. The pharmaceutical formulation of claim 15, wherein the antioxidant is present at a concentration of 0.01 mM to about 50 mM.

18. The pharmaceutical formulation of claim 1, further comprising a preservative present at a concentration of 0.001% w / v to 10% w / v, wherein the preservative is selected from the group consisting of phenol, m-cresol, benzyl alcohol, benzalkonium chloride, benzyl chloride, phenoxyethanol, and methylparaben.

19. The pharmaceutical preparation of claim 1, wherein the preparation is suitable for oral, rectal, mucosal, intestinal or parenteral administration.

20. The pharmaceutical preparation of claim 19, wherein parenteral administration is selected from intravenous, intra-arterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular, intraosseous, intradermal, or subcutaneous administration.

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