Antibody formulations

Abstract

The invention relates to a pharmaceutical preparation of an alpha synuclein antibody.

Description

[0001] This invention relates to a pharmaceutical formulation of an α-synuclein antibody.

[0002] Parkinson's disease (PD) is a slow, chronic, progressive neurodegenerative disease that is estimated to affect 7-10 million people worldwide. In the United States, an estimated 725,000 people are affected, with more than 50,000 new cases reported each year. Although 5% to 10% of patients are diagnosed before the age of 50, PD is generally considered a disease that affects older adults, affecting one in 100 people over the age of 60, and it is more common in men than women.

[0003] Alpha-synuclein is a protein commonly associated with synapses and is thought to play a role in neuroplasticity, learning, and memory. Under pathological conditions, alpha-synuclein can aggregate to form insoluble fibrils and is a major component of the pathology characterizing a variety of neurodegenerative diseases, including Parkinson's disease. Soluble oligomers of alpha-synuclein may be neurotoxic. The accumulation of alpha-synuclein with similar morphological and neurological changes in species and animal models such as humans, mice, and flies suggests that this molecule contributes to the development of Parkinson's disease. Antibodies against alpha-synuclein may be able to reduce alpha-synuclein deposition and symptoms in Parkinson's disease.

[0004] As part of the protein drug family, antibody molecules are highly susceptible to physical and chemical degradation. Chemical degradation encompasses any process involving the modification of proteins via bond formation or cleavage to produce new chemical entities. A variety of chemical reactions are known to affect proteins. These reactions may involve hydrolysis (including the cleavage of peptide bonds) as well as deamidation, isomerization, oxidation, and decomposition. Physical degradation refers to changes in higher-order structures and includes denaturation, adsorption to surfaces, aggregation, and precipitation. Protein stability is influenced by both intrinsic protein characteristics (e.g., amino acid sequence, glycosylation pattern) and external factors such as temperature, solvent pH, excipients, interfacial processes, or shear rates. Therefore, determining optimal formulation conditions to protect proteins from degradation reactions during manufacturing, storage, and administration is crucial. (Manning, MC, et al. (1989), "Stability of protein pharmaceuticals", Pharm Res 6(11), 903-918; Zheng, JY, Janis, LJ (2005), "Influence of pH, buffer species, and storage temperature on physicochemical stability of a humanized monoclonal antibody LA298", Int. J.Pharmaceutics 308, 46-51). Obtaining stable liquid formulations of therapeutic antibodies is particularly difficult when the formulation should contain high concentrations of antibody.

[0005] Therefore, one object of the present invention is to provide a stable formulation of an α-synuclein antibody having as few necessary excipients as possible, which enables desired administration and allows convenient delivery of the antibody to a patient. Summary of the Invention

[0006] In a first aspect, the present invention relates to a pharmaceutical preparation comprising:

[0007] 120 to 200 mg / ml of α-synuclein antibody;

[0008] 1 to 50 mM buffer;

[0009] 0.01% to 1% (w / v) of surfactant;

[0010] At least one stabilizer of 1 to 50 mM;

[0011] 100 to 150 mM tension agent,

[0012] pH ranges from 5.0 to 7.0.

[0013] In embodiments of the present invention, the concentration of the anti-synuclein antibody is in the range of 150 to 200 mg / ml, preferably 180 mg / ml.

[0014] In an embodiment of the present invention, the buffer is a histidine buffer, preferably a histidine acetate buffer.

[0015] In an embodiment of the present invention, the buffer concentration is 10 to 40 mM.

[0016] In embodiments of the invention, the buffer provides a pH of 5.0 to 6.0, preferably 5.5.

[0017] In an embodiment of the present invention, the surfactant is polysorbate, preferably polysorbate 20.

[0018] In embodiments of the present invention, the surfactant concentration is 0.01% to 0.1% (w / v).

[0019] In embodiments of the present invention, at least one stabilizer is selected from the group consisting of salts, sugars, and amino acids, preferably histidine.

[0020] In embodiments of the present invention, at least one stabilizer concentration is 5 to 50 mM.

[0021] In an embodiment of the present invention, the concentration of the tensioning agent is 120 to 150 mM, preferably 130 mM.

[0022] In an embodiment of the present invention, the tensioning agent is arginine-HCl.

[0023] In an embodiment of the present invention, the formulation comprises:

[0024] 150 to 180 mg / ml α-synuclein antibody;

[0025] 15 to 30 mM L-histidine / acetate buffer;

[0026] 0.02% to 0.05% (w / v) polysorbate 20;

[0027] 120 to 180 mM arginine-HCl;

[0028] 5 to 25 mM L-methionine;

[0029] The pH is 5.5 ± 0.5.

[0030] In an embodiment of the present invention, the formulation comprises:

[0031] 180 mg / ml α-synuclein antibody,

[0032] 20 mM L-histidine / acetate buffer,

[0033] 0.04% (w / v) Polysorbate 20,

[0034] 130 mM arginine-HCl

[0035] 10 mM methionine, pH 5.5.

[0036] In an embodiment of the present invention, the anti-α synuclein antibody comprises: a VH domain having an amino acid sequence of Seq. Id. No. 1; and a VL domain having an amino acid sequence of Seq. Id. No. 2.

[0037] In an embodiment of the present invention, the α-synuclein antibody comprises: a heavy chain having an amino acid sequence of Seq. Id. No. 3; and a light chain having an amino acid sequence of Seq. Id. No. 4.

[0038] In an embodiment of the present invention, the α-synuclein antibody is an antibody containing INN prasinezumab.

[0039] In embodiments of the present invention, the formulation is in liquid form, in lyophilized form, or in liquid form reconstituted from lyophilized form. Detailed Implementation

[0040] As used herein, the term "buffer" refers to a pharmaceutically acceptable agent that stabilizes the pH of a pharmaceutical preparation. Suitable buffers are well known in the art and can be found in the literature. For example, citrate, acetate, histidine, succinate, malate, phosphate, or lactate, and / or their corresponding free acids or bases, as well as various salts and / or mixtures of acids and bases, can be used. Preferred pharmaceutically acceptable buffers include, but are not limited to, histidine buffers, citrate buffers, succinate buffers, acetate buffers, and phosphate buffers. The preferred buffer used in this invention is a histidine buffer, i.e., a buffer having histidine (typically L-histidine) as a buffer. Most preferably is an L-histidine / HCl buffer, which contains L-histidine or a mixture of L-histidine and L-histidine hydrochloride, and pH adjustment is achieved with hydrochloric acid. Unless otherwise stated, the term "L-histidine" as used herein to describe a buffer refers to an L-histidine / HCl buffer. The L-histidine / HCl buffer can be prepared by dissolving an appropriate amount of L-histidine and L-histidine hydrochloride in water, or by dissolving an appropriate amount of L-histidine in water and adjusting the pH to the desired value by adding hydrochloric acid. The buffer is typically used at a concentration of about 1 mM to about 100 mM, preferably about 10 mM to about 50 mM, more preferably about 15 to 30 mM, and most preferably 20 mM. Regardless of the buffer used, acids or bases known in the art, such as hydrochloric acid, acetic acid, phosphoric acid, sulfuric acid and citric acid, sodium hydroxide and potassium hydroxide, can be used to adjust the pH to a value in the range of about 4.0 to about 7.0, preferably about 5.0 to about 6.0, and most preferably about 5.5.

[0041] "Stable" formulations are those in which proteins (such as antibodies) retain their physical and chemical stability and thus their biological activity during storage.

[0042] "Stable liquid antibody formulations" are liquid antibody formulations that show no significant changes after being stored at refrigerated temperatures (2°C to 8°C) for at least 12 months, particularly 2 years, and even more particularly 3 years. The criteria for stability are as follows: no more than 10%, particularly 5%, of antibody monomers are degraded, as measured by size exclusion chromatography (SEC-HPLC). Furthermore, the solution is colorless or transparent to slightly milky white by visual analysis. The protein concentration of the formulation changes by no more than + / - 10%. No more than 10%, particularly 5%, of aggregates are formed. Stability is measured by methods known in the art, such as UV spectroscopy, size exclusion chromatography (SEC-HPLC), ion exchange chromatography (IE-HPLC), turbidimetry, and visual inspection.

[0043] As used herein, the term "surfactant" refers to a pharmaceutically acceptable surfactant. Preferably, nonionic surfactants are used. Examples of pharmaceutically acceptable surfactants include, but are not limited to, polyoxyethylene sorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenyl polyoxyethylene ethers (Triton X), polyoxyethylene-polyoxypropylene copolymers (poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). Preferred polyoxyethylene sorbitan fatty acid esters are polysorbate 20 (polyoxyethylene sorbitan monolaurate, sold under the trademark Tween 20™) and polysorbate 80 (polyoxyethylene sorbitan monooleate, sold under the trademark Tween 80™). Preferred polyoxyethylene-polyoxypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188™. Preferred polyoxyethylene alkyl ethers are those sold under the trademark Brij™. Preferred alkylphenyl polyoxyethylene ethers are sold under the trademark Triton X, with the most preferred being p-tert-octylphenoxypolyethoxyethanol (sold under the trademark Triton X-100™). Preferred surfactants used in this invention are polyoxyethylene sorbitan fatty acid esters, preferably polysorbate 20 or polysorbate 80, most preferably polysorbate 20. When polysorbate 20 (Tween 20™) and polysorbate 80 (Tween 80™) are used, they are typically used at concentrations ranging from about 0.001% to about 1%, preferably from about 0.01% to about 0.1%, more preferably from about 0.02% to about 0.05%, and most preferably about 0.05%. In the formulations of this invention, surfactant concentrations are described as percentages, expressed as weight / volume (w / v).

[0044] As used herein, the term "stabilizer" refers to a pharmaceutically acceptable agent that protects an active pharmaceutical ingredient and / or formulation from chemical and / or physical degradation during manufacturing, storage, and application. Stabilizers include, but are not limited to, sugars, amino acids, polyols (e.g., mannitol, sorbitol, xylitol, dextran, glycerol, araitol, propylene glycol, polyethylene glycol), cyclodextrins (e.g., hydroxypropyl-β-cyclodextrin, sulfobutylethyl-β-cyclodextrin, β-cyclodextrin), polyethylene glycols (e.g., PEG 3000, PEG 3350, PEG 4000, PEG 6000), albumins (e.g., human serum albumin (HSA), bovine serum albumin (BSA)), salts (e.g., sodium chloride, magnesium chloride, calcium chloride), and chelating agents (e.g., EDTA), as defined below. As described above, the stabilizer may be present in the formulation in an amount of about 1 to about 500 mM, preferably in an amount of about 10 to about 300 mM, and more preferably in an amount of about 120 mM to about 300 mM. More than one stabilizer selected from the same or different groups may be present in the formulation.

[0045] As used herein, the term "sugar" includes both monosaccharides and oligosaccharides. Monosaccharides are monomeric carbohydrates that cannot be hydrolyzed by acid, including monosaccharides and their derivatives, such as amino sugars. Sugars typically exist in their D conformation. Examples of monosaccharides include glucose, fructose, galactose, mannose, sorbose, ribose, deoxyribose, and neuraminic acid. Oligosaccharides are carbohydrates composed of more than one monomeric sugar unit linked by branched or straight-chain glycosidic bonds. The monomeric sugar units within an oligosaccharide can be the same or different. Depending on the number of monomeric sugar units, oligosaccharides are disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, etc. Unlike polysaccharides, monosaccharides and oligosaccharides are water-soluble. Examples of oligosaccharides include sucrose, trehalose, lactose, maltose, and raffinose. Preferred sugars used in this invention are sucrose and trehalose (i.e., α,α-D-trehalose), with sucrose being the most preferred. Trehalose can be obtained in the form of trehalose dihydrate. The sugar may be present in the formulation in an amount of about 100 to about 500 mM, preferably in an amount of about 200 to about 300 mM, more preferably in an amount of about 220 to about 250 mM, particularly in an amount of about 230 mM or about 240 mM, and most preferably in an amount of about 230 mM.

[0046] Punizumab (PRX002 / RG7935 / R07046015 / NEOD002) is a humanized monoclonal antibody (mAb) of immunoglobulin G1 (IgG1) derived from the mouse parent antibody 9E4 (produced from a hybridoma of ATCC accession number PTA-8221), and targets the C-terminal epitope (amino acids 118-126) of human synuclein. Punizumab binds to both soluble and insoluble forms of human α-synuclein in biochemical and biophysical experiments, exhibiting greater relative affinity / binding affinity for α-synuclein aggregated in monomeric form. The amino acid sequence of the heavy chain variable region (VH) of punizumab is shown in Seq. Id. No. 1, and the amino acid sequence of the light chain variable region (VL) of punizumab is shown in Seq. Id. No. 2. The heavy chain amino acid sequence of punizumab is shown in Seq. Id. No. 3, with or without a C-terminal lysine, and the light chain amino acid sequence is as shown in Seq. Id. No. 4.

[0047] As used herein, the term "amino acid" refers to a pharmaceutically acceptable organic molecule having an amino group at the α-position of a carboxyl group. Examples of amino acids include, but are not limited to, arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, and proline. In each case, the amino acid used is preferably L-type. Basic amino acids, such as arginine, histidine, or lysine, are preferably used in the form of their inorganic salts (advantageously in the form of hydrochloride, i.e., as amino acid hydrochloride). The preferred amino acid used in this invention is methionine. Methionine is preferably used at a concentration of about 5 to about 25 mM, most preferably about 10 mM.

[0048] One subgroup of stabilizers is antioxidants. The term "antioxidant" refers to a pharmaceutically acceptable excipient that prevents the oxidation of the active pharmaceutical ingredient. Antioxidants include, but are not limited to, ascorbic acid, glutathione, cysteine, methionine, citric acid, and EDTA. Antioxidants can be used in amounts from about 0.01 to about 100 mM, preferably from about 5 to about 50 mM, and more preferably from about 5 to about 25 mM.

[0049] The formulations according to the invention may also contain one or more tonic agents. The term "tonic agent" refers to a pharmaceutically acceptable excipient used to adjust the tension of the formulation. The formulation may be hypotonic, isotonic, or hypertonic. Isotonicity is generally related to the osmotic pressure of the solution, typically to the osmotic pressure of human serum (about 250 to 350 mOsmol / kg). The formulations according to the invention may be hypotonic, isotonic, or hypertonic, but are preferably isotonic. An isotonic formulation is a liquid or a liquid reconstituted from a solid form, such as from a lyophilized form, and indicates a solution having the same tension as some other solutions being compared, such as physiological saline solutions and serum. Suitable tonic agents include, but are not limited to, sodium chloride, potassium chloride, glycerol, and any component selected from the group consisting of amino acids or sugars, particularly glucose. Tonic agents are typically used in amounts from about 5 mM to about 500 mM.

[0050] Among stabilizers and tension agents, there exists a group of compounds that can function in both ways; that is, they can act as both stabilizers and tension agents simultaneously. Examples can be found in the group consisting of sugars, amino acids, polyols, cyclodextrins, polyethylene glycols, and salts. Trehalose is an example of a sugar that can act as both a stabilizer and a tension agent.

[0051] The formulation may also contain adjuvants, such as preservatives, wetting agents, emulsifiers, and dispersants. The absence of microorganisms can be ensured by both sterilization procedures and the addition of various antimicrobial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, etc.). Preservatives are typically used in amounts from about 0.001 to about 2% (w / v). Preservatives include, but are not limited to, ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methylparaben or propylparaben, and benzalkonium chloride.

[0052] As used herein, the term “antigenic determinant” is synonymous with “antigen” and “epitope”, and refers to a site on a polypeptide macromolecule to which the antigen-binding moiety binds, forming an antigen-binding moiety-antigen complex (e.g., a continuous extension of amino acids or a conformational configuration consisting of different regions of discontinuous amino acids). For example, available antigenic determinants may be present on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, on the surface of immune cells, not in serum, and / or in the extracellular matrix (ECM).

[0053] As used herein, the term "antigen-binding moiety" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, the antigen-binding moiety is capable of directing its attached entity (e.g., a second antigen-binding moiety) to a target site, such as to specific types of tumor cells carrying the antigenic determinant. In another embodiment, the antigen-binding moiety is capable of activating signal transduction via its target antigen (e.g., a T-cell receptor complex antigen). The antigen-binding moiety comprises an antibody and a fragment thereof as further defined herein. A particular antigen-binding moiety comprises an antigen-binding domain of the antibody, which includes a variable region of the antibody heavy chain and a variable region of the antibody light chain. In some embodiments, the antigen-binding moiety may comprise a constant region of the antibody as further defined herein and known in the art. Available heavy chain constant regions include any of the following five isoforms: α, δ, ε, γ, or μ. Available light chain constant regions include any of the following two isoforms: κ and λ.

[0054] "Specific binding" refers to binding that is selective to the antigen and can distinguish unwanted or non-specific interactions. The ability of an antigen-binding moiety to bind to a specific antigenic determinant can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) techniques (e.g., analysis on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and conventional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the degree of binding of the antigen-binding moiety to unrelated proteins is less than about 10% of the degree of binding of the antigen-binding moiety to the antigen, for example, as measured by SPR. In some embodiments, the antigen-binding moiety binding to the antigen or the antibody containing the antigen-binding moiety has a concentration of ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g., 10 μM). -8 M or smaller, such as 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 The dissociation constant (K) of M) D ).

[0055] “Affinity” refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). Unless otherwise stated, as used herein, “binding affinity” refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., the antigen-binding moiety and the antigen or receptor and its ligand). The affinity of molecule X for its partner Y can generally be expressed by the dissociation constant (K). D This indicates that the dissociation constant is the sum of the dissociation rate constant and the association rate constant (k, k ... off and k on The ratio of the rate constants is given by the ratio of the rate constants to the rate constants. Therefore, equivalent affinity can include different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by established methods known in the art, including those described herein. A specific method used for measuring affinity is surface plasmon resonance (SPR).

[0056] As used herein, the term "valence" indicates the presence of a specified number of antigen-binding sites in an antibody. Therefore, the term "monovalently bound to antigen" means that the antibody contains one (and no more than one) antigen-binding site that is specific to the antigen.

[0057] The term “antibody” in this article is used in the broadest sense and covers a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0058] The terms “full-length antibody,” “intact antibody,” and “complete antibody” are used interchangeably in this document and refer to antibodies that have a structure that is substantially similar to that of natural antibodies.

[0059] "Antibody fragment" refers to a molecule other than the complete antibody, which contains a portion of the complete antibody that binds to the antigen bound by the complete antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, bifunctional antibodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and single-domain antibodies. For a review of some antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see, for example, Pluckthün, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments containing rescue receptor-binding epitope residues and having an increased in vivo half-life, see U.S. Patent No. 5,869,046. Bifunctional antibodies are antibody fragments having two antigen-binding sites (which may be bivalent or bispecific). See, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Trifunctional and tetrafunctional antibodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Single-domain antibodies are antibody fragments containing all or part of the variable domain of the antibody's heavy chain or all or part of the variable domain of the antibody's light chain. In some embodiments, single-domain antibodies are human single-domain antibodies (Domantis, Inc., Waltham, MA; see, for example, U.S. Patent No. 6,248,516 B1). Antibody fragments can be manufactured using various techniques, including but not limited to proteolytic digestion of intact antibodies as described herein and the generation of recombinant host cells (e.g., E. coli or bacteriophages).

[0060] The term "variable region" or "variable domain" refers to a domain of the antibody heavy or light chain involved in antibody-antigen binding. The variable domains of the heavy and light chains (VH and VL, respectively) of natural antibodies typically have similar structures, with each domain containing four conserved frame regions (FRs) and three hypervariable regions (HVRs). See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. As used herein, the "Kabat number" associated with the variable region sequence refers to the numbering system shown by Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0061] As used herein, the amino acid positions of all constant regions and constant domains of the heavy and light chains are numbered according to the Kabat numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), and are referred to herein as “according to Kabat numbering” or “Kabat numbering”. Specifically, the Kabat numbering system (see pages 647–660 of Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)) is used for the κ and λ isoform light chain constant domain CL, and the Kabat EU index numbering system (see pages 661–723) is used for the heavy chain constant domains (CH1, hinge, CH2, and CH3), which is further clarified herein by referring to this case as “according to Kabat EU index numbering”.

[0062] An antibody or immunoglobulin "class" refers to the type of constant domain or region possessed by its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chain corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0063] A “Fab molecule” is a protein composed of the VH and CH1 domains of the heavy chain (“Fab heavy chain”) and the VL and CL domains of the light chain (“Fab light chain”) of immunoglobulins.

[0064] The term "immunoglobulin molecule" refers to a protein with the structure of naturally occurring antibodies. For example, IgG immunoglobulins are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. Each heavy chain has a variable domain (VH), also called the heavy chain variable domain or heavy chain variable region, from the N-terminus to the C-terminus, followed by three constant domains (CH1, CH2, and CH3), also called the heavy chain constant region. Similarly, each light chain has a variable domain (VL), also called the light chain variable domain or light chain variable region, from the N-terminus to the C-terminus, followed by a constant light chain (CL) domain, also called the light chain constant region. The heavy chains of immunoglobulins can be classified into one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further subdivided into subtypes, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). Based on the amino acid sequence of their constant domains, the light chains of immunoglobulins can be classified into one of two types, called kappa (κ) and lamuda (λ). Immunoglobulins are essentially composed of two Fab molecules linked by an immunoglobulin hinge region and an Fc domain.

[0065] The term "Fc domain" or "Fc region" used in this document is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. This term includes both native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of the IgG heavy chain may vary slightly, the Fc region of the human IgG heavy chain is generally defined as extending from Cys226 or Pro230 to the C-terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids at the C-terminus of the heavy chain. Therefore, antibodies produced by host cells by expressing a specific nucleic acid molecule encoding the full-length heavy chain may include the full-length heavy chain or a cleaved variant of it. This could be the case where the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, according to the Kabat EU index number). Therefore, the C-terminal lysine (Lys447) or C-terminal glycine (Gly446) and lysine (K447) of the Fc region may or may not be present. Unless otherwise stated herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index, as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (see above). As used herein, the “subunit” of the Fc domain refers to one of the two polypeptides that form the dimer Fc domain, namely a polypeptide containing the C-terminal constant region of the immunoglobulin heavy chain that is stably self-associating. For example, the subunit of the IgG Fc domain contains the IgG CH2 and IgG CH3 constant domains.

[0066] The term "effective function" refers to those biological activities attributed to the Fc region of an antibody, which vary across antibody isotypes. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), cytokine secretion, antigen-presenting cell-mediated antigen uptake by immune complexes, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0067] The formulation may also contain adjuvants, such as preservatives, wetting agents, emulsifiers, and dispersants. The absence of microorganisms can be ensured by both sterilization procedures and the addition of various antimicrobial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, etc.). Preservatives are typically used in amounts from about 0.001 to about 2% (w / v). Preservatives include, but are not limited to, ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methylparaben or propylparaben, and benzalkonium chloride.

[0068] Pharmaceutical formulations may also contain adjuvants, such as preservatives, wetting agents, emulsifiers, and dispersants. The absence of microorganisms can be ensured through both sterilization procedures and the addition of various antimicrobial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, etc.). Preservatives are typically used in amounts from about 0.001 to about 2% (w / v). Preservatives include, but are not limited to, ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methylparaben or propylparaben, and benzalkonium chloride.

[0069] The formulations of the present invention can be administered by a variety of methods known in the art. As those skilled in the art will understand, the route and / or mode of administration will vary depending on the desired outcome. To administer the formulations of the present invention via certain routes of administration, it may be necessary to dilute the formulation in a diluent. Pharmaceutically acceptable diluents include saline, glucose, Ringer's solution, and aqueous buffer solutions.

[0070] Preferably, the formulation according to the invention is administered intravenously (IV), subcutaneously (SC), or by any other parenteral administration method (such as those known in the pharmaceutical field). In a preferred aspect, the pharmaceutical formulation is administered by IV infusion. When administered via intravenous injection, it can be administered as a bolus or as a continuous infusion. For example, the pharmaceutical formulation of the invention can be diluted with sterile saline solution and administered using an infusion pump commonly used in clinical settings.

[0071] As used in this article, the terms “parenteral administration” and “post-gastrointestinal administration” refer to administration methods other than enteral and local administration, usually by injection, and including but not limited to intravenous, intramuscular, intra-arterial, intrathecal, extracapsular, infraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions.

[0072] The pharmaceutical preparations described in this invention are suitable for administration to a patient once or in a series of treatments, and can be administered to the patient at any time from the onset of diagnosis; they can be administered as a single treatment or in combination with other drugs or therapies that can be used to treat the conditions described herein.

[0073] An antibody targeting α-synuclein may be the sole active ingredient in a liquid pharmaceutical composition. Alternatively, an antibody targeting α-synuclein may be administered in combination with one or more other therapeutic active ingredients (e.g., simultaneously, sequentially, or separately). As used herein, the term "active ingredient" refers to an ingredient that has a pharmacological effect (such as a therapeutic effect) at relevant doses.

[0074] The pharmaceutical composition suitably comprises a therapeutically effective amount of antibody. As used herein, the term "therapeutically effective amount" refers to the amount of therapeutic agent required to treat, improve, or prevent a target disease or condition, or to exhibit a detectable therapeutic, pharmacological, or preventative effect. For any antibody, the therapeutically effective amount can initially be estimated in cell culture assays or animal models, typically rodents, rabbits, dogs, pigs, or primates. Animal models can also be used to determine appropriate concentration ranges and routes of administration. This information can then be used to determine the useful dose and route of administration to humans.

[0075] The precise therapeutically effective dose for human subjects will depend on the severity of the disease state, the subject's overall health, age, weight and sex, diet, timing and frequency of administration, drug combination, sensitivity to response, and tolerance / response to the therapy. This dose can be determined through routine laboratory testing and is within the judgment of the clinician. In a particular aspect, antibodies against α-synuclein are administered at a (fixed) dose of 600 to 1200 mg, particularly at a dose of 750 to 1100 mg, more particularly at a dose of 750 to 1000 mg, and even more particularly at a dose of 900 to 1000 mg. In a preferred aspect, the dose is 1000 mg.

[0076] Stable formulations intended for in vivo administration must be sterile. This can be easily achieved through filtration using a sterile filter membrane. The formulation must be fluid, to the extent that it can be delivered via a syringe or infusion system. In addition to water, the carrier can be isotonic buffered saline solution, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Due to its high stability, the pharmaceutical formulations according to the invention can be administered intravenously without an in-line filter and are therefore easier to handle than conventional formulations that require in-line filtration. In-line filters such as Sterifix® must be installed in the infusion line for intravenous medications to prevent administration of any particles, air, or microorganisms that may be present in the intravenous solution or line. Particles with a size of 5 to 20 micrometers and larger have the ability to impede blood flow through pulmonary capillaries, which can lead to complications such as pulmonary embolism. Foreign particles can also cause phlebitis at the injection site, and filters may help reduce the incidence of phlebitis.

[0077] The stable pharmaceutical formulations according to the present invention can be prepared by methods known in the art, such as ultrafiltration-percolation, dialysis, addition and mixing, lyophilization, reconstitution, and combinations thereof. Examples of the preparation of the formulations according to the present invention can be found below.

[0078] Therefore, the present invention includes a method for preparing a formulation according to the invention. The method includes buffer exchange of an antibody with a percolation buffer containing a desired buffer composition, and, if necessary, concentration of the antibody by percolation, followed by the addition of an excipient (e.g., sucrose, sodium chloride, methionine) as a stock solution to the antibody solution, followed by the addition of a surfactant as a stock solution to the antibody / excipient solution, and finally adjusting the antibody concentration to a desired final concentration using a buffer solution, thereby also achieving the final excipient and surfactant concentrations.

[0079] Alternatively, excipients may also be added as solids to the starting solution containing the antibody. If the antibody is in solid form, such as a lyophilized product, the formulation according to the invention can be prepared by first dissolving the antibody in an aqueous or buffer solution optionally containing one or more excipients, and then adding other excipients as a stock solution or solid. The antibody can also advantageously be dissolved directly in a solution containing all other excipients. One or more excipients present in the formulation according to the invention may have already been added during or at the end of the antibody preparation process, for example by dissolving the antibody directly in a solution containing one, more than one, or preferably all of the excipients in the final purification step after antibody preparation. If the solution containing the antibody and excipients does not yet have the desired pH, this is adjusted by adding an acid or base, preferably an acid or base already present in the buffer system. Sterile filtration is then performed.

[0080] The stable liquid pharmaceutical formulations according to the present invention may also be in lyophilized form or in a liquid form reconstituted from a lyophilized form. The “lyophilized form” is manufactured by a freeze-drying method known in the art. The lyophilized product typically has a residual moisture content of about 0.1% to 5% (w / w) and exists as a powder or a physically stable cake. The “reconstituted form” can be obtained from the lyophilized product by rapid dissolution upon addition of a reconstituted medium. Suitable reconstituted media include, but are not limited to, water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solutions (e.g., 0.9% (w / v) NaCl), glucose solutions (e.g., 5% (w / v) glucose), surfactant-containing solutions (e.g., 0.01% (w / v) polysorbate 20), and pH buffer solutions (e.g., phosphate buffer solutions).

[0081] In another preferred embodiment, the buffer contained in the formulation according to the invention is a histidine buffer, preferably an L-histidine / HCl buffer. In particular, an L-histidine / acetate buffer (i.e., L-histidine as the buffer) is preferred.

[0082] Preferably, the buffer provides a pH of 5.0 to 6.0, more preferably 5.5 ± 0.5, and most preferably 5.5 ± 0.3.

[0083] In a preferred embodiment, the surfactant contained in the formulation according to the invention is polysorbate, preferably polysorbate 20 or polysorbate 80, and most preferably polysorbate 20.

[0084] Preferably, the surfactant concentration is 0.01% to 0.1% (w / v), more preferably 0.02% to 0.05%, and most preferably 0.04%.

[0085] Preferably, the concentration of at least one stabilizer is 120 to 300 mM, more preferably 220 to 250 mM, and most preferably 230 to 240 mM.

[0086] The formulations according to the present invention may be in liquid form, in lyophilized form, or in a liquid form reconstituted from a lyophilized form. In some embodiments, the formulation is in liquid form.

[0087] As used herein in connection with formulations according to the invention, the term "liquid" means a formulation that is liquid at atmospheric pressure and at a temperature of at least about 2 to about 8°C.

[0088] In this document, the term "lyophilized" in relation to formulations according to the present invention refers to a formulation manufactured by a lyophilization method known in the art itself. Solvents (e.g., water) are removed by freezing followed by sublimation of the ice under vacuum and desorption of residual water at elevated temperatures. Lyophilized products typically have a residual moisture content of about 0.1 to 5% (w / w) and are present as a powder or a physically stable cake. Lyophilized products are characterized by rapid dissolution upon addition of a reconstitution medium.

[0089] In this document, the term "reconstituted form" in relation to formulations according to the present invention refers to a lyophilized formulation that has been redissolved by adding a reconstitution medium. Suitable reconstitution media include, but are not limited to, water for injection (WFI), water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), surfactant-containing solution (e.g., 0.01% polysorbate 20), and pH buffer solution (e.g., phosphate buffer solution).

[0090] The formulations according to the present invention exhibit good physiological tolerance, can be easily prepared, can be precisely dispensed, and are stable against decomposition products and aggregates during storage, repeated freeze-thaw cycles, and mechanical stress.

[0091] The present invention further includes a method for preparing a formulation according to the invention. The method includes buffer exchange of an α-synuclein antibody with a percolation buffer containing a desired buffer composition, and, if necessary, concentration of the antibody by percolation, followed by the addition of an excipient (e.g., trehalose dihydrate, sucrose, arginine, sodium chloride, methionine) as a stock solution to the antibody solution, followed by the addition of a surfactant as a stock solution to the antibody / excipient solution, and finally adjusting the antibody concentration to a desired final concentration using a buffer solution, thereby also achieving the final excipient and surfactant concentrations.

[0092] Alternatively, excipients may be added as solids to the starting solution containing α-synuclein antibody. If the α-synuclein antibody is in solid form, such as a lyophilized product, the formulation according to the invention can be prepared by first dissolving the bispecific antibody in an aqueous or buffer solution optionally containing one or more excipients, and then adding other excipients as a stock solution or solid. The α-synuclein antibody can also advantageously be dissolved directly in a solution containing all other excipients. One or more excipients present in the formulation according to the invention may have already been added during or at the end of the α-synuclein antibody preparation process, for example by directly dissolving the α-synuclein antibody in a solution containing one, more than one, or preferably all of the excipients in the final purification step following the preparation of the α-synuclein antibody. If the solution containing the α-synuclein antibody and excipients does not yet have the desired pH, this is adjusted by adding an acid or base, preferably an acid or base already present in the buffer system. Sterile filtration is then performed.

[0093] The present invention further includes the use of the formulations according to the invention for treating diseases, or the use of the formulations according to the invention in the preparation of medicaments for treating diseases in which CEA is expressed, particularly cell proliferation disorders, especially where CEA is abnormally expressed (e.g., overexpressed) compared to normal tissues of the same cell type. Such disorders include different types of cancer, such as colorectal cancer, lung cancer, pancreatic cancer, breast cancer, and gastric cancer. CEA expression levels can be determined by methods known in the art, such as immunohistochemistry, immunofluorescence assays, immunoenzyme assays, ELISA, flow cytometry, radioimmunoassay, Western blotting, ligand binding, kinase activity, etc. The present invention also includes methods for treating diseases as described above, comprising administering the formulations according to the invention to an individual in need.

[0094] To administer the formulations of the present invention via certain routes of administration, it may be necessary to dilute the formulations in a diluent. Pharmaceutically acceptable diluents include saline, glucose, Ringer's solution, and aqueous buffer solutions.

[0095] Preferably, the formulation according to the invention is administered intravenously (iv), subcutaneously (sc), or by any other parenteral administration method (such as those known in the pharmaceutical field).

[0096] As used in this article, the terms “parenteral administration” and “post-gastrointestinal administration” refer to modes of administration other than enteral and local administration, usually by injection, and including but not limited to intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions.

[0097] The formulations according to the present invention can be prepared by methods known in the art, such as ultrafiltration-percolation, dialysis, addition and mixing, lyophilization, reconstitution, and combinations thereof. Examples of the preparation of the formulations according to the present invention can be found below.

[0098] punizumab amino acid sequence

[0099]

[0100] These examples illustrate the invention in more detail, but should not be construed as limiting the scope of the invention. All disclosures of patents and scientific literature cited herein are expressly incorporated herein by reference.

[0101] Example

[0102] The anti-synuclein α antibody formulation according to the present invention was developed based on the experimental results provided below, using the general preparation and analytical methods and assays outlined below.

[0103] The liquid pharmaceutical product formulation according to the present invention is developed as follows.

[0104] Example 1: Preparation of liquid formulations

[0105] To prepare the liquid formulation, punizumab is buffer-exchanged with percolation buffer containing the desired buffer composition, and, if necessary, concentrated by percolation to an antibody concentration approximately 20% to 30% above the target concentration. After percolation, excipients (e.g., sucrose, sodium chloride, and methionine) are added to the antibody solution as a stock solution. A surfactant is then added as a stock solution at a concentration of 50 to 200 times. Finally, the protein concentration is adjusted with buffer to a final punizumab concentration of approximately 150 mg / ml.

[0106] All formulations were aseptically filtered through a 0.22 µm low-protein-binding filter and aseptically filled into sterile 6 ml glass vials, sealed with ETFE (ethylene-tetrafluoroethylene) coated rubber stoppers and aluminum crimp caps. The filling volume was approximately 2.7 ml. These formulations were stored at different ICH climatic conditions (5 °C, 25 °C, and 40 °C) for varying time intervals and pressurized by shaking (at a shaking frequency of 200 min⁻¹ for one week at 5 °C and 25 °C) and freeze-thaw stress methods. Samples before and after the stress tests were analyzed using the following analytical methods:

[0107] The following analytical methods were used to analyze the samples before and after the stress test.

[0108] ●UV spectrophotometry

[0109] ● Size Exclusion Chromatography (SEC)

[0110] ●Imaging capillary electrophoresis / capillary isoelectric focusing (iCE / cIEF) ion exchange chromatography (IEC)

[0111] ● Clarity and turbidity of the solution

[0112] ●Analytical Protein A Chromatography

[0113] ●Visual inspection

[0114] UV spectroscopy used to determine protein content was performed on a Perkin Elmer λ35 UV spectrophotometer, with wavelengths ranging from 240 nm to 400 nm. Pure protein samples were diluted to approximately 0.5 mg / mL with the appropriate formulation buffer. Protein concentration was calculated according to Equation 1.

[0115] Formula 1: Protein content =

[0116] The UV light absorption at 280 nm is corrected for the light scattering at 320 nm and multiplied by a dilution factor determined based on the weight and density of the pure sample and the dilution buffer. The molecule is then divided by the product of the cuvette path length d and the extinction coefficient ε.

[0117] Size exclusion chromatography (SEC) was used to detect soluble high molecular weight substances (aggregates) and low molecular weight hydrolysis products (LMWs) in formulations. This method was performed on a Waters Alliance 2695 HPLC instrument equipped with a Waters W2487 dual absorbance detector and a Waters BioSuite 250 column. A mobile phase of 0.2 M K₂HPO₄ / 0.25 M KCl at pH 7.0 was used. Intact monomers, aggregates, and hydrolysis products were separated by isocratic elution and detected at a wavelength of 280 nm.

[0118] Imaging capillary electrophoresis / capillary isoelectric focusing (iCE / cIEF) was used to characterize the different charge classes of the antibody punizumab in the formulation. The instrument used for formulation F1 was an ICE3 IEF analyzer from Convergent Bioscience (ProteinSimple). The instruments used for F4n, F5n, F8n, and F9n were iCE280 IEF analyzers from the same manufacturer. FC-coated capillary cartridges (code 101701). Pre-focusing: 1 min / 1500 V (all). Focusing: 11 min / 3000 V, all without Hb standard (4.5 min / 3000 V). Detection wavelength: 280 nm. Low pI label: 8.18 / 0.01 mg / ml. High pI label: 9.77 / 0.01 mg / ml. Pharmalyte: 8 – 10.5 / 3%. Biolyte: 7 – 9 / 1%. Sample pre-dilution: 2.5 mg / ml, containing Mil-Q water. Centrifugation: 10,000 rpm before loading. Sample temperature: 10°C. Standard: Hemoglobin stock solution, Bioscience, catalog number 101801. Sample concentration: 0.25 mg / ml.

[0119] Clarity and turbidity were measured by turbidity determination and expressed in formalin turbidity units (FTU). The pure sample was transferred to a transparent glass tube with a diameter of 11 mm and placed in a HACH 2100AN turbidimeter.

[0120] Analytical protein A chromatography was performed to monitor the oxidation state of four conserved methionine side chains in the Fc moiety of α-synuclein antibody. This method was performed on a Waters Alliance 2695 HPLC instrument equipped with a Waters W2487 dual absorbance detector (detection wavelength 280 nm) and a Poros A720 4.6 mm x 50 mm column from Applied Biosystems, USA. PBS from Gibco and Invitrogen, and 0.1 M acetic acid and 0.15 M sodium chloride, pH 2.8, were used as mobile phases A and B, respectively, at a flow rate of 2.0 mL / min.

[0121] The presence of visible particles in the sample was checked using the Simplex ampoule test apparatus OPTIMA I (European Pharmacopoeia (EP) kit method).

[0122] The stability test results for formulations F4n to F9n and F1 are provided in the table below. Formulation F1 was determined to be the most favorable antibody formulation for obtaining maximum antibody concentration, maximum antibody stability, and no particles.

[0123]

Claims

1. A pharmaceutical preparation comprising: 120 to 200 mg / ml of α-synuclein antibody; 1 to 50 mM buffer; 0.01% to 1% (w / v) of surfactant; At least one stabilizer of 1 to 50 mM; 100 to 150 mM tension agent, Its pH ranges from 5.0 to 7.

0.

2. The formulation according to claim 1, wherein the concentration of the anti-synuclein antibody is in the range of 150 to 200 mg / ml, preferably 180 mg / ml.

3. The formulation according to claim 1 or 2, wherein the buffer is a histidine buffer, preferably a histidine acetate buffer.

4. The formulation according to any one of claims 1 to 3, wherein the concentration of the buffer is 10 to 40 mM.

5. The formulation according to any one of claims 1 to 4, wherein the buffer provides a pH of 5.0 to 6.0, preferably 5.

5.

6. The formulation according to any one of claims 1 to 5, wherein the surfactant is polysorbate, preferably polysorbate 20.

7. The formulation according to any one of claims 1 to 6, wherein the concentration of the surfactant is 0.01% to 0.1% (w / v).

8. The formulation according to any one of claims 1 to 7, wherein at least one stabilizer is selected from the group consisting of salts, sugars and amino acids, preferably methionine.

9. The formulation according to any one of claims 1 to 8, wherein the concentration of the at least one stabilizer is 5 to 50 mM.

10. The formulation according to any one of claims 1 to 9, wherein the concentration of the tensioning agent is 120 to 150 mM, preferably 130 mM.

11. The formulation according to claim 10, wherein the tensile agent is arginine-HCl.

12. The formulation according to any one of claims 1 to 11, comprising: 150 to 180 mg / ml α-synuclein antibody; 15 to 30 mM L-histidine / acetate buffer; 0.02% to 0.05% (w / v) polysorbate 20; 120 to 180 mM arginine-HCl; 5 to 25 mM L-methionine; Its pH is 5.5 ± 0.

5.

13. The formulation according to any one of claims 1 to 12, comprising: 180 mg / ml α-synuclein antibody, 20 mM L-histidine / acetate buffer, 0.04% (w / v) Polysorbate 20, 130 mM arginine-HCl 10 mM methionine at pH 5.

5.

14. The formulation according to any one of claims 1 to 13, wherein the anti-α synuclein antibody comprises: a VH domain having an amino acid sequence of Seq. Id. No. 1; and a VL domain having an amino acid sequence of Seq. Id. No.

2.

15. The formulation according to any one of claims 1 to 14, wherein the α-synuclein antibody comprises: a heavy chain having an amino acid sequence of Seq. Id. No. 3; and a light chain having an amino acid sequence of Seq. Id. No.

4.

16. The formulation according to any one of claims 1 to 15, wherein the α-synuclein antibody is an antibody having INN pronizumab.

17. The formulation according to any one of claims 1 to 16, wherein it is in liquid form, in lyophilized form, or in liquid form reconstituted from lyophilized form.

18. The formulation according to claim 16, wherein the formulation is a liquid formulation, preferably for intravenous and / or subcutaneous administration.

Images