RU2764521C2 - AQUEOUS PHARMACEUTICAL COMPOSITION OF A RECOMBINANT MONOCLONAL ANTIBODY TO TNF-α - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: group of inventions relates to improved aqueous pharmaceutical compositions of adalimumab. The aqueous pharmaceutical composition for treating TNF-α mediated diseases for intravenous or subcutaneous administration includes adalimumab at a concentration of 50 to 200 mg/ml; a buffering agent or buffer system based on acetate ions, wherein the concentration of the acetate-based buffering solution is from 1 to 100 mm; trehalose at a concentration of 25 to 150 mg/ml and/or proline at a concentration of 5 to 40 mg/ml, or trehalose at a concentration of 25 to 150 mg/ml and arginine at a concentration of 0.05 to 0.5 mg/ml; polysorbate 20 at a concentration of 0.05 mg/ml to 10 mg/ml, polysorbate 80 at a concentration of 0.05 mg/ml to 10 mg/ml, or poloxamer 188 at a concentration of 0.05 mg/ml to 10 mg/ml or a combination thereof, wherein the composition has a pH of 5.5. Also disclosed are a method for treating TNF-α-mediated diseases, an application of the composition for treating TNF-α-mediated diseases, and a method for producing the composition.

EFFECT: group of inventions prevents physico-chemical instability expressed in the formation of aggregates and fragments of proteins or modification of proteins in the solution.

19 cl, 8 dwg, 11 tbl, 5 ex

Description

The technical field to which the invention belongs

The invention relates to medical pharmacology and concerns, in particular, an aqueous pharmaceutical composition of human antibodies that specifically bind or neutralize TNFα, and its use.

State of the art

Tumor necrosis factor alpha (TNFα) is a natural mammalian cytokine produced by various cell types, including monocytes and macrophages, in response to endotoxin or other stimuli. TNFα is a major mediator of inflammatory, immunological and pathophysiological responses (Grell, M., et al. (1995) Cell, 83: 793-802).

Soluble TNFα is generated by cleavage of a transmembrane precursor protein (Kriegler, et al. (1988) Cell 53: 45-53) and secreted 17 kilodalton (kDa) polypeptides are assembled into soluble homotrimeric complexes (Smith, et al. (1987), J. Biol Chem 262: 6951-6954; Butler, et al. (1986) Nature 320:584; Old (1986) Science 230: 630). These complexes then bind to receptors found on many cells. Binding provides a number of pro-inflammatory effects including (i) release of other pro-inflammatory cytokines such as interleukin (IL)-6, IL-8, and IL-1, (ii) release of matrix metalloproteinases, and (iii) increased expression of endothelial adhesion molecules, further enhancing inflammatory and immune cascades by attracting leukocytes to extravascular tissues.

There are many disorders associated with elevated levels of TNFα. For example, TNFα expression has been shown to be elevated in a number of human diseases, including chronic diseases such as rheumatoid arthritis (RA), inflammatory bowel disorders including Crohn's disease and ulcerative colitis, sepsis, congestive heart failure, asthma, and multiple sclerosis. TNFα is also referred to as a pro-inflammatory cytokine.

Physiologically, TNFα is also associated with protection against specific infections (Cerami. et al. (1988), Immunol. Today 9:28). TNFα is released by macrophages activated by lipopolysaccharides of Gram-negative bacteria. As such, TNFα appears to be a very important endogenous mediator involved in the development and pathogenesis of endotoxic shock associated with bacterial sepsis.

Adalimumab (Humira®, AbbVie, Inc.) is a recombinant human IgGl monoclonal antibody specific for human TNF. This antibody is also known as D2E7. Adalimumab consists of 1330 amino acids and is described and patented in US Pat. Typically, adalimumab is produced by recombinant DNA technology in an expression system with mammalian cells such as, for example, Chinese hamster cells. Adalimumab specifically binds to TNF and neutralizes the biological functions of TNF by blocking its interaction with TNF receptors p55 and p75 on the cell surface.

Various formulations of adalimumab are known in the art (See, for example, international applications WO2004016286 and WO2012065072).

W02004016286 describes a liquid composition for stabilizing antibodies used in the treatment of TNF-mediated diseases, comprising an antibody, for example, adalimumab, a buffer system, polysorbate, and isotonic agents, for example, mannitol and sodium chloride. The composition uses a citrate-phosphate buffer (composition Humira 1). The above composition has a number of disadvantages: 1) insufficient colloidal stability of the antibody at high concentrations, 2) insufficient stability under heat stress, and 3) aggregation during long-term storage.

WO2012065072 describes a liquid composition for stabilizing antibodies used in the treatment of TNFα-mediated diseases, comprising an antibody, for example, adalimumab, as well as mannitol, polybrother 80 (composition of Humira 2). The above composition has a number of disadvantages: 1) pronounced instability during freezing and thawing, and 2) low aggregation temperature.

Thus, there is a need to create a new drug containing an antibody that binds TNFα, such as adalimumab.

The invention relates to stable aqueous compositions containing adalimumab, which ensure its long-term storage, and also easily tolerate freezing-thawing and thermal stress.

Brief description of the invention

In one aspect, the present invention relates to an aqueous pharmaceutical composition for intravenous, subcutaneous or intramuscular administration, containing a recombinant anti-TNFα monoclonal antibody, an acetate ion buffering agent (or buffer system), trehalose or proline, or a combination thereof, or trehalose and arginine, polysorbate 20, polysorbate 80, or poloxamer 188, or a combination thereof.

In some embodiments, the recombinant anti-TNFα monoclonal antibody is adalimumab.

In some embodiments of the composition, the concentration of the monoclonal antibody is from 50 to 200 mg/ml.

In some embodiments of the composition, the concentration of the acetate buffer solution is from 1 to 100 mm.

In some embodiments, the composition has a pH of 4 to 7.

In some embodiments of the composition, the concentration of trehalose is from 25 to 150 mg/ml.

In some embodiments of the composition, the concentration of proline is from 5 to 40 mg/ml.

In some embodiments of the composition, the concentration of arginine is from 0.05 to 1.0 mg/ml.

In some embodiments of the composition, the concentration of polysorbate 20 is from 0.05 mg/ml to 10 mg/ml.

In some embodiments of the composition, the concentration of polysorbate 80 is from 0.05 mg/ml to 10 mg/ml.

In some embodiments of the composition, the concentration of poloxamer 188 is from 0.05 mg/ml to 10 mg/ml.

In some embodiments, the composition contains from 50 to 200 mg/ml of a recombinant anti-TNFα monoclonal antibody, from 0.4 to 1.2 mg/ml of sodium acetate trihydrate, from 25 to 150 mg/ml of trehalose and/or from 5 to 40 mg/ml of proline, or from 25 to 150 mg/ml trehalose and 0.05 to 1.0 mg/ml arginine, acetic acid, ice. up to pH 5.0 - 6.0, from 0.1 mg/ml to 1 mg/ml polysorbate 80.

In some embodiments, the recombinant anti-TNFα monoclonal antibody is adalimumab.

In some embodiments, the composition contains from 50 to 150 mg/ml adalimumab, 0.436 mg/ml sodium acetate trihydrate, 80 mg/ml trehalose dihydrate, acetic acid ice. to pH 5.5, 0.5 mg/ml polysorbate 80.

In some embodiments, the composition contains from 50 to 150 mg/ml adalimumab, 0.436 mg/ml sodium acetate trihydrate, 80 mg/ml trehalose dihydrate, 0.1 mg/ml arginine hydrochloride, acetic acid ice. to pH 5.5, 0.5 mg/ml polysorbate 80.

In some embodiments, the composition contains from 50 to 150 mg/ml adalimumab, 0.436 mg/ml sodium acetate trihydrate, 27 mg/ml proline, acetic acid ice. to pH 5.5, 0.5 mg/ml polysorbate 80.

In some embodiments, the composition contains from 50 to 150 mg/ml adalimumab, 0.436 mg/ml sodium acetate trihydrate, 40 mg/ml trehalose dihydrate, 13.5 mg/ml proline, acetic acid ice. to pH 5.5, 0.5 mg/ml polysorbate 80.

In some embodiments, the composition contains from 50 to 200 mg/ml of a recombinant anti-TNFα monoclonal antibody, from 0.4 to 1.2 mg/ml of sodium acetate trihydrate, from 25 to 150 mg/ml of trehalose and/or from 5 to 40 mg/ml of proline, or from 25 to 150 mg/ml trehalose and 0.05 to 1.0 mg/ml arginine, acetic acid, ice. to pH 5.5, from 0.1 mg/ml to 1 mg/ml polysorbate 20.

In some embodiments, the recombinant anti-TNFα monoclonal antibody is adalimumab.

In some embodiments, the composition contains from 50 to 150 mg/ml adalimumab, 0.436 mg/ml sodium acetate trihydrate, 80 mg/ml trehalose dihydrate, acetic acid ice. to pH 5.5, 0.5 mg/ml polysorbate 20.

In some embodiments, the composition contains from 50 to 150 mg/ml adalimumab, 0.436 mg/ml sodium acetate trihydrate, 80 mg/ml trehalose dihydrate, 0.1 mg/ml arginine hydrochloride, acetic acid ice. to pH 5.5, 0.5 mg/ml polysorbate 20.

In some embodiments, the composition contains from 50 to 150 mg/ml adalimumab, 0.436 mg/ml sodium acetate trihydrate, 27 mg/ml proline, acetic acid ice. to pH 5.5, 0.5 mg/ml polysorbate 20.

In some embodiments, the composition contains from 50 to 150 mg/ml adalimumab, 0.436 mg/ml sodium acetate trihydrate, 40 mg/ml trehalose dihydrate, 13.5 mg/ml proline, acetic acid ice. to pH 5.5, 0.5 mg/ml polysorbate 20.

In some embodiments, the composition contains from 50 to 200 mg/ml of a recombinant anti-TNFα monoclonal antibody, from 0.4 to 1.2 mg/ml of sodium acetate trihydrate, from 25 to 150 mg/ml of trehalose and/or 5 to 40 mg/ml of proline, or from 25 up to 150 mg / ml of trehalose and from 0.05 to 1.0 mg / ml of arginine, acetic acid ice. up to pH 5.0 – 6.0, from 0.1 mg/ml to 1 mg/ml poloxamer 188.

In some embodiments, the recombinant anti-TNFα monoclonal antibody composition is adalimumab.

In some embodiments, the composition contains from 50 to 150 mg/ml adalimumab, 0.436 mg/ml sodium acetate trihydrate, 80 mg/ml trehalose dihydrate, acetic acid ice. to pH 5.5, 1 mg/ml poloxamer 188.

In some embodiments, the composition contains from 50 to 150 mg/ml adalimumab, 0.436 mg/ml sodium acetate trihydrate, 80 mg/ml trehalose dihydrate, 0.1 mg/ml arginine hydrochloride, acetic acid ice. to pH 5.5, 1 mg/ml poloxamer 188.

In some embodiments, the composition contains from 50 to 150 mg/ml adalimumab, 0.436 mg/ml sodium acetate trihydrate, 27 mg/ml proline, acetic acid ice. to pH 5.5, 1 mg/ml poloxamer 188.

In some embodiments, the composition contains from 50 to 150 mg/ml adalimumab, 0.436 mg/ml sodium acetate trihydrate, 40 mg/ml trehalose dihydrate, 13.5 mg/ml proline, acetic acid ice. to pH 5.5, 1 mg/ml poloxamer 188.

In one aspect, the present invention relates to a method of treating diseases mediated by TNFα, comprising the introduction of the above compositions in an effective amount.

In some embodiments, the TNF-mediated disease is selected from active rheumatoid arthritis (moderate to severe), active psoriatic arthritis, active ankylosing spondylitis, chronic plaque psoriasis (moderate or severe), ulcerative colitis (moderate to severe), axial spondyloarthritis, active purulent hidradenitis, juvenile idiopathic arthritis, Crohn's disease (moderate or severe), uveitis, active enthesitis-associated arthritis.

In one aspect, the present invention relates to the use of the above compositions for the treatment of diseases mediated by TNFα.

In some applications, the disease is selected from active rheumatoid arthritis (moderate to severe), active psoriatic arthritis, active ankylosing spondylitis, chronic plaque psoriasis (moderate or severe), ulcerative colitis (moderate to severe), axial spondyloarthritis, active suppurative hydradenitis , juvenile idiopathic arthritis, Crohn's disease (moderate or severe), uveitis, active enthesitis-associated arthritis.

In one aspect, the present invention relates to a method for preparing the above compositions, which includes adding acetate buffering agents to the aqueous phase, followed by adding, in any order, the following components: an osmolytic and/or a stabilizer selected from the group: trehalose, proline, or combinations thereof , a recombinant monoclonal antibody to TNFα, a surfactant selected from the group: polysorbate 20, polysorbate 80, poloxamer 188, or combinations thereof.

Brief description of the drawings

Fig.1. The dependence of the optical density of solutions at 400 nm on the concentration of PEG 6000 after preparation.

Fig.2. The dependence of the optical density of solutions at 400 nm on the concentration of PEG 6000 after preparation.

Fig.3. The dependence of the optical density of solutions at 400 nm on the concentration of PEG 6000 after preparation.

Fig.4. Adalimumab aggregation point in acetate buffer, pH 5.0.
_Tag 74.0°С. Dark gray: Z-average, nm, light gray: intensity, kcps.

Fig.5. Adalimumab aggregation point in citrate buffer, pH 5.0.
_Tag 69.5°С. Dark gray: Z-average, nm, light gray: intensity, kcps.

Fig.6. Adalimumab aggregation point in histidine buffer solution, pH 6.0.
_Tag 69.5°С. Dark gray: Z-average, nm, light gray: intensity, kcps.

Fig.7. Aggregation point of adalimumab in phosphate buffer solution, pH 6.0.
T _ag 71.0 ° C. Dark gray: Z-average, nm, light gray: intensity, kcps.

Fig.8. Adalimumab aggregation point in Humira 1, pH 5.2. _Tag 69.5°С. Dark gray: Z-average, nm, light gray: intensity, kcps.

Description of the invention

Definitions and Common Methods

The terms used in the present description, as a rule, have their usual meanings for this field within the context of the invention and in the specific context where each term is used. Below, or elsewhere in the description, certain terms that are used to describe the invention are defined to provide the practice with additional guidance in relation to the description of the invention. Some synonyms are given. The presentation of one or more synonyms does not preclude the use of other synonyms. The use of examples anywhere in this specification, including examples of any terms described herein, is for illustrative purposes only and does not in any way limit the scope or meaning of the invention or any object illustrated. The invention is not limited to the various embodiments described in the present description.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by a person skilled in the art to which the invention belongs. In case of conflict, this document, including the definitions, shall govern.

The term "adalimumab" is synonymous with the active pharmaceutical ingredient in Humira®, as well as the protein considered or implied by its biosimilar or bioimproved variants. Adalimumab is a recombinant human IgGl monoclonal antibody specific for human TNF. Adalimumab is also known as D2E7. Adalimumab contains two light chains with a molecular weight of approximately 24 kDa each and two IgGl heavy chains with a molecular weight of approximately 49 kDa each. Each light chain consists of 214 amino acid residues, and each heavy chain consists of 451 amino acid residues. Thus, adalimumab consists of 1330 amino acids. The term adalimumab is also intended to include the so-called biosimilar or biosimilar variants of the adalimumab protein used in the commercially available Humira®. For example, a variant of commercial Humira® may be acceptable to the FDA when it has substantially the same pharmacological effect as commercially available Humira®, even though it may exhibit certain physical characteristics, such as a glycosylation profile and an acid-base profile, that may be , if not identical to Humira®, then similar to them.

For the purposes of this application, the term "adalimumab" also includes adalimumab with minor modifications in amino acid structure (including deletions, introductions and/or amino acid substitutions) or in glycosylation and acid-base profile properties that do not significantly affect the function of the polypeptide. The term "adalimumab" includes all forms and formulations of Humira®, including, but not limited to, concentrated formulations, injectable ready-to-use formulations; formulations reconstituted with water, alcohol and/or other ingredients, and others.

The term "human TNFα", which is also known as hTNFα or hTNF or TNFα, is intended to refer to a human cytokine that exists as a 17 kDa secreted form and a 26 kDa membrane-associated form, the biologically active form of which consists of a trimer of non-covalently bound molecules of mass 17 kDa. The structure of TNFα is further described, for example, in Pennica, D., et al. (1984) Nature 312:724-729; Davis, J.M., et al. (1987) Biochemistry 26:1322-1326 and Jones, E. Y., et al. (1989) Nature 338:225-228. The term human rhTNFa is intended to include recombinant human TNFα, which can be produced by standard recombinant expression methods or purchased commercially (R&D Systems, cat. no. 210-TA, Minneapolis, Minn.).

As used herein, the term "antibody" refers to immunoglobulin-type molecules consisting of four polypeptide chains, two heavy (H) chains, and two light (L) chains linked together by internal disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region consists of a single domain, CL. The VH and VL regions can be further divided into regions of hypervariability, referred to as complementarity determining regions (CDRs), interspersed with regions that are more conserved, referred to as framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, N-terminal to C-terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In one of the embodiments of the invention, the composition contains an antibody with sequences of CDR1, CDR2 and CDR3, similar to those described in US patent No. 6090382; 6258562 and 8216583.

The antibody, or antigen-binding portion thereof, may be part of a larger immunoadhesion molecule formed by covalent or non-covalent associations of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of the streptavidin core region to produce a tetramer scFv molecule (Kipriyanov, S. M., et al. (1995) Human Antibodies and Hybridomas 6:93-101) and the use of a cysteine residue, a marker peptide, and a C-terminal polyhistidine labels to produce bivalent and biotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol. Immunol. 31:1047-1058). Whole antibodies can be prepared from whole antibodies by conventional methods such as papain or pepsin digestion, such as Fab and F(ab') ₂ fragments of whole antibodies, respectively. In addition, antibodies, antibody moieties, and immunoadhesion molecules can be produced by standard recombinant DNA techniques as described herein.

As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds to TNFα is substantially free of antibodies that specifically bind to antigens, other than TNFα). However, an isolated antibody that specifically binds to TNFα may be cross-reactive with other antigens, such as TNFα molecules from other species. In addition, the isolated antibody may be substantially free of other cellular materials and/or chemicals.

The term "antibody to TNFα" refers to a human anti-TNFα antibody described in the present description, as well as described in US patent No. 6090382; 6258562; 6509015; 7,223,394 and 6,509,015. The term anti-TNFα antibody as used herein is an anti-TNFα antibody or fragment thereof, including infliximab (Remicade®, Johnson and Johnson; described in US Pat. No. 5,656,272); CDP571 (humanized anti-TNF-alpha IgG4 monoclonal antibody); CDP870 (a fragment of a humanized monoclonal antibody against TNF-alpha); anti-TNF dAb (Peptech); CNTO148 (golimumab; Centocor, see WO 02/12502 and U.S. 7521206 and U.S. 7250165); and adalimumab (HUMIRA® Abbott Laboratories, human anti-TNF mAb described in US 6,090,382 as D2E7). Additional anti-TNF antibodies that can be used in the invention are described in US Pat. Nos. 6,593,458; 6498237; 6451983 and 6448380. In another embodiment, the TNFα inhibitor is a TNF fusion protein, eg etanercept (Enbrel®, Amgen; described in WO 91/03553 and WO 09/406476). In another embodiment, the TNFα inhibitor is recombinant TNF binding protein (r-TBP-I) (Serono).

The term "long-term storage" or "long-term stability" should be understood as meaning that the pharmaceutical composition can be stored for three months or more, for six months or more, and preferably for one year or more, most preferably with a minimum period storage in stable condition for at least two years. In general, the terms "long-term storage" and "long-term stability" further include steady-state storage times that are at least comparable to or better than the steady-state shelf life typically required for currently available commercial loss-free adalimumab formulations. in stability, which may render the formulation unsuitable for its intended pharmaceutical use. Extended storage should also be understood to mean that the pharmaceutical composition is stored either as a liquid at
2-8°C, or frozen, for example, at -18°C or less. It is also contemplated that the composition may be frozen and thawed at least once.

The term "stable state" in relation to long-term storage should be understood to mean that adalimumab contained in pharmaceutical compositions loses no more than 20%, or more preferably 15%, or even more preferably 10%, and most preferably 5% of its activity relative to activity of the composition at the beginning of storage.

The term "excipient" or "adjuvant" is used herein to describe any ingredient other than those previously described in this invention.

The term "sugar" refers to monosaccharides, disaccharides and polysaccharides or mixtures thereof. Non-limiting examples of sugars include sucrose, trehalose, glucose, dextrose, and others.

The term "polyol" as used herein refers to an excipient with multiple hydroxyl groups and includes sugar alcohols and sugar acids. Non-limiting examples of polyols include mannitol (mannitol), sorbitol (sorbitol), and others.

The term "buffer", "buffer composition", "buffer agent" as used herein, refers to an added composition that allows the liquid antibody preparation to exhibit resistance to changes in pH, usually through the action of its acid-base conjugate components. When reference is made to the concentration of a buffer, it is understood that said concentration is the sum of the molar concentrations of the free acid and free base forms of that buffer. Examples of buffers that are known to those skilled in the art and can be found in the literature include, but are not limited to, histidine, citrate, succinate, acetate, phosphate, phosphate-salt, citrate-phosphate buffers, as well as buffers based on tromethamine and the like, or their suitable mixtures.

The terms "tonicity agent" or "tonicity control agent" and "osmolytic" or "osmotic agent" as used herein refer to an excipient that can apply the osmotic pressure of a liquid antibody formulation. In certain embodiments, the tonicity agent may adjust the osmotic pressure of the liquid antibody preparation to isotonic such that the antibody preparation is physiologically compatible with the tissue cells of the subject's body. In yet another embodiment, a "tonicity agent" can help improve the stability of the antibodies described herein. An "isotonic" drug is a drug that has an osmotic pressure equivalent to that of human blood. Isotonic preparations typically have an osmotic pressure of about 250 to 350 mOsm. The term "hypotonic" describes a preparation with an osmotic pressure less than that of human blood. Accordingly, the term "hypertonic" is used to describe a drug with an osmotic pressure greater than that of human blood. Isotonicity can be measured using, for example, a steam or cryoscopic osmometer. The tonicity agent may be in enantiomeric (eg L- or D-enantiomer) or racemic form; in the form of isomers such as alpha or beta, including alpha, alpha; or beta, beta; or alpha, beta; or beta, alpha; in the form of free acid or free base; in the form of salt; in hydrated form (eg monohydrate) or in anhydrous form.

The term "surfactant" (also known as a surfactant or detergent, or surfactant), as used herein, refers to an excipient that can change the surface tension of a liquid antibody preparation. In certain embodiments, the surfactant reduces the surface tension of the liquid antibody preparation. In other embodiments, a "surfactant" may help improve the colloidal stability or solubility of any antibody in a given formulation. The surfactant may reduce aggregation of the formulated antibody preparation and/or minimize the formation of particles in the preparation and/or reduce adsorption. The surfactant can also improve the stability of the antibody during, including after freezing/thawing and shaking. Surfactants may be ionic or non-ionic. Illustrative non-ionic surfactants that can be included in the compositions of the present invention include, for example, alkyl poly(ethylene oxide), alkyl polyglucosides (e.g., octyl glucoside and decyl maltoside), fatty alcohols such as cetyl alcohol and oleyl alcohol, cocamide-MEA, cocamide -DEA and cocamide-TEA. Specific non-ionic surfactants that may be included in the compositions of the present invention include, for example, polysorbates such as polysorbate 20 (Tween 20), polysorbate 28, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80 (Tween 80), polysorbate 81 and polysorbate 85; poloxamers such as poloxamer 188 (Kolliphor P188), poloxamer 407; polyethylene polypropylene glycol or polyethylene glycol (PEG), copolymers of ethylene and propylene glycol (eg Pluronics PF68, etc.).

The term "lyophilized" as used herein refers to a formulation that has been subjected to a process known in the art as freeze drying, which involves freezing the formulation and then removing ice from the frozen contents.

The term "amino acid" as used herein means an amino acid (a free amino acid, i.e., not an amino acid in a peptide or protein sequence). The amino acids used in the present invention include, but are not limited to, for example, arginine, glycine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tryptophan, serine, cysteine, methionine, and proline.

"Pharmaceutical composition" means a composition comprising an antibody according to the invention and at least one of the components selected from the group consisting of pharmaceutically acceptable and pharmacologically compatible excipients, solvents, diluents, carriers, auxiliary, dispensing, delivery vehicles, such as preservatives, stabilizers, emulsifiers, suspending agents, thickeners, delayed delivery regulators, the choice and ratio of which depends on the nature and method of administration and dosage. Examples of suspending agents are ethoxylated isostearyl alcohol, polyoxyethylene, sorbitol and sorbitol ether, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, as well as mixtures of these substances. Protection against the action of microorganisms can be provided by a variety of antibacterial and antifungal agents, such as parabens, chlorobutanol, sorbic acid, and the like. The composition may also include isotonic agents such as sugars, sodium chloride and the like. Prolonged action of the composition can be provided by agents that slow down the absorption of the active principle, for example, aluminum monostearate and gelatin. Examples of suitable carriers, solvents, diluents and delivery vehicles are water, ethanol, polyalcohols and mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). Examples of fillers are lactose, milk sugar, sodium citrate, calcium carbonate, calcium phosphate and the like. A pharmaceutical composition for oral, sublingual, transdermal, intramuscular, intravenous, subcutaneous, topical or rectal administration of the active principle, alone or in combination with another active principle, can be administered to animals and humans in standard form of administration as a mixture with conventional pharmaceutical carriers. Suitable standard administration forms include parenteral forms, implants, and transdermal systems.

"Drug (preparation)" - a substance (or a mixture of substances in the form of a pharmaceutical composition) in the form of tablets, capsules, powders, lyophilisates, injections, infusions, ointments and other finished forms, intended to restore, correct or change physiological functions in humans and animals, as well as for the treatment and prevention of diseases, diagnostics, anesthesia, contraception, cosmetology and other things.

"Treat", "treatment" and "therapy" refer to a method of alleviating or eliminating a biological disorder and/or at least one of its accompanying symptoms. Used herein to "alleviate" a disease, disease, or condition means to reduce the severity and/or frequency of occurrence of symptoms of the disease, disorder, or condition. In addition, references herein to "treatment" include references to curative, palliative, and prophylactic therapy.

In one aspect, the subject of treatment or patient is a mammal, preferably a human subject. The above subject may be male or female of any age.

The term "disorder" means any condition that can be improved as a result of the treatment of the present invention. The definition of this term includes chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to the occurrence of this disorder. Non-limiting examples of diseases to be treated include benign and malignant tumors; leukemias and lymphoid malignancies, in particular cancer of the breast, ovary, stomach, endometrium, salivary gland, lung, kidney, colon, thyroid, pancreas, prostate or bladder; neuronal, glial, astrocytic, hypothalamus and other glandular, macrophage, epithelial, stromal and blastocele disorders; inflammatory, angiogenic and immunological disorders. The preferred disorder to be treated according to the invention are autoimmune diseases.

The terms "immune response", "autoimmune reaction", "autoimmune inflammation" refer to, for example, the action of lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by said or liver cells (including antibodies, cytokines, and complement, resulting from the selective damage, destruction, or elimination from the human body of invasive pathogens, pathogen-infected cells or tissues, cancer cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues).

The term "autoimmune disease" in the context of the present description means a non-malignant disease or disorder that occurs and is directed against an individual's own (auto) antigens and/or tissues.

This definition includes, but is not limited to, rheumatoid arthritis, arthrosis, juvenile chronic arthritis, septic arthritis, Lyme arthrosis, psoriatic arthritis, reactive arthritis, spondyloarthropathy, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, inflammatory bowel disease, diabetes mellitus, thyroiditis, asthma, allergic diseases, psoriasis, atopic dermatitis, scleroderma, graft-versus-host disease, graft rejection, transplant-related acute or chronic immune diseases, sarcoidosis, Kawasaki disease, Graves' disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Henoch-Schönlein purpura, microscopic renal vasculitis, chronic active hepatitis, uvenita, septic shock, toxic shock syndrome, septic syndrome, cachexia, acquired immunodeficiency syndrome, acute transverse myelitis, Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke, primary biliary c irrosis, hemolytic anemia, adult (acute) respiratory distress syndrome, alopecia, alopecia areata, seronegative arthropathy, arthropathy, Reiter's disease, psoriatic arthropathy, ulcerative colitis-associated arthropathy, atopic allergies, autoimmune bullous diseases, pemphigus vulgaris, pemphigus foliaceus, disease pemphigoid, linear IgA, autoimmune hemolytic anemia, Coombs positive hemolytic anemia, pernicious anemia, juvenile malignant anemia, arthritis, primary sclerosing hepatitis A, cryptogenic autoimmune hepatitis, fibrosing lung diseases, cryptogenic fibrous alveolitis, post-inflammatory interstitial lung diseases, interstitial chronic pneumonia, postinfectious interstitial lung disease, gouty arthritis, autoimmune hepatitis, type I autoimmune hepatitis (classic autoimmune hepatitis or lipoid), type II autoimmune hepatitis, osteoarthritis inflammation, primary sclerosing cholangitis, psoriasis type I, psoriasis type II, idiopathic leukopenia, autoimmune neutropenia, renal NOS disease [renal NOS-disease], glomerulonephritis, microscopic renal vasculitis, discoid lupus erythematosis, idiopathic or male NOS fertility, [autoimmunity to sperm ], all subtypes of multiple sclerosis, sympathetic ophthalmia, secondary pulmonary hypertension in connective tissue diseases, Goodpasture's syndrome, pulmonary manifestation of nodular arthritis, acute rheumatic fever, rheumatoid spondylitis, ankylosing spondylitis, Still's disease, systemic sclerosis, Shengren's syndrome, Takayasu's syndrome, autoimmune thrombocytopenia , idiopathic thrombocytopenia, autoimmune thyroiditis, hyperthyroidism, Hoshimoto's disease, autoimmune atrophic hypothyroidism, primary mexidema, phacogenic uveitis, primary vasculitis, vitiligo, acute liver disease, chronic liver disease, allergies, asthma, mental illness disorders (including depression and schizophrenia), Th2-type and Th1-type mediated diseases, conjunctivitis, allergic contact dermatitis, allergic rhinitis, alpha-1 antitrypsin deficiency, amyotrophic lateral sclerosis, anemia, cystic fibrosis, cytokine therapy-associated diseases, demyelinating diseases, dermatitis, iridocyclitis/uveitis/optic neuritis, ischemic reperfusion injury, ischemic stroke, juvenile rheumatoid arthritis, autoimmune enteropathy, autoimmune hearing loss, autoimmune lymphoproliferative syndrome, autoimmune myocarditis, autoimmune premature ovarian failure, and blepharitis. The antibody may also treat any combination of the disorders listed above.

As used herein, the term "disorder in which TNFα activity is detrimental" is meant to include diseases and other disorders in which the presence of TNFα in a subject suffering from the disorder has been shown or suspected to either contribute to the pathophysiology of the disorder or is a factor that aggravates the disorder. Accordingly, a disorder in which TNFα activity is detrimental is a disorder in which inhibition of TNFα activity is expected to reduce the symptoms and/or development of the disorder. Such disorders can be detected, for example, by an increase in the concentration of TNFα in the body fluid of the subject suffering from the disorder (for example, an increase in the concentration of TNFα in the serum, plasma, synovial fluid, etc. of the subject), which can be determined, for example, using anti-TNFα antibodies as described above. There are many examples of disorders in which TNFα activity is detrimental. The use of antibodies and antibody fragments corresponding to a disorder in the treatment of specific disorders is further discussed below:

A. Sepsis

Tumor necrosis factor plays an established role in the pathophysiology of sepsis with biological effects including hypotension, myocardial weakening, vascular leak syndrome, organ necrosis, stimulation of the release of toxic secondary mediators, and activation of the blood coagulation cascade (see, e.g., Moeller A., et al.( 1990), Cytokine 2:162-169; US Patent No. 5231024 Moeller et at.; European Patent No. 260610 B1 Moeller A.; Tracey KJ and Cerami A. (1994) Annu. Rev. Med. 45:491-503; Russel D. and Thompson RC (1993) Curr Opin Biotech 4:714-721). Accordingly, human antibodies and antibody fragments of the invention can be used to treat sepsis in any of its clinical manifestations, including septic shock, endotoxic shock, Gram-negative bacterial sepsis, and toxic shock syndrome.

Moreover, for the treatment of sepsis, anti-TNFα antibodies and antibody fragments of the invention can be administered together with one or more therapeutic agents that can further alleviate sepsis, such as an interleukin-1 inhibitor (described in PCT Publications No WO 92/16221 and WO 92/17583), the cytokine interleukin-6 (see, for example, PCT Publication No WO 93/11793) or a platelet activating factor antagonist (see, for example, European Patent Application No EP 374510). Other combination therapies for the treatment of sepsis are discussed below in subsection III.

In addition, in a preferred embodiment, the anti-TNFα antibody and antibody fragments of the invention are administered to a human from a subgroup of patients with sepsis having a serum or plasma concentration of IL-6 greater than 500 pg/mL and more preferably 1000 pg/mL during treatment ( see PCT Publication No WO 95/20978 Daum L et al.).

B. Autoimmune diseases

Tumor necrosis factor has been shown to play a role in the pathophysiology of a number of autoimmune diseases. For example, TNFα has been implicated in activating tissue inflammation and causing joint destruction in rheumatoid arthritis (see, e.g., Moeller A. et al. (1990), Cytokine 2:162-169; US Pat. 260610 B1 Moeller A.; Tracey KJ and Cerami A., supra; end WP and Dayer JM (1995) ath. Rheum. 38:151-160; Fava RA et al. (1993) Clin. Exp. Immunol. 94:261 -266). TNFα has also been implicated in promoting cell islet death and in the formation of insulin resistance in diabetes (see, for example, Tracey and Cerami, supra; PCT Publication No WO 94/08609). TNFα has also been implicated in oligodendrocyte cytotoxicity and induction of inflammatory plaques in multiple sclerosis (see, for example, Tracey and Cerami, supra). Chimeric and humanized mouse anti-TNFα antibodies have been clinically tested for the treatment of rheumatoid arthritis (see, e.g., Elliot MJ et al. (1994); Lancet 344:1125-1127; Elliot MJ et al. (1994) Lancet 344:1105-1110 Rankin EC et al (1995) Br J Rheumatol 34:334-342).

Human antibodies and antibody fragments of the invention can be used to treat autoimmune diseases, in particular those associated with inflammation, including rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis and gouty arthritis, allergies, multiple sclerosis, autoimmune diabetes, autoimmune uveitis, and renal syndrome. Typically, the antibody or antibody fragment is applied systemically, although for some disorders topical application of the antibody or antibody fragment to the site of inflammation (e.g., topical injection in rheumatoid arthritis or topical application in diabetic ulcers, alone or in combination with a cyclohexanylidene derivative) may be successful. , as described in PCT Publication No WO 93/19751). The antibody and antibody fragment of the invention may also be used with one or more additional therapeutic agents useful in the treatment of autoimmune diseases, as described further in subsection III.

C. Infectious diseases

Tumor necrosis factor has been implicated in the biological effects seen in a number of infectious diseases. For example, TNFα has been implicated in the development of brain inflammation and capillary thrombosis and infarction in malaria. TNFα was also involved in the development of brain inflammation, the induction of the destruction of the blood-brain barrier, the development of septic shock syndrome, and the activation of venous infarction in meningitis. TNFα has also been implicated in the induction of cachexia, stimulation of viral proliferation and the formation of lesions in the central nervous system in acquired immunodeficiency syndrome (AIDS). Accordingly, the antibodies and antibody fragments of the invention can be used to treat infectious diseases, including bacterial meningitis (see, for example, European Patent Application No. EP 585705), cerebral malaria, AIDS, and AIDS-associated complex (αC) (see, eg, European Patent Application No EP 230574), as well as secondary infection with cytomegalovirus during transplantation (see, for example, Fietze E., et al. (1994) Transplantation 58:675-680). Antibodies and antibody fragments of the invention can be used to alleviate symptoms associated with infectious diseases, including fever and myalgia due to infection (such as influenza) and secondary cachexia in infection (eg, secondary to AIDS or αC).

D. Transplantation

Tumor necrosis factor has been considered as a key mediator in graft rejection and graft-versus-host disease (GVHD) and in producing an adverse reaction that was noted when a rat OCTZ antibody directed against the T-cell receptor-CDR complex was used to inhibit renal transplant rejection (see e.g., Eason JD et al (1995) Transplantation 59:300-305; Suthanthiran M. and Strom T. B. (1994) New Engl J. Med 331:365-375). Accordingly, the antibodies and antibody fragments of the invention can be used to inhibit transplant rejection, including allograft and xenograft rejection, and to inhibit GVHD. Although the antibody or antibody fragment can be used alone, it is more preferred to use it in combination with one or more agents that inhibit the immune response against the allograft or inhibit GVHD. For example, in one embodiment, an antibody or antibody fragment of the invention is used in combination with one or more antibodies directed to other targets involved in the regulation of immune responses, such as cell surface molecules CD25 (interleukin-2 receptor-β), CD11a (LFA-1), CD54 (ICAM-1), CD4, CD45, CD28/CTLA4, CD80 (B7-1) and/or CD86 (B7-2). In yet another embodiment, an antibody or antibody fragment of the invention is used in combination with one or more major immunosuppressive agents such as cyclosporine A or FK506.

E. Malignant neoplasms

Tumor necrosis factor was involved in the induction of cachexia, stimulation of tumor growth, enhancement of metastatic potential and the appearance of cytotoxicity in malignant neoplasms. Accordingly, the antibodies or antibody fragments of the invention are useful in the treatment of malignancies to inhibit tumor growth or metastases and/or to alleviate secondary cachexia in malignancies. The antibody or antibody fragment can be administered systemically or topically to the tumor site.

F. Pulmonary disorders

Tumor necrosis factor has been implicated in the pathophysiology of adult respiratory distress syndrome (αDS), including stimulation of endothelial leukocyte activation, targeting pneumocyte cytotoxicity, and induction of vascular leak syndrome. Accordingly, antibodies or antibody fragments of the invention can be used in the treatment of various pulmonary diseases, including adult respiratory distress syndrome (see, for example, PCT Publication No WO 91/04054), pulmonary shock, chronic pulmonary inflammatory disease, pulmonary sarcoidosis, pulmonary fibrosis and silicosis. An antibody or antibody fragment of the invention may be administered with one or more additional therapeutic agents useful in the treatment of pulmonary disorders, as described below in section III.

G. Intestinal disorders

Tumor necrosis factor has been implicated in the pathophysiology of inflammatory bowel disorders (see, e.g., Tracy KJ, et al. (1986) Science 234:470-474; Sun XM., et al. (1988) J. Clin. Invest. 81:1328 -1331 MacDonald TT, et al (1990) Clin Exp Immunol 81: 301-305). Chimeric mouse anti-TNFα antibodies have been clinically tested for the treatment of Crohn's disease (van Dullemen H.M. et al. (1995) Gastroenterology 109:129-135). A human antibody or antibody fragment of the invention may also be used to treat intestinal disorders such as idiopathic inflammatory bowel disease, which includes two syndromes, Crohn's disease and ulcerative colitis. The antibody or antibody fragment of the invention may also be administered with one or more additional therapeutic agents used in the treatment of intestinal disorders, as described below in subsection III.

H. Cardiac disorders

Antibodies or antibody fragments of the invention may also be used to treat various cardiac disorders, including cardiac ischemia (see, for example, European Patent Application No. EP 453898) and heart failure (weakness of the heart muscle) (see, for example, PCT Publication No WO 94/20139).

I. Others

The antibodies or antibody fragments of the invention may also be used to treat various disorders in which TNFα activity is detrimental. Examples of other diseases and disorders in which TNFα activity is involved in the pathophysiology and which can thus be treated using an antibody or antibody fragment of the invention include inflammatory bone disorders and diseases associated with bone resorption (see, for example, Bertolini DR, et al (1986) Nature 319:516-518 Konig A., et al (1988) J Bone Miner Res 3:621-627 Lerner UH and Ohiin A (1993) J Bone Miner Res 8:147-155 and Shanka G. and Stern PH (1993) Bone 14:871-876), hepatitis, including alcoholic hepatitis (see, for example, McClain CJ and Cohen DA (1989) Hepatology 9:349-351; Felver ME et al (1990) Alcohol Clin Exp Res 14:255-259 and Hansen J et al (1994) Hepatology 20:461-474), viral hepatitis (Sheron N et al (1991) J. Hepatol 12:241-245 and Hussain MJ et al (1994) J. Clin Pathol 47:1112-1115) and fulminant hepatitis; coagulation disorders (see, for example, van der Poll T. et al. (1990) N. Engl. J. Med. 322:1622-1627; van der Poll T. et al. (1991) Prog. Clin. Biol. Res. 367:55-60; burns (see, for example, Giroir BP et al. (1994) Am. J. Physiol. 267:H118-124; Liu XS et al. (1994) Burns 20:40-44) , reperfusion injury (see e.g. Scales WE et al. (1994) Am. J. Physiol. 26:1122-1127; Serrick C. et al. (1994) Transplantation 58:1158-1162; Yao YM et al. (1995) Resuscitation 29:157-168), keloid formation (see e.g. McCauley R. L et al. (1992) J. Clin. Immunol. 12:300-308), scar tissue formation, hyperthermia, periodontal disease , obesity and radiation toxicity.

A "therapeutically effective amount" is the amount of a therapeutic agent administered during treatment that will relieve to some extent one or more of the symptoms of the disease being treated.

The term "chronic" use refers to the continuous (long-term) use of the agent(s), as opposed to the acute (short-term) route of administration, so as to maintain the initial therapeutic effect (activity) over a long period of time.

"Intermittent" use means a treatment that is not carried out sequentially without interruption, but which is rather intermittent in nature.

In the present description and in the following claims, unless the context otherwise requires, the words "have", "include" and "comprise" or variations thereof, such as "has", "having", "includes", "including", " contains" or "comprising" should be understood to mean the inclusion of the specified integer or group of integers, but not the exclusion of any other integer or group of integers.

Detailed description of the invention

water composition

The present invention relates to new, improved aqueous compositions, which may optionally be lyophilized, comprising a suitable amount of a recombinant anti-TNFα monoclonal antibody, in a suitable buffer(s), one or more suitable stabilizers and other excipients, which are selected from suitable surfactants. active substances and means to maintain isotonicity. Said composition prevents the formation of protein (antibody) aggregates and maintains the efficacy and stability of the therapeutic compound for the desired period of time.

Adalimumab and infliximab are examples of recombinant anti-TNFα monoclonal antibodies. Infliximab is an example of a chimeric antibody. Adalimumab is an example of a human antibody. In a preferred embodiment, the antibodies used in the composition are human antibodies. In a more preferred embodiment, the antibodies used in the composition are human anti-human TNFα antibodies. In yet another embodiment, the composition includes D2E7 and a combination of D2E7 with other antibodies. The D2E7 antibody, known by its generic name as adalimumab, with its high binding affinity for TNFα, low dissociation kinetics and high neutralizing ability. Adalimumab is available on the pharmaceutical market under the brand name Humira®. As used herein, the names adalimumab and D2E7 represent the same human monoclonal antibody. In a preferred embodiment, the monoclonal antibody is adalimumab or an antigen-binding portion thereof.

In some embodiments, the anti-TNFα recombinant monoclonal antibodies, or antigen-binding portions thereof, are typically present in a therapeutic amount up to 200 mg/mL. In a preferred embodiment, the therapeutic amount is from about 1 mg/ml to about 150 mg/ml. In a more preferred embodiment, the therapeutic amount is from about 1 mg/mL to about 100 mg/mL. In a more preferred embodiment, the therapeutic amount is from about 50 mg/ml to about 100 mg/ml. In an even more preferred embodiment, the therapeutic amount is about 100 mg/ml.

The aqueous composition in question comprises a suitable buffer solution in combination with other pharmaceutically acceptable excipients which stabilize the pharmaceutical formulation. Suitable buffer solutions that may be used are selected from a group known to those skilled in the art and can be found in the literature. In one embodiment, suitable buffer solutions include, but are not limited to, an acetate ion buffer agent (or buffer system). In a preferred embodiment, a suitable buffer includes an acetate buffer. In an even more preferred embodiment, the acetate buffer is selected from sodium acetate trihydrate in combination with acetic acid.

Buffer solutions are generally used at concentrations from about 1 mM to about 100 mM. In a preferred embodiment, the buffer concentration is from about 1 mM to about 50 mM. In a more preferred embodiment, the buffer concentration is from about 1 mM to about 20 mM. In an even more preferred embodiment, the buffer concentration is about 5 mM.

In some embodiments, sodium acetate trihydrate is present in an amount up to 1.2 mg/mL. In a preferred embodiment, the amount of sodium acetate trihydrate is from about 0.4 mg/mL to about 1.2 mg/mL. In a more preferred embodiment, the amount of sodium acetate trihydrate is from about 0.4 mg/mL to about 0.5 mg/mL. In a more preferred embodiment, the amount of sodium acetate trihydrate is 0.436 mg/ml.

In one embodiment, the liquid composition maintains a pH value in the range of 4.0 to about 7.0, depending on the monoclonal antibody used. In a preferred embodiment, the buffer used maintains the pH of the composition in the range of about 4.5 to 6.0. In a more preferred embodiment, the pH is maintained at about 5.0 to 6.0. In a more preferred embodiment, the pH is maintained at about 5.5.

Said aqueous composition includes suitable surfactants which are pharmaceutically acceptable excipients used to protect protein compositions from various stresses such as agitation, shear, high temperature, freezing, etc. Suitable surfactants include, but are not limited to, polysorbates or poloxamers, or a combination of polysorbate and poloxamer. In a preferred embodiment, a suitable surfactant is polysorbate. In a preferred embodiment, a suitable surfactant is poloxamer. In a preferred embodiment, a combination of polysorbate and poloxamer is a suitable surfactant. In a more preferred embodiment, the polysorbate is polysorbate 20 or polysorbate 80 (including those sold under the brand name Tween® 20 or Tween® 80). In a more preferred embodiment, the poloxamer is poloxamer 188 (also sold under Kolliphor P188 ^® trademark). In another preferred embodiment, a suitable surfactant is a combination of polysorbate 20/polysorbate 80 and poloxamer 188.

In some embodiments, polysorbate 20 is present in an amount up to 10 mg/mL. In a preferred embodiment, the amount of polysorbate 20 is from about 0.05 mg/mL to about 10 mg/mL. In a more preferred embodiment, the amount of polysorbate 20 is from about 0.1 mg/mL to about 1 mg/mL. In a more preferred embodiment, the amount of polysorbate 20 is about 0.5 mg/ml.

In some embodiments, polysorbate 80 is present in an amount up to 10 mg/mL. In a preferred embodiment, the amount of polysorbate 80 is from about 0.05 mg/mL to about 10 mg/mL. In a more preferred embodiment, the amount of polysorbate 80 is from about 0.1 mg/mL to about 1 mg/mL. In a more preferred embodiment, the amount of polysorbate 80 is about 0.5 mg/ml.

In some embodiments, poloxamer 188 is present in an amount up to 10 mg/mL. In a preferred embodiment, the amount of poloxamer 188 is from about 0.05 mg/mL to about 10 mg/mL. In a more preferred embodiment, the amount of poloxamer 188 is from about 0.1 mg/mL to about 1 mg/mL. In a more preferred embodiment, the amount of poloxamer 188 is about 1.0 mg/mL.

In some embodiments, the combination of polysorbate 20/polysorbate 80 and poloxamer 188 is present in an amount up to 10 mg/mL of polysorbate 80 and up to 10 mg/mL of poloxamer 188. In a preferred embodiment, the amount of poloxamer 188 is from about 0.05 mg/mL to about 10 mg/ml and the amount of polysorbate 20/polysorbate 80 is from about 0.05 mg/ml to about 10 mg/ml. In a more preferred embodiment, the amount of poloxamer 188 is from about 0.1 mg/ml to about 1 mg/ml and the amount of polysorbate 20/polysorbate 80 is from about 0.1 mg/ml to about 1 mg/ml. In a more preferred embodiment, the amount of polysorbate 20/polysorbate 80 is about 0.5 mg/mL and the amount of poloxamer 188 is about
1.0 mg/ml.

The aqueous composition includes one or more suitable stabilizers which are pharmaceutically acceptable excipients and which protect the pharmaceutically active ingredient from chemical and/or physical degradation during manufacture, storage and use. In one embodiment, stabilizers include, but are not limited to, amino acids, oligosaccharides, or suitable derivatives or mixtures thereof. In another preferred embodiment, a suitable stabilizer is trehalose. In another preferred embodiment, proline is a suitable stabilizer. In another preferred embodiment, a suitable stabilizer is a combination of trehalose and proline. In another preferred embodiment, a combination of trehalose and arginine is a suitable stabilizer.

In another embodiment, oligosaccharides that can also be used as stabilizers include trehalose.

In some embodiments, the trehalose is present in an amount up to 150 mg/mL. In a preferred embodiment, the amount of trehalose is from about 25 mg/ml to about 150 mg/ml. In a more preferred embodiment, the amount of trehalose is from about 50 mg/ml to about 100 mg/ml. In a more preferred embodiment, the amount of trehalose dihydrate is about 80 mg/ml.

In another embodiment, a combination of trehalose and arginine is chosen as the stabilizer. In some embodiments, trehalose is present in an amount up to 150 mg/ml and arginine in an amount up to 1.0 mg/ml. In a preferred embodiment, the amount of trehalose is from about 25 mg/ml to about 150 mg/ml and the amount of arginine is from 0.05 to 1.0 mg/ml. In a more preferred embodiment, the amount of trehalose is from about 50 mg/ml to about 100 mg/ml and the amount of arginine is from 0.05 to 0.2 mg/ml. In a more preferred embodiment, the amount of trehalose dihydrate is about 80 mg/ml and the amount of arginine hydrochloride is about 0.1 mg/ml.

In another such embodiment, an amino acid that can be used as stabilizers is proline.

In some embodiments, proline is present in an amount up to 40 mg/mL. In a preferred embodiment, the amount of proline is from about 5 mg/ml to about 40 mg/ml. In a more preferred embodiment, the amount of proline is from about 20 mg/ml to about 35 mg/ml. In a more preferred embodiment, the amount of proline is about 27 mg/ml.

In some embodiments, the combination of trehalose and proline is present in an amount up to 150 mg/ml trehalose and up to 40 mg/ml proline. In a preferred embodiment, the amount of trehalose is from about 25 mg/ml to about 150 mg/ml and the amount of proline is from about 5 mg/ml to about 40 mg/ml. In a more preferred embodiment, the amount of trehalose is from about 20 mg/ml to about 50 mg/ml and the amount of proline is from about 10 mg/ml to about 20 mg/ml. In a more preferred embodiment, the amount of trehalose dihydrate is about 40 mg/ml and the amount of proline is about 13.5 mg/ml.

In some embodiments, the aqueous composition contains 50 to 200 mg/mL of an anti-TNFα recombinant monoclonal antibody, 0.4 to 1.2 mg/mL of sodium acetate trihydrate, 25 to 150 mg/mL of trehalose, and/or 5 to 40 mg/mL of proline or 25 to 150 mg/ml trehalose and 0.05 to 0.5 mg/ml arginine, ice acetic acid. to pH 5.0 - 6.0, from 0.1 mg/ml to 1 mg/ml polysorbate 20. In a preferred embodiment of the aqueous formulation, the anti-TNFα recombinant monoclonal antibody is adalimumab. In a more preferred embodiment, the composition contains 50 to 150 mg/ml adalimumab, 0.436 mg/ml sodium acetate trihydrate, 80 mg/ml trehalose dihydrate, acetic acid, ice. to pH 5.5, 0.5 mg/ml polysorbate 20. In a more preferred embodiment, the composition contains 50 to 150 mg/ml adalimumab, 0.436 mg/ml sodium acetate trihydrate, 80 mg/ml trehalose dihydrate, 0.1 mg/ml arginine hydrochloride, acetic acid ice. to pH 5.5, 0.5 mg/ml polysorbate 20. In a more preferred embodiment, the composition contains 50 to 150 mg/ml adalimumab, 0.436 mg/ml sodium acetate trihydrate, 27 mg/ml proline, acetic acid ice. to pH 5.5, 0.5 mg/ml polysorbate 20. In a more preferred embodiment, the composition contains 50 to 150 mg/ml adalimumab, 0.436 mg/ml sodium acetate trihydrate, 40 mg/ml trehalose dihydrate, 13.5 mg/ml proline, acetic acid ice. to pH 5.5, 0.5 mg/ml polysorbate 20.

In some embodiments, the aqueous composition contains 50 to 200 mg/mL of an anti-TNFα recombinant monoclonal antibody, 0.4 to 1.2 mg/mL of sodium acetate trihydrate, 25 to 150 mg/mL of trehalose, and/or 5 to 40 mg/mL of proline or 25 to 150 mg/ml trehalose and 0.05 to 0.5 mg/ml arginine, ice acetic acid. to pH 5.0 - 6.0, from 0.1 mg/ml to 1 mg/ml polysorbate 80. In a preferred embodiment of the aqueous composition, the recombinant anti-TNFα monoclonal antibody is adalimumab. In a more preferred embodiment, the composition contains 50 to 150 mg/ml adalimumab, 0.436 mg/ml sodium acetate trihydrate, 80 mg/ml trehalose dihydrate, acetic acid ice. to pH 5.5, 0.5 mg/ml polysorbate 80. In a more preferred embodiment, the composition contains 50 to 150 mg/ml adalimumab, 0.436 mg/ml sodium acetate trihydrate, 80 mg/ml trehalose dihydrate, 0.1 mg/ml arginine hydrochloride, acetic acid ice. to pH 5.5, 0.5 mg/ml polysorbate 80. In a more preferred embodiment, the composition contains 50 to 150 mg/ml adalimumab, 0.436 mg/ml sodium acetate trihydrate, 27 mg/ml proline, acetic acid ice. to pH 5.5, 0.5 mg/ml polysorbate 80. In a more preferred embodiment, the composition contains 50 to 150 mg/ml adalimumab, 0.436 mg/ml sodium acetate trihydrate, 40 mg/ml trehalose dihydrate, 13.5 mg/ml proline, acetic acid ice. to pH 5.5, 0.5 mg/ml polysorbate 80.

In some embodiments, the aqueous composition contains 50 to 200 mg/mL of an anti-TNFα recombinant monoclonal antibody, 0.4 to 1.2 mg/mL of sodium acetate trihydrate, 25 to 150 mg/mL of trehalose, and/or 5 to 40 mg/mL of proline or 25 to 150 mg/ml trehalose and 0.05 to 0.5 mg/ml arginine, ice acetic acid. to pH 5.0 - 6.0, from 0.1 mg/ml to 1 mg/ml poloxamer 188. In a preferred embodiment, the recombinant anti-TNFα monoclonal antibody is adalimumab. In a more preferred embodiment, the composition contains 50 to 150 mg/ml adalimumab, 0.436 mg/ml sodium acetate trihydrate, 80 mg/ml trehalose dihydrate, acetic acid, ice. to pH 5.5, 1 mg/ml poloxamer 188. In a more preferred embodiment, the composition contains 50 to 150 mg/ml adalimumab, 0.436 mg/ml sodium acetate trihydrate, 80 mg/ml trehalose dihydrate, 0.1 mg/ml arginine hydrochloride, acetic acid ice. to pH 5.5, 1 mg/ml poloxamer 188. In a more preferred embodiment, the composition contains 50 to 150 mg/ml adalimumab, 0.436 mg/ml sodium acetate trihydrate, 27 mg/ml proline, acetic acid ice. to pH 5.5, 1.0 mg/ml poloxamer 188. In a more preferred embodiment, the composition contains 50 to 150 mg/ml adalimumab, 0.436 mg/ml sodium acetate trihydrate, 40 mg/ml trehalose dihydrate, 13.5 mg/ml proline, acetic acid ice. to pH 5.5, 1.0 mg/ml poloxamer 188.

Said composition may furthermore include one or more other suitable excipients which are well known to those skilled in the art.

The above compositions are suitable for intravenous, subcutaneous or intramuscular administration.

In some embodiments, the liquid composition is storage stable in the sense that it does not undergo any further protein aggregation or modifications compared to time zero stability.

Methods of treatment and use of the aqueous composition

In another embodiment, the invention relates to a method of treating a mammal, comprising administering to the mammal a therapeutically effective amount of the pharmaceutical compositions of the invention, wherein the mammal may have a disease or disorder that can be effectively treated with adalimumab.

In a preferred embodiment, the mammal is a human.

Diseases or disorders that can be treated with the provided compositions, as non-limiting examples: active rheumatoid arthritis (moderate to severe), active psoriatic arthritis, active ankylosing spondylitis, chronic plaque psoriasis (moderate to severe), ulcerative colitis (moderate to severe) ), axial spondyloarthritis, active purulent hidradenitis, juvenile idiopathic arthritis, Crohn's disease (moderate or severe), uveitis, active enthesitis-associated arthritis. Additional diseases or disorders that may be treated with the compositions of the present invention include those described in US Pat. Nos. 6,090,382 and 8,216,583, the respective portions of which are incorporated herein by reference.

The provided pharmaceutical compositions can be administered to an individual in need of treatment by systemic injection, for example, by intravenous or subcutaneous or intramuscular injection; or by injection or application to an appropriate site, for example, by direct injection or direct application to the site, when the site is accessible by surgery; or through topical application.

In one embodiment, the invention relates to a method of treating and/or preventing rheumatoid arthritis, comprising administering to a mammal in need thereof a therapeutically effective amount of one of the provided adalimumab compositions.

The therapeutically effective amount of adalimumab in the compositions provided depends on the condition being treated, the severity of the condition, prior therapy, and the patient's medical history and response to the therapeutic agent. A suitable dose may be adjusted at the discretion of the attending physician such that it may be administered to the patient once or over multiple administrations.

In one embodiment, the effective amount of adalimumab per dose for an adult is from about 1 - 500 mg / m ^2, or about 1 - 200 mg / m ^2, or about 1 - 40 mg / ^m2 or about 5 - 25 mg / m ² .

Alternatively, you can enter a constant dose, the amount of which may be in the range of 2-500 mg/dose, 2-100 mg/dose, or about 10-80 mg/dose.

If the dose is to be administered more than once per week, an exemplary dosage range is the same as or less than the above dosage ranges, and is preferably administered two or more times per week with a dosage range of 25-100 mg/dose.

In another embodiment, an acceptable dose for administration by injection contains 80-100 mg/dose, or alternatively contains 80 mg per dose.

The dose can be administered weekly, biweekly, or separated by several weeks (eg, 2 to 8).

In one embodiment, adalimumab is administered at a dose of 40 mg via a single subcutaneous (SC) injection.

In some cases, improvement in the patient's condition can be achieved by administering a dose of the pharmaceutical composition up to about 100 mg once to three times a week for a period of at least three weeks. Treatment for longer periods may be necessary to achieve the desired degree of improvement. For incurable chronic conditions, the treatment regimen can be continued indefinitely. For pediatric patients (age 4 to 17 years), a suitable treatment regimen may include the administration of a dose of adalimumab from 0.4 mg/kg to 4 mg/kg once or several times a week.

In another embodiment, the pharmaceutical formulations of the invention may be prepared as a bulk formulation, and as such, the components of the pharmaceutical composition are present in amounts greater than may be required for administration and are suitably diluted prior to administration.

Pharmaceutical compositions can be administered as a single therapeutic agent or in combination with additional therapeutic agents as needed. Thus, in one embodiment, the provided methods of treatment and/or prophylaxis are used in combination with the administration of a therapeutically effective amount of another active agent. The other active agent may be administered before, during or after administration of the pharmaceutical compositions of the present invention. The other active agent may be administered as part of the provided composition or, alternatively, as a separate formulation.

Administration of the provided pharmaceutical compositions may be by various routes, including parenteral, oral, buccal, nasal, rectal, intraperitoneal, intradermal, transdermal, subcutaneous, intravenous, intraarterial, intracardiac, intraventricular, intracranial, intratracheal, intrathecal, intramuscular injection, intravitreal injection, and topical administration. introduction.

The pharmaceutical compositions of the present invention are particularly suitable for parenteral administration, i. subcutaneously, intramuscularly, intravenously, intraperitoneally, into the spinal cord, into the joints, intrasynovially and / or intrathecally. Parenteral administration may be by bolus injection or continuous infusion. Pharmaceutical compositions for injection may be in unit dosage form, such as, but not limited to, ampoules, vials, pre-filled syringes or multi-dose containers with an added preservative. In addition, a number of recent drug delivery approaches have been developed and the pharmaceutical compositions of the present invention are suitable for administration by these novel routes, e.g. Inject-ease®, Genject®, injector pens such as GenPen®, and needleless devices such as MediJector® and BioJector®. The pharmaceutical composition of the present invention can also be adapted to as yet undiscovered routes of administration. See also Langer, 1990, Science, 249:1527-1533.

The pharmaceutical compositions provided may also be formulated as a depot formulation. Such long acting formulations may be administered by implantation (eg subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, formulations can be modified using suitable polymeric or hydrophobic materials (eg, as an emulsion in a suitable oil), or ion exchange resins, or as sparingly soluble derivatives, eg, as a sparingly soluble salt.

Pharmaceutical compositions, if desired, may be provided in a vial, pack, or dispenser device, which may contain one or more unit dosage forms containing the active ingredient. In one embodiment, the dispenser device may comprise a syringe containing a single dose of a liquid formulation ready for injection. The syringe may be accompanied by instructions for administration.

In another embodiment, the present invention relates to a kit or container containing an aqueous pharmaceutical composition of the invention. The concentration of the polypeptide in the aqueous pharmaceutical composition may vary over a wide range, but generally within the range of about 1 to about 200 mg/ml of the aqueous composition. The kit may also be accompanied by instructions for use.

How to obtain

The method for obtaining the above compositions includes adding acetate buffer agents to the aqueous phase, followed by the addition, in any order, of the following components: an osmolytic agent and / or a stabilizer selected from the group: trehalose, proline or a combination thereof, a recombinant monoclonal antibody to TNFα, a surfactant a substance selected from the group: polysorbate 20, polysorbate 80, poloxamer 188 or combinations thereof.

Techniques

1. Determination of the protein concentration in the test samples.

The protein concentration was determined using the method of UV spectrophotometry at a wavelength of 280 nm in plates for UV spectrophotometry.

Each sample was diluted with the appropriate placebo solution to a concentration
~ 0.5 mg/ml. 150 μl of the diluted sample was placed in the well of the UV spectrophotometry plate. The optical density of the solutions placed in the plate was measured on a plate spectrophotometer at a wavelength of 280 nm. The corresponding placebo solution was used as a reference solution.

Protein concentration (C) in mg/ml was calculated by the formula:

A ₂₈₀ - the value of optical density at a wavelength of 280 nm;

ε is the extinction coefficient of the studied protein;

b is the total dilution factor of the sample;

l is the thickness of the layer in the well of the plate; for 150 µl l = 0.42 cm.

2. Changing the buffer solution and concentrating the samples.

Diafiltration and concentration of the samples were carried out in Stirred Cells (Millipore) under pressure.

3. "PEG aggregation".

Preparation of PEG 6000 solutions.

A PEG 6000 solution was prepared with a mass concentration of 20–25% in the studied composition of excipients. The final solutions were filtered through a Durapore 0.45 µm filter.

The calculated amount of the sample, solution of excipients and 20-25% PEG 6000 solution was transferred into 96-well plates for UV spectrophotometry so that the concentration of PEG6000 in each well was from 0 to 18%, and the protein concentration in each well was 1 mg / ml. All solutions obtained in the wells were mixed well by pipetting.

After that, the degree of turbidity of the solutions was assessed visually, and the optical density of the solutions was measured at a wavelength of 400 nm. The less stable the sample, the lower concentration of PEG 6000 it will form visible aggregates (opalescence). The degree of turbidity of the solutions was also assessed a day or several days after the preparation of the solutions.

4. Determination of protein homogeneity and aggregation point using dynamic light scattering (DLS) method .

The determination of the homogeneity of the studied samples was carried out on a Zetasizer Nano ZSP instrument in the Size measurement mode. For this, 0.05 ml of the solution was placed into a dust-free disposable plastic cuvette.

• Analytical model: Protein analysis.

• Keeping at temperature before starting measurement for 30 seconds.

• At each point, the average value of 13 measurements in 3 replicates.

The determination of the aggregation point of the studied proteins was carried out on a Zetasizer Nano ZSP instrument. To do this, the solution was placed in a dust-free quartz cuvette, which was gradually heated in the device while continuously measuring the scattered light intensity in the Temperature trend measurement mode.

• Analytical model: Protein analysis.

• Temperature trend mode, mod: Protein aggregation point. From 50 to 85°С at a heating step of 1.5°С.

• Keeping at temperature before starting measurement for 30 seconds.

• At each point, the average value of 15 measurements in 1 repetition.

The temperature trend was built using the instrument software, which automatically calculates the protein aggregation point (Aggregation point).

5. Determination of thermal stability by the "Thermostress 50°C" method.

The test samples were divided into 2 parts and placed in separate tubes: 1 tube for each composition was placed in a refrigerator for storage at +4°C, the rest were placed in a thermostat and incubated at 50°C for the specified time. After the end of heating, the tubes were removed from the thermostat, kept at room temperature for about 15 min, and transferred for analysis according to the specification.

6. Determination of colloidal stability by the "shake test" method.

The test samples were divided into 2 parts of 200 µl and placed in glass vials, 1 vial for each composition was placed in a refrigerator for storage at +4°C, the rest were placed in a thermoshaker and shaken at a speed of 800 rpm at a temperature of 2–8° C during the specified time. After the end of the stress, the tubes were removed from the thermoshaker and transferred for analysis according to the specification.

7. Determination of colloidal stability by cryoconcentration.

The test samples were divided into 2 parts and placed in polymer tubes: 1 tube for each composition was placed in a refrigerator for storage at +4°C, the rest were placed in a freezer and stored at a temperature of minus 16–20°C for the specified time. After the end of the stress, the tubes were removed from the freezer, kept at room temperature until the contents were completely thawed, the solutions were mixed with a Vortex, and submitted for analysis according to the specification.

8. Determination of the purity of samples using the method of size exclusion high performance liquid chromatography (E HPLC) .

Column Tosoh TSK-GelG3000SW _XL 7.8 mm ID × 30 cm, cat. No. 08541.

Column temperature: 25 ºС

Mobile phase flow rate: 0.7 ml/min.

Injection volume: 20 µl.

Sample concentration: 5 mg/ml.

Detector wavelength: 220 nm.

Elution time: 23 min.

Mobile phase: Disodium hydrophosphate b / in 7.1 mg / ml.

Sodium chloride 17.54 mg/ml.

The pH of the mobile phase was adjusted to 7.0 with phosphoric acid.

9. Determination of the acid-base profile of the samples on the instrument Caliper LabChip GX II .

9.1. Sample preparation.

Samples were diluted to a concentration of 1 mg/ml. To 200 μl of the resulting solution, 2 μl of a carboxypeptidase B (CpB) solution at a concentration of 5 mg/ml was added. Mixed and incubated for 2 hours at 37°C. Test samples were dialyzed against three changes of water in Amicon Ultra centrifuge tubes and concentrated to 2 mg/mL.

9.2. Preparation of working solutions.

Working solutions and a plate with analyzed samples were prepared in accordance with the manufacturer's method using the HT Protein Charge Variant Labeling Kit.

9.3. Chip preparation.

The preparation of the chip and tubes with buffer was carried out according to the manufacturer's method using buffer solutions from the Protein Charge Variant Buffer Kit.

Running an analysis is a standard operation. Method of Analysis Protein Charge Variant 68s.

10. Determination of the purity of samples on the instrument Caliper Labchip GX II in reducing and non-reducing conditions .

10.1. Preparation of analyzed samples.

For the preparation of denaturing and reducing solutions, 700 µl of HT Protein Express Sample Buffer was used with the addition of 24.5 µl of 1 M DTT for buffer under reducing conditions and 24.5 µl of 1 M IAM for buffer under non-reducing conditions. Samples were diluted to a concentration of 2 mg/ml. Two microtubes were prepared for each sample: 35 µl of denaturing buffer was added to one, and 35 µl of reducing buffer was added to the other. The samples were denatured at 100°C for 5 minutes. The tubes were vortexed, then 70 µl of water was added to each tube and mixed. 44 μl of each sample was transferred to a 96-well assay plate.

10.2. Preparation of working solutions and filling the chip.

Working solutions were prepared and the chip was prepared according to the standard procedure using the HT Protein Express Reagent Kit. Running an analysis is a standard operation. Method of analysis HT Protein Express 200.

11. Determination of the acid-base profile of samples using ion-exchange (IE) HPLC.

Speaker: DIONEX ProPack WCX-10 4 mm x 250 mm (Tosoh bioscience, Japan); Precolumn: Dionex ProPack WCX-10G Mobile phase: Eluent A: 0.01 M 2-(N-morpholino)ethanesulfonic acid (MESA), pH 6.0 Eluent B: 0.01 M MESA, 0.4 M sodium chloride, pH 5.5; Volume of injected sample: 40 µl; Flow rate: 0.5 ml/min; Column temperature: 25ºС; Autosampler temperature: 5ºС; Detector wavelength: 280 nm. Gradient: Phase A 95 - 0 - 95%.

12. Determination of purity by polyacrylamide gel electrophoresis (PAAG) under reducing and non-reducing conditions.

PAAG was prepared in glass plates in the presence of sodium dodecyl sulfate, consisting of a concentrating layer - 4% PAAG and a separating layer: under reducing conditions - 12.5% PAAG, under non-reducing conditions - 8% PAAG.

The electrophoresis chamber was assembled and installed in accordance with the operating instructions for the device for vertical electrophoresis. Samples were prepared by diluting samples with purified water to a final concentration of 1 mg/mL. A volume equivalent to 40 μg was taken and prepared samples of the test sample were mixed in a ratio of 3:1 (v/v) with a 4-fold sample application buffer containing 2-mercaptoethanol (reducing conditions) and not containing 2-mercaptoethanol (non-reducing conditions) , stirred. The resulting solutions were incubated at (99 ± 1)°С for 3 min (samples containing 2-mercaptoethanol) and at (99 ± 1)°С for 1 min (samples not containing 2-mercaptoethanol). The solutions were cooled to room temperature, mixed, and applied to the wells of PAAG under a layer of electrode buffer solution.

Electrophoresis was carried out in direct current mode using a water cooling system. The operating parameters of the power source were set: when the dye front passed through the concentrating gel, the voltage was 110 V. After the dye front entered the lower separating gel by 5–7 mm, the voltage was increased to 180 V. The power source was turned off when the dye front reached the lower boundary of the gel .

After the end of electrophoresis, the gels were separated from the slides and the proteins were fixed in a fixing solution for 16–18 hours at room temperature. The gels were then stained (in Acid Blue 83) and washed until the bands were clearly visualized. The gels were scanned. The purity and impurities in the test samples were evaluated using GelPro software.

13. Determination of specific activity.

The specific activity of the samples was assessed by the ability to specifically bind and neutralize TNFα, which in turn has a cytotoxic effect and causes the death of the culture WEHI-13 var (fibrosarcoma, Mus musculus), a culture variant that has an increased sensitivity to TNFα in the presence of Actinomycin D. Sample preparation was carried out using a TecanEvo 200 robotic station, RPMI1640, 2 mM Gln, 10% FBS, actinomycin D 2.5 µg/ml, gentamicin 5 µg/ml were used as CSE (quantification medium).

The antibody sample under study was diluted to 50 μg/ml with a dilution step of no more than 20 and placed in a robotic station. Using TecanEvo200 in culture plates, three independent dilutions of CO and IO were prepared in the range of 10,000–0.5 ng/mL; a TNFα solution of 500 pg/mL was added to the prepared dilutions. The resulting mixture was stirred and incubated at room temperature for 1 hour.

After incubation, a cell suspension of WEHI-13 var (0.5 ± 0.1) × 10 ⁶ cells/ml was added. The plates were placed in a CO ₂ incubator and incubated at (37 ± 1) ºС, 5% CO ₂ in humidified air for 20–24 h.

At the end of the incubation period, the vital dye Alamar Blue was added to the culture plates and the plates were incubated under the same conditions until the color developed. The fluorescence intensity was evaluated at an excitation/emission wavelength of 544/590 nm using an Infinite M200Pro reader. Using the Magellan 7.2 software, curves of the dependence of the fluorescence intensity on the protein concentration were plotted; the parallelism of the obtained curves was evaluated. The relative specific activity of the studied samples was determined as the ratio of the ED50 of the standard sample to the ED50 of the test, expressed as a percentage.

For a better understanding of the invention, the following examples are given. These examples are provided for illustrative purposes only and should not be construed as limiting the scope of the invention in any form.

All publications, patents, and patent applications referenced in this specification are incorporated herein by reference. Although the above invention has been described in some detail by way of illustration and example in order to avoid ambiguity, it will be quite clear to those skilled in the art based on the ideas disclosed in this invention that certain changes and modifications can be made without deviating from the essence and scope of the attached options. implementation of the invention.

Examples

Example 1. Choice of the nature of the buffer solution.

Investigated compositions.

In the present study, four buffer solutions were chosen, as well as the original formulations of Humira preparation (for formulations with a protein content of 50 mg/ml and 100 mg/ml) as controls.

Humira 1 Disodium hydrophosphate dihydrate 1.530 mg/ml
Sodium dihydrophosphate dihydrate 0.860 mg/ml
Mannitol 12.0 mg/ml
Citric acid monohydrate 1.305 mg/ml
Polysobrate 80 1.0 mg/ml
Sodium citrate 0.305 mg/ml
Sodium chloride 6.165 mg/ml
Sodium hydroxide up to pH 5.2 Humira 2 Mannitol 42.0 mg/ml
Polysobrate 80 1.0 mg/ml Acet, pH 5.0 Sodium acetate t/g 0.436 mg/ml
Acetic acid ice. up to pH 5.0 Cit, pH 5.0 Sodium citrate d/g 0.932 mg/ml
Citric acid b / in 0.352 mg / ml His, pH 6.0 L-histidine 0.40 mg/ml
Histidine hydrochloride m/g 0.40 mg/ml PB, pH 6.0 Sodium dihydrogen phosphate m/g 1.21 mg/ml
Sodium hydrophosphate b / in 0.17 mg / ml

Determination of colloidal stability by PEG aggregation.

The PEG aggregation test was performed in triplicate for each sample. The analysis was performed according to method 3. The data of the average optical density of the solutions are presented in table 1. The results are also presented in FIG. one.

Table 1. Average optical density of solutions after preparation.

Determination of thermal stability by the point of protein aggregation by dynamic light scattering (DLS).

The analysis was performed according to method 4. The results are presented in Table 2 and in FIG. 4 - 8.

Determination of thermal stability during prolonged exposure by the Thermostress 50ºС method.

The analysis was performed according to method 5. The results are presented in table 2.

Results.

Table 2. Summary of results on the choice of the nature of the buffer solution.

Conclusions from example 1.

According to the results of this study, the recommended storage composition (excluding additional excipients):

Acet, pH 5.0 Sodium acetate t/g 0.436 mg/ml
Acetic acid ice. up to pH 5.0

This composition showed the best stabilizing properties among all the studied samples: the smallest increase in impurities during thermal stress (0.02% on a par with the Humira 2 composition), the absence of aggregation at 18% PEG 6000, and the highest aggregation temperature (74°C).

Adalimumab in Humira 1 has less thermal stability than the recommended formulation. The minimum colloidal and thermal stability was noted for the composition based on 5 mM citrate buffer solution with pH 5.0.

Example 2 Optimization of the pH and Buffer Capacity of the Composition.

According to the results of the first part of the study, adalimumab showed the best stability in an acetate buffer solution with pH 5.0. As the state of the art has shown, most patents and patent applications for pharmaceutical compositions containing adalimumab protect solutions with a pH of 4.0 to 8.0. Thus, the purpose of this section was to investigate the possibility of obtaining stable compositions containing acetate ions with a pH less than 4.

2.1. Determination of colloidal stability by PEG aggregation.

The PEG aggregation test was performed in triplicate for each sample. The analysis was performed according to method 3. The data of the average optical density of the solutions are presented in table 3. The results are also presented in FIG. 2.

Table 3. Average optical density (400 nm) of solutions after preparation.

2.2. Determination of thermal stability during prolonged exposure by the Thermostress 50ºС method.

The analysis was performed according to method 5.

2.3. Determination of colloidal stability by the shake test method.

The analysis was performed according to method 6.

2.4. Determination of colloidal stability by cryoconcentration.

The analysis was performed according to method 7.

The overall results are presented in Table 4, which clearly reflects the quality indicators of the samples before and after thermal stress, shake test and cryoconcentration.

2.5. Results.

Table 4. Summary results of sample quality indicators before and after stresses.

2.6. Conclusions from example 2.

• The study showed that the presence of adalimumab in acidic placebo solutions resulted in a significant increase in pH. In addition, compositions with a pH less than 5.0 showed poor stability during dialysis (low purity on Labchip Caliper under reducing conditions) and thermal stress (low purity after stress on all used methods of analysis).

• The most stable sample among all studied is adalimumab in 5 mM acetate buffer pH 5.0 – 6.0. They demonstrated minimal change in purity as well as acid-base profile during stresses. However, to create an adalimumab composition, it is possible to use solutions with a higher buffer capacity.

Example 3 Choice of Osmotic Agent and Stabilizers.

According to the results of the first part of the study, adalimumab showed the best stability in an acetate buffer solution with a pH of 5.0 - 6.0. The composition with pH 5.5 was taken as the basis for the choice of osmotic agent and stabilizers.

Investigated compositions.

Humira 1 Disodium hydrophosphate dihydrate 1.530 mg/ml
Sodium dihydrophosphate dihydrate 0.860 mg/ml
Mannitol 12.0 mg/ml
Citric acid monohydrate 1.305 mg/ml
Polysobrate 80 1.0 mg/ml
Sodium citrate dihydrate 0.305 mg/ml
Sodium chloride 6.165 mg/ml
Sodium hydroxide up to pH 5.2 Humira 2 Mannitol 42.0 mg/ml
Polysobrate 80 1.0 mg/ml Acet, pH 5.5 Sodium acetate trihydrate 0.436 mg/ml
Acetic acid ice. up to pH 5.5 Acet + NaCl, pH 5.5 Sodium acetate trihydrate 0.436 mg/ml
Acetic acid ice. up to pH 5.5
Sodium chloride 9 mg/ml Acet + Tre, pH 5.5 Sodium acetate trihydrate 0.436 mg/ml
Acetic acid ice. up to pH 5.5
Trehalose dihydrate 100 mg/ml Acet + Suc, pH 5.5 Sodium acetate trihydrate 0.436 mg/ml
Acetic acid ice. up to pH 5.5
Sucrose 100 mg/ml Acet + Mannitol, pH 5.5 Sodium acetate trihydrate 0.436 mg/ml
Acetic acid ice. up to pH 5.5
Mannitol 50 mg/ml Acet + Sorb, pH 5.5 Sodium acetate trihydrate 0.436 mg/ml
Acetic acid ice. up to pH 5.5
Sorbitol 50 mg/ml Acet + 100mM Gly, pH 5.5 Sodium acetate trihydrate 0.436 mg/ml
Acetic acid ice. up to pH 5.5
Glycine 7.5 mg/ml Acet + 50 mM Arg, pH 5.5 Sodium acetate trihydrate 0.436 mg/ml
Acetic acid ice. up to pH 5.5
Arginine hydrochloride 10.5 mg/ml Acet + 10 mM Meth, pH 5.5 Sodium acetate trihydrate 0.436 mg/ml
Acetic acid ice. up to pH 5.5
Methionine 1.5 mg/ml Acet + 250 mM Prol, pH 5.5 Sodium acetate trihydrate 0.436 mg/ml
Acetic acid ice. up to pH 5.5
L-Proline 27.0 mg/ml Acet + 10 mM EDTA, pH 5.5 Sodium acetate trihydrate 0.436 mg/ml
Acetic acid ice. up to pH 5.5
EDTA disodium salt dihydrate 3.72 mg/ml Acet + 100mM Lys, pH 5.5 Sodium acetate trihydrate 0.436 mg/ml
Acetic acid ice. up to pH 5.5
Lysine 14.6 mg/ml Acet + 100mM Guan, pH 5.5 Sodium acetate trihydrate 0.436 mg/ml
Acetic acid ice. up to pH 5.5
Guanidine hydrochloride 9.55 mg/ml Acet + 100mM Glu, pH 5.5 Sodium acetate trihydrate 0.436 mg/ml
Acetic acid ice. up to pH 5.5
Glucose monohydrate 19.82 mg/ml Acet + 100mM Lact, pH 5.5 Sodium acetate trihydrate 0.436 mg/ml
Acetic acid ice. up to pH 5.5
Lactose monohydrate 36.03 mg/ml Acet + 100mM Mannose, pH 5.5 Sodium acetate trihydrate 0.436 mg/ml
Acetic acid ice. up to pH 5.5
Mannose 18.02 mg/ml Acet + Tre + 0. Cyst, pH 5.5 Sodium acetate trihydrate 0.436 mg
Acetic acid ice. up to pH 5.5
Trehalose dihydrate 100 mg
Cysteine hydrochloride monohydrate 0.088 mg Acet + Tre + 2. Cyst, pH 5.5 Sodium acetate trihydrate 0.436 mg
Acetic acid ice. up to pH 5.5
Trehalose dihydrate 100 mg
Cysteine hydrochloride monohydrate 0.44 mg Acet + 10mM Cyst, pH 5.5 Sodium acetate trihydrate 0.436 mg/ml
Acetic acid ice. up to pH 5.5
Cysteine hydrochloride monohydrate 1.76 mg/ml Acet + Tween20 0.5, pH 5.5 Sodium acetate trihydrate 0.436 mg/ml
Acetic acid ice. up to pH 5.5
Polysorbate 20 0.5 mg/ml Acet + Tween20 1.0, pH 5.5 Sodium acetate trihydrate 0.436 mg/ml
Acetic acid ice. up to pH 5.5
Polysorbate 20 1.0 mg/ml Acet + Tween80 0.5, pH 5.5 Sodium acetate trihydrate 0.436 mg/ml
Acetic acid ice. up to pH 5.5
Polysorbate 80 0.5 mg/ml Acet + Tween80 1.0, pH 5.5 Sodium acetate trihydrate 0.436 mg/ml
Acetic acid ice. up to pH 5.5
Polysorbate 80 1.0 mg/ml Acet + Pol.188 0.5, pH 5.5 Sodium acetate trihydrate 0.436 mg/ml
Acetic acid ice. up to pH 5.5
Poloxamer 188 0.5 mg/ml Acet + Pol.188 1.0, pH 5.5 Sodium acetate trihydrate 0.436 mg/ml
Acetic acid ice. up to pH 5.5
Poloxamer 188 1.0 mg/ml

3.1. Determination of colloidal stability by PEG aggregation.

The PEG aggregation test was performed in triplicate for each sample. The analysis was performed according to method 3. The data of the average optical density of the solutions are presented in table 6. The results are also presented in FIG. 3.

Table 6 Average absorbance of adalimumab solutions after preparation.

3.2. Determination of thermal stability by the point of protein aggregation by dynamic light scattering (DLS).

The analysis was performed according to method 4.

3.3. Determination of thermal stability during prolonged exposure by the Thermostress 50ºС method.

The analysis was performed according to method 5.

3.4. Results.

3.5. Conclusions from example 3.

Based on the results of this study, promising excipients for adalimumab formulations are: trehalose dihydrate, glycine, proline, methionine, and arginine hydrochloride. The following were taken as surfactants for screening the final pharmaceutical composition: polysorbate 80, polysorbate 20, poloxamer 188.

The colloidal stability of adalimumab is negatively affected by: sodium chloride, arginine, EDTA, lysine, guanidine.

A negative effect on the thermal stability of adalimumab is exerted by: sodium chloride, lysine, guanidine. The content of cysteine in the composition leads to complete fragmentation of the antibody. A shift in the acid-base profile of the protein was noted when EDTA was added to the composition of the excipient.

Example 4 Selection of the final pharmaceutical composition.

According to the results of the third part of the study, trehalose dihydrate, glycine, proline, methionine and arginine hydrochloride were selected as promising excipients. The following were taken as surfactants for screening the final pharmaceutical composition: polysorbate 80, polysorbate 20, poloxamer 188.

4.1. Determination of thermal stability during prolonged exposure by the Thermostress 50ºС method.

The analysis was performed according to method 5.

4.2. Determination of colloidal stability by the shake test method.

The analysis was performed according to method 6.

4.3. Determination of colloidal stability by cryoconcentration.

The analysis was performed according to method 7.

The overall results are presented in tables 8 and 9, which reflect the quality indicators of the samples before and after thermal stress, shake test and cryoconcentration.

4.4. Results.

Conclusions from example 4.

As a result of the study, it was shown that adalimumab in the composition of Humira 1 is the most thermolabile of all the studied samples. Thermal stress of 50°C for 120 hours leads to the maximum increase in impurities (1.2%) on the E HPLC, as well as to the maximum change in the acid-base profile (the total absolute change of all fractions is more than 33% on the LabChip Caliper instrument). The colloidal stability of the sample in Humira 1 is comparable to alternative formulations.

The composition of Humira 2 has thermal stability comparable to alternative compositions (loss of purity during thermal stress 0.38%). However, during freezing-thawing of adalimumab in this composition, a noticeable protein aggregation occurs: the increase in aggregates on size-exclusion HPLC is more than 7%, which makes the use of the drug unsuitable if it is accidentally frozen. A noticeable increase in impurities after freezing was also noted using DLS.

Alternative compositions based on an acetate buffer solution with the addition of a number of excipients showed good thermal and colloidal stability during stresses. Size exclusion HPLC showed no difference in the growth of impurities of these compositions. Also, there was no significant effect of surfactants on the stability of adalimumab at a concentration of 15 mg/ml.

Analysis of stressed samples using DLS showed that the presence of arginine hydrochloride reduces the increase in impurities during the shake test, and the presence of methionine increases the accumulation of impurities during shaking.

The minimum change in the acid-base profile was noted for formulations 12 - 29 (compositions containing trehalose and arginine hydrochloride, as well as those containing L-proline). Based on the results of this study, promising formulations for adalimumab are:

Sodium acetate trihydrate 0.436 mg/ml
Acetic acid ice. up to pH 5.0-6.0
Trehalose dihydrate 80 mg/ml Sodium acetate trihydrate 0.436 mg/ml
Acetic acid ice. up to pH 5.0-6.0
Trehalose dihydrate 80 mg/ml
Polysorbate 20 / Polysorbate 80 / Poloxamer 188 0.5 – 1.0 mg/ml Sodium acetate trihydrate 0.436 mg/ml
Acetic acid ice. up to pH 5.0-6.0
Trehalose dihydrate 80 mg/ml
Arginine hydrochloride 0.1 mg/ml Sodium acetate trihydrate 0.436 mg/ml
Acetic acid ice. up to pH 5.0-6.0
Trehalose dihydrate 80 mg/ml
Arginine hydrochloride 0.1 mg/ml
Polysorbate 20 / Polysorbate 80 / Poloxamer 188 0.5 – 1.0 mg/ml Sodium acetate trihydrate 0.436 mg/ml
Acetic acid ice. up to pH 5.0-6.0
L-Proline 27 mg/ml Sodium acetate trihydrate 0.436 mg/ml
Acetic acid ice. up to pH 5.0-6.0
L-Proline 27 mg/ml
Polysorbate 20 / Polysorbate 80 / Poloxamer 188 0.5 – 1.0 mg/ml

Example 5 Confirmation of the final pharmaceutical composition of highly concentrated adalimumab.

To confirm the stability of the recommended pharmaceutical formulations, adalimumab at 50 mg/mL, 100 mg/mL, and 150 mg/mL was heat stressed at 50°C for 6 days.

5.1. Determination of thermal stability during prolonged exposure by the Thermostress 50ºС method.

The analysis was performed according to method 5.

5.2. Results.

General conclusions

1. Screening of a stable pharmaceutical composition of adalimumab was carried out in several stages: the choice of the nature of the buffer solution, the choice of pH and buffer capacity of the solution, the choice of osmolytics and stabilizers, the choice of the final pharmaceutical composition, and the confirmation of the final pharmaceutical composition of highly concentrated adalimumab.

2. In the course of the work, the thermal and colloidal stability of adalimumab in more than 90 formulations was studied using the following methods: PEG aggregation, shake test, freeze-thaw, thermal stress, followed by analysis of the degree of turbidity (UV spectrophotometry), purity (exclusion HPLC , dynamic light scattering and gel electrophoresis, including using the LabChip, Caliper system), as well as acid-base profile (LabChip, Caliper and ion-exchange HPLC).

3. As a result of the study, low thermal stability of the original composition of the Humira preparation (Humira 1) used in the commercial preparation with a concentration of adalimumab 50 mg / ml was revealed, compared with the compositions of the present invention.

4. During the experiment, a significant effect of freezing and thawing processes on the aggregation of adalimumab in the original composition of the Humira preparation (Humira 2) used in the commercial preparation of adalimumab 100 mg/ml was revealed in comparison with the compositions of the present invention.

5. Pharmaceutical compositions of adalimumab based on an acetate buffer solution with pH 5.0 - 6.0 with the addition of trehalose dihydrate, arginine hydrochloride, proline, as well as surfactants from the group of polysorbate 20, polysorbate 80 and poloxamer 188 exhibit greater thermal and colloidal stability at concentrations of 50 – 150 mg/ml compared to the original formulations and are the subject of the present invention.

The optimal composition of adalimumab excipients was chosen for solutions with pH 5.5. The stability of adalimumab in formulations with a pH range of 5.0 - 6.0 was confirmed on samples at protein concentrations of 50, 100 and 150 mg/ml.

6. In the course of the technological process when obtaining pharmaceutical compositions of the present invention with an adalimumab concentration of 150 mg/ml, concentration is possible up to 200 mg/ml without loss of protein quality for subsequent dilution when adding surfactants, as well as when washing the ultrafiltration unit after concentration.

Claims (134)

1. An aqueous pharmaceutical composition for the treatment of TNF-mediated diseases for intravenous or subcutaneous administration, containing: a) adalimumab at a concentration of 50 to 200 mg/ml; b) buffer agent or buffer system based on acetate ions, where the concentration of the acetate buffer solution is from 1 to 100 mm; c) trehalose at a concentration of 25 to 150 mg/ml and/or proline at a concentration of 5 to 40 mg/ml or trehalose at a concentration of 25 to 150 mg/ml and arginine at a concentration of 0.05 to 0.5 mg/ml; d) polysorbate 20 at a concentration of 0.05 mg/ml to 10 mg/ml, polysorbate 80 at a concentration of 0.05 mg/ml to 10 mg/ml or poloxamer 188 at a concentration of 0.05 mg / ml to 10 mg / ml or a combination thereof, where the composition has a pH of 5.5. 2. Composition according to claim 1, containing: a) 50 to 200 mg/ml adalimumab; b) 0.4 to 1.2 mg/ml sodium acetate trihydrate; c) 25 to 150 mg/ml trehalose and/or 5 to 40 mg/ml proline, or 25 to 150 mg/ml trehalose and 0.05 to 0.5 mg/ml arginine; d) acetic acid ice. up to pH 5.5; e) 0.1 mg/ml to 1 mg/ml polysorbate 80. 3. Composition according to claim 2, containing: a) 50 to 150 mg/ml adalimumab; b) 0.436 mg/ml sodium acetate trihydrate; c) 80 mg/ml trehalose dihydrate; d) acetic acid ice. up to pH 5.5; e) 0.5 mg/ml polysorbate 80. 4. Composition according to claim 2, containing: a) from 50 to 150 mg/ml of adalimumab; b) 0.436 mg/ml sodium acetate trihydrate; c) 80 mg/ml trehalose dihydrate and 0.1 mg/ml arginine hydrochloride; d) acetic acid ice. up to pH 5.5; e) 0.5 mg/ml polysorbate 80. 5. Composition according to claim 2, containing: a) 50 to 150 mg/ml adalimumab; b) 0.436 mg/ml sodium acetate trihydrate; c) 27 mg/ml proline; d) acetic acid ice. up to pH 5.5; e) 0.5 mg/ml polysorbate 80. 6. Composition according to claim 2, containing: a) 50 to 150 mg/ml adalimumab; b) 0.436 mg/ml sodium acetate trihydrate; c) 40 mg/ml trehalose dihydrate; d) 13.5 mg/ml proline; e) acetic acid ice. up to pH 5.5; f) 0.5 mg/ml polysorbate 80. 7. Composition according to claim 1, containing: a) 50 to 200 mg/ml adalimumab; b) 0.4 to 1.2 mg/ml sodium acetate trihydrate; c) 25 to 150 mg/ml trehalose and/or 5 to 40 mg/ml proline, or 25 to 150 mg/ml trehalose and 0.05 to 0.5 mg/ml arginine; d) acetic acid ice. up to pH 5.5; e) 0.1 mg/ml to 1 mg/ml polysorbate 20. 8. Composition according to claim 7, containing: a) 50 to 150 mg/ml adalimumab; b) 0.436 mg/ml sodium acetate trihydrate; c) 80 mg/ml trehalose dihydrate; d) acetic acid ice. up to pH 5.5; e) 0.5 mg/ml polysorbate 20. 9. Composition according to claim 7, containing: a) 50 to 150 mg/ml adalimumab; b) 0.436 mg/ml sodium acetate trihydrate; c) 80 mg/ml trehalose dihydrate and 0.1 mg/ml arginine hydrochloride; d) acetic acid ice. up to pH 5.5; e) 0.5 mg/ml polysorbate 20. 10. Composition according to claim 7, containing: a) 50 to 150 mg/ml adalimumab; b) 0.436 mg/ml sodium acetate trihydrate; c) 27 mg/ml proline; d) acetic acid ice. up to pH 5.5; e) 0.5 mg/ml polysorbate 20. 11. Composition according to claim 7, containing: a) 50 to 150 mg/ml adalimumab; b) 0.436 mg/ml sodium acetate trihydrate; c) 40 mg/ml trehalose dihydrate; d) 13.5 mg/ml proline; e) acetic acid ice. up to pH 5.5; f) 0.5 mg/ml polysorbate 20. 12. Composition according to claim 1, containing: a) 50 to 200 mg/ml adalimumab; b) 0.4 to 1.2 mg/ml sodium acetate trihydrate; c) 25 to 150 mg/ml trehalose and/or 5 to 40 mg/ml proline, or 25 to 150 mg/ml trehalose and 0.05 to 0.5 mg/ml arginine d) acetic acid ice. up to pH 5.5; e) 0.1 mg/ml to 1 mg/ml poloxamer 188. 13. Composition according to claim 12, containing: a) 50 to 150 mg/ml adalimumab; b) 0.436 mg/ml sodium acetate trihydrate; c) 80 mg/ml trehalose dihydrate; d) acetic acid ice. up to pH 5.5; e) 1.0 mg/ml poloxamer 188. 14. Composition according to claim 12, containing: a) 50 to 150 mg/ml adalimumab; b) 0.436 mg/ml sodium acetate trihydrate; c) 80 mg/ml trehalose dihydrate and 0.1 mg/ml arginine hydrochloride; d) acetic acid ice. up to pH 5.5; e) 1.0 mg/ml poloxamer 188. 15. Composition according to claim 12, containing: a) 50 to 150 mg/ml adalimumab; b) 0.436 mg/ml sodium acetate trihydrate; c) 27 mg/ml proline; d) acetic acid ice. up to pH 5.5; e) 1.0 mg/ml poloxamer 188. 16. Composition according to claim 12, containing: a) 50 to 150 mg/ml adalimumab; b) 0.436 mg/ml sodium acetate trihydrate; c) 40 mg/ml trehalose dihydrate; d) 13.5 mg/ml proline; e) acetic acid ice. up to pH 5.5; f) 1.0 mg/ml poloxamer 188. 17. A method for the treatment of diseases mediated by TNF, including the introduction of the composition according to any one of paragraphs. 1-16 in an effective amount, wherein the TNF-mediated disease is selected from the group: a) active rheumatoid arthritis, b) active psoriatic arthritis, c) active ankylosing spondylitis, d) chronic plaque psoriasis, e) ulcerative colitis, f) axial spondyloarthritis, g) active purulent hydradenitis, h) juvenile idiopathic arthritis, i) Crohn's disease, j) uveitis, k) active enthesitis-associated arthritis. 18. The use of a composition according to any one of paragraphs. 1-16 for the treatment of TNF-mediated diseases, wherein the disease is selected from the group: a) active rheumatoid arthritis, b) active psoriatic arthritis, c) active ankylosing spondylitis, d) chronic plaque psoriasis, e) ulcerative colitis, f) axial spondyloarthritis, g) active purulent hydradenitis, h) juvenile idiopathic arthritis, i) Crohn's disease, j) uveitis, k) active enthesitis-associated arthritis. 19. The method of obtaining a composition according to any one of paragraphs. 1-16, which includes the addition of acetate buffer agents to the aqueous phase, followed by the addition of the following components in any sequence: a) an osmolytic and/or stabilizer selected from the group: trehalose, proline, arginine, or combinations thereof; b) adalimumab; c) a surfactant selected from the group: polysorbate 20, polysorbate 80, poloxamer 188, or combinations thereof.

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