RU2665966C2 - Recombinant monoclonal antibody to tnf-alpha aqueous pharmaceutical composition - Google Patents

Abstract

FIELD: pharmaceuticals.SUBSTANCE: invention relates to the medical pharmacology, in particular to the recombinant monoclonal antibody to TNFα (adalimumab) improved aqueous pharmaceutical compositions and to their preparation method. Invention also relates to the use recombinant monoclonal antibody to TNFα (adalimumab) improved aqueous pharmaceutical compositions for the TNFα mediated diseases treatment.EFFECT: proposed invention allows to prevent the physic-chemical instability, expressed in the aggregates and protein fragments formation or proteins modification in the solution, and also prevents instability during the freezing and thawing, stirring and shaking.22 cl, 9 dwg, 10 tbl, 5 ex

Description

FIELD OF THE INVENTION

The invention relates to medical pharmacology, and relates, in particular, to 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 naturally occurring mammalian cytokine produced by various types of cells, including monocytes and macrophages, in response to endotoxin or other stimuli. TNFα is a major mediator of inflammatory, immunological and pathophysiological reactions (Grell, M., et al. (1995) Cell, 83: 793-802).

Soluble TNFα is formed by cleaving the transmembrane precursor protein (Kriegler, et al. (1988) Cell 53: 45-53), and secreted polypeptides weighing 17 kilodaltons (kDa) 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). Then these complexes bind to receptors located on many cells. Binding provides a number of pro-inflammatory effects, including (i) the release of other pro-inflammatory cytokines, such as interleukin (IL) -6, IL-8 and IL-1, (ii) the release of matrix metalloproteinases and (iii) enhancing the expression of endothelial adhesion molecules, further enhancing the expression of inflammatory and immune cascades, attracting white blood cells into extravascular tissues.

There are many disorders associated with elevated levels of TNFα. For example, TNFα expression has been shown to increase 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, bronchial 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 release macrophages activated by lipopolysaccharides of gram-negative bacteria. Essentially, TNFα appears to be a very important endogenous neurotransmitter 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 is composed of 1330 amino acids, and its molecular weight is approximately 148 kDa. Adalimumab is described and patented in US patent No. 6090382 (international application WO9729131), the description of which, therefore, is incorporated by reference in its entirety. As a rule, adalimumab is obtained 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, blocking its interaction with TNF receptors p55 and p75 on the cell surface.

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

W02004016286 describes a liquid composition for stabilizing antibodies used to treat diseases mediated by TNFα, including an antibody, for example, adalimumab, a buffer system, polysorbate and means for maintaining the isotonicity of the solution, for example, mannitol and sodium chloride. The composition uses citrate-phosphate buffer (Humir composition 1). The above composition has several disadvantages: 1) insufficient colloidal stability of the antibody in high concentrations, 2) insufficient stability under thermal stress, and 3) aggregation during prolonged storage.

WO2012065072 describes a liquid composition for stabilizing antibodies used to treat diseases mediated by TNFα, including an antibody, for example, adalimumab, as well as mannitol, polysobrate 80 (Humir composition 2). The above composition has several disadvantages: 1) pronounced instability during freezing and thawing and 2) low aggregation temperature.

Thus, there is a need to create a new drug product containing an antibody that binds TNFα, for example, 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.

SUMMARY OF THE INVENTION

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

In some embodiments of the composition, the recombinant monoclonal anti-TNFα 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 acetate buffer solution is from 1 to 100 mm.

In some embodiments, the composition has a pH of from 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 proline concentration is from 5 to 40 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 comprises from 50 to 200 mg / ml of a recombinant monoclonal anti-TNFα 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 25 to 150 mg / ml trehalose and from 0.05 to 1.0 mg / ml arginine, acetic acid ice. to pH 5.0, from 0.1 mg / ml to 1 mg / ml of polysorbate 80.

In some embodiments of the composition, the recombinant monoclonal anti-TNFα 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. up to pH 5.0, 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. up to pH 5.0, 0.5 mg / ml polysorbate 80.

In some embodiments, the composition comprises from 50 to 150 mg / ml adalimumab, 0.436 mg / ml sodium acetate trihydrate, 27 mg / ml proline, acetic acid ice. up to pH 5.0, 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. up to pH 5.0, 0.5 mg / ml polysorbate 80.

In some embodiments, the composition comprises from 50 to 200 mg / ml of a recombinant monoclonal anti-TNFα 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 25 to 150 mg / ml trehalose and from 0.05 to 1.0 mg / ml arginine, acetic acid ice. to pH 5.0, from 0.1 mg / ml to 1 mg / ml of polysorbate 20.

In some embodiments of the composition, the recombinant monoclonal anti-TNFα 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 5.0, 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 5.0, 0.5 mg / ml polysorbate 20.

In some embodiments, the composition comprises from 50 to 150 mg / ml adalimumab, 0.436 mg / ml sodium acetate trihydrate, 27 mg / ml proline, acetic acid ice. to pH 5.0, 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 5.0, 0.5 mg / ml polysorbate 20.

In some embodiments, the composition comprises from 50 to 200 mg / ml of a recombinant monoclonal anti-TNFα 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 25 up to 150 mg / ml trehalose and from 0.05 to 1.0 mg / ml arginine, acetic acid ice. to pH 5.0, from 0.1 mg / ml to 1 mg / ml of poloxamer 188.

In some embodiments, the recombinant monoclonal anti-TNFα 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 5.0, 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 5.0, 1 mg / ml poloxamer 188.

In some embodiments, the composition comprises from 50 to 150 mg / ml adalimumab, 0.436 mg / ml sodium acetate trihydrate, 27 mg / ml proline, acetic acid ice. to pH 5.0, 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 5.0, 1 mg / ml poloxamer 188.

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

In some embodiments of the treatment method, the disease mediated by TNFα 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 spondylitis, active purulent hydradenitis, 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 and severe), active psoriatic arthritis, active ankylosing spondylitis, chronic plaque psoriasis (moderate or severe), ulcerative colitis (moderate and severe), axial active spondylitis , 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 producing the above compositions, which comprises adding acetate buffering agents to the aqueous phase, followed by adding, in any sequence, the following components: an osmolytic and / or stabilizer selected from the group: trehalose, proline, or a combination thereof , a recombinant monoclonal antibody to TNFα, a surfactant selected from the group: polysorbate 20, polysorbate 80, poloxamer 188, or a combination thereof.

Brief Description of the Drawings

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

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

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

Figure 4. The dependence of the optical density of solutions at 400 nm on the concentration of PEG 6000 after 24 hours of incubation.

Figure 5. The aggregation point of adalimumab in acetate buffer solution, pH 5.0. T _ag = 74.0 ° C.

6. The aggregation point of adalimumab in citrate buffer solution, pH 5.0. T _ag = 69.5 ° C.

7. The aggregation point of adalimumab in histidine buffer solution, pH 6.0. T _ag = 69.5 ° C.

Fig. 8. Aggregation point of adalimumab in phosphate buffered saline, pH 6.0. T _ag = 71.0 ° C.

Fig.9. The aggregation point of adalimumab as part of Humir 1, pH 5.2. T _ag = 69.5 ° C.

Description of the invention

Definitions and General Methods

The terms used in the present description, as a rule, have their usual meanings in this area within the context of the invention and in the specific context where each term is used. Below, or elsewhere in the specification, certain terms are defined that are used to describe the invention, to provide the practice of additional guidance regarding the description of the invention. Synonyms of some terms are given. A statement of one or more synonyms does not preclude the use of other synonyms. The use of examples anywhere in the present description, including examples of any of the terms described herein, is illustrative only and in no way limits the scope and meaning of the invention or any illustrated object. The invention is not limited to the various embodiments described herein.

Unless otherwise specified, all technical and scientific terms used in this document have the same meaning, which is usually understood by a specialist in the field to which the invention belongs. In case of conflict, this document should be guided by, including definitions.

The term "human TNFα", also known as hTNFα or hTNF, or TNFα, is intended to refer to a human cytokine that exists in the form of a secreted form with a mass of 17 kDa and a membrane-associated form with a mass of 26 kDa, 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 obtained by standard recombinant expression methods or purchased commercially (R&D Systems, catalog 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 interconnected by internal disulfide bonds. Each heavy chain consists of a variable region of the heavy chain (abbreviated herein as HCVR or VH) and a constant region of the heavy chain. The constant region of the heavy chain consists of three domains, CH1, CH2 and CHZ. Each light chain consists of a variable region of the light chain (abbreviated herein as LCVR or VL) and a constant region of the light chain. The constant region of the light chain consists of one domain, CL. The VH and VL regions can be further divided into hypervariability regions, denoted by complementarity determining regions (CDRs), alternated by regions that are more conservative, called framework regions (FR). Each VH and VL consists of three CDRs and four FRs, located from the N-end to the C-end in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In one embodiment, the composition comprises an antibody with the sequences CDR1, CDR2, and CDR3 similar to those described in US Pat. Nos. 6,090,382; 6258562 and 8216583.

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

As used herein, the term "isolated antibody" refers to an antibody that essentially does not contain other antibodies with other antigenic specificities (for example, an isolated antibody that specifically binds to TNFα, essentially does not contain antibodies that specifically bind to antigens, different from TNFα). However, an isolated antibody that specifically binds to TNFα may have cross-reactivity with other antigens, such as other types of TNFα molecules. In addition, the isolated antibody may essentially not contain other cellular materials and / or chemicals.

The term "anti-TNFα antibody" refers to a human anti-TNFα antibody described herein, as well as described in US Pat. Nos. 6,090,382; 6,258,562; 6,509,015; 7223394 and 6509015. The term anti-TNFα antibody used in the invention is an anti-TNFα antibody or fragment thereof, including infliximab (remicade®, Johnson and Johnson; described in US Pat. No. 5,656,272); CDP571 (humanized monoclonal antibody IgG4 against TNF-alpha); CDP870 (fragment of a humanized monoclonal antibody against TNF-alpha); dAb vs TNF (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 6090382 as D2E7). Additional anti-TNF antibodies that can be used in the invention are described in US Pat. Nos. 6,593,458; 6,498,237; 6451983 and 6448380. In another embodiment, the TNFα inhibitor is a TNF fusion protein, for example, etanercept (Enbrel®, Amgen; described in WO 91/03553 and WO 09/406476). In another embodiment, the TNFα inhibitor is a recombinant TNF binding protein (r-TBP-I) (Serono).

The term “adalimumab” is synonymous with the active pharmaceutical ingredient in Humira®, as well as a protein considered or implied by its bio-analogous or bio-enhanced options. 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, and its total molecular weight is approximately 148 kDa. The term adalimumab is also intended to include the so-called bio-analogous or bio-enhanced variants of the adalimumab protein used in the commercially available Humira®. For example, a commercial Khumira® variant may be acceptable to the FDA when it has substantially the same pharmacological effect as the commercially available Humira®, even if it can exhibit certain physical characteristics, such as a glycosylation profile and an acid-base profile, which may be , if not identical to Khumira®, then similar to them.

For the purposes of this application, the term “adalimumab” also includes adalimumab with minor modifications in the structure of amino acids (including deletions, introductions and / or substitutions of amino acids) or in the properties of glycosylation and acid-base profile, which do not significantly affect the function of the polypeptide. The term “adalimumab” includes all forms and compositions 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 "long-term storage" or "long-term stability" should be understood as a designation 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 stable storage durations that are at least comparable to or better than the stable storage period, typically necessary for the lossless commercial adalimumab formulations currently available stability, which may render the composition unsuitable for a particular pharmaceutical use. Long-term storage should also be understood as an indication that the pharmaceutical composition is stored either in liquid form 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 as meaning 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 “excipient” is used herein to describe any ingredient other than previously described in this invention.

The term “sugar” refers to monosaccharides, disaccharides and polysaccharides or mixtures thereof. Examples of sugars by way of non-limiting examples include sucrose, trehalose, glucose, dextrose and others.

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

The term “buffer”, “buffer composition”, “buffer agent”, as used here, refers to an added composition that enables a liquid preparation of an antibody to exhibit resistance to pH changes, usually through the action of components of its acid-base conjugate. When reference is made to the concentration of the buffer, it is understood that the concentration is the total molar concentration of the free acid and free base form of the buffer. Examples of buffers that are known to those skilled in the art and may be found in the literature include, but are not limited to, histidine, citrate, succinate, acetate, phosphate, phosphate buffered saline, citrate phosphate buffers, as well as trometamine-based buffers and the like, or the like suitable mixtures.

The terms “tonicity agent” or “tonicity agent”, as well as “osmolytic” or “osmotic agent” as used herein, refer to an excipient that can apply the osmotic pressure of a liquid antibody preparation. In certain embodiments, the tonicity agent can adjust the osmotic pressure of the liquid antibody preparation to isotonic so that the antibody preparation is physiologically compatible with the tissue cells of the subject's body. In yet another embodiment, a “tonicity agent” can improve stability of the antibodies described herein. An “isotonic” drug is a drug that has an osmotic pressure equivalent to human blood. Isotonic preparations typically have an osmotic pressure of about 250 to 350 mOsm. The term "hypotonic" describes a drug with an osmotic pressure lower than the osmotic pressure of human blood. Accordingly, the term "hypertonic" is used to describe a drug with an osmotic pressure greater than the osmotic pressure 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 a free acid or free base; in salt form; in hydrated form (e.g. monohydrate) or in anhydrous form.

The term “surfactant” (another name for surfactant or detergent, or surfactant), as used here, refers to an excipient that can alter the surface tension of a liquid antibody preparation. In certain embodiments, the surfactant reduces the surface tension of a liquid antibody preparation. In other embodiments, a “surfactant” can help improve the colloidal stability or solubility of any antibody in a given preparation. A surfactant can reduce the aggregation of the prepared 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 may be included in the compositions of the present invention include, for example, alkyl polyols (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 nonionic surfactants that can 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 (for example, Pluronic PF68, etc.).

The term “lyophilized” as used herein refers to a preparation that has undergone a process known in the art as freeze-drying, which involves freezing the preparation and then removing ice from the frozen contents.

The term “amino acid” as used herein means an amino acid (free amino acid, ie, not an amino acid in a peptide or in a protein sequence). 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, distributing, delivery vehicles, such as preservatives, stabilizers, emulsifiers, suspending agents, thickeners, prolonged delivery regulators, the choice and ratio of which depends on the nature and method of administration and doses ings. 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 achieved using a variety of antibacterial and antifungal agents, for example, such as parabens, chlorobutanol, sorbic acid and the like. The composition may also include isotonic agents, for example, sugars, sodium chloride and the like. The prolonged action of the composition can be achieved using 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 also mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). Examples of excipients are lactose, milk sugar, sodium citrate, calcium carbonate, calcium phosphate and the like. The pharmaceutical composition for oral, sublingual, transdermal, intramuscular, intravenous, subcutaneous, local or rectal administration of the active principle, alone or in combination with another active principle, can be administered to animals and humans in a standard administration form in the form of a mixture with traditional pharmaceutical carriers. Suitable unit dosage forms include parenteral forms, implants and transdermal systems.

“Medicinal product (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 formulations intended to restore, correct, or change physiological functions in humans and animals, as well as for the treatment and prevention of diseases, diagnosis, 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. As used herein to “alleviate” a disease, disease, or condition, it means reducing the severity and / or frequency of symptoms of the disease, disorder, or condition. In addition, references to “treatment” contained herein include references to therapeutic, palliative, and prophylactic therapy.

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

The term "violation" means any condition that can be improved as a result of treatment of the present invention. The definition of this term includes chronic and acute disorders or diseases, including pathological conditions that cause a mammal's predisposition to the occurrence of this violation. Non-limiting examples of diseases to be treated include benign and malignant tumors; leukemia and lymphoid malignancies, in particular breast, ovarian, stomach, endometrial, salivary, lung, kidney, colon, thyroid, pancreas, prostate or bladder cancers; neural, glial, astrocytal, hypothalamic and other glandular, macrophage, epithelial, stromal and blastocellular 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, for example, to the action of lymphocytes, antigen-presenting cells, phagocytic cells, granulocytes and soluble macromolecules produced by these cells or liver cells (including antibodies, cytokines and complement, invasive pathogens, cells or tissues infected with pathogens, cancer cells or, in cases of autoimmunity or pa, resulting from selective damage, destruction or elimination from the human body ologicheskogo inflammation, normal human cells or tissues).

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

This definition covers, but is not limited to, rheumatoid arthritis, arthrosis, juvenile chronic arthritis, septic arthritis, Lyme arthrosis, psoriatic arthritis, reactive arthritis, spondylarthropathy, systemic lupus erythematosus, Crohn’s disease, ulcerative colitis, inflammatory bowel disease, diabetes mellitus, thyroiditis, asthma, allergic diseases, psoriasis, atopic dermatitis, scleroderma, transplant versus host disease, transplant rejection, acute or chronic immune diseases associated with transplant tacia, sarcoidosis, Kawasaki disease, Graves disease, nephrotic syndrome, chronic fatigue syndrome, Wegener granulomatosis, Genoch-Shenlein 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 cirrhosis, hemolytic anemia, adult (acute) respiratory distress syndrome, al pestia, focal alopecia, seronegative arthropathy, arthropathy, Reiter’s disease, psoriatic arthropathy associated with ulcerative colitis, arthropathy, atopic allergies, autoimmune bullous diseases, pemphigus vulgaris, foliate pemphigus, hematolytic anemic hemolytic anemic, linear IgE pernicious anemia, juvenile pernicious anemia, arthritis, primary sclerosing hepatitis A, cryptogenic autoimmune hepatitis, fibrosing lung diseases, creep toogenic fibrotic alveolitis, post-inflammatory interstitial lung disease, interstitial pneumonitis, chronic eosinophilic pneumonia chronic, postinfectious interstitial lung disease, gouty arthritis, autoimmune hepatitis, autoimmune hepatitis type I, hepatitis, hepatitis, lipitis hepatitis or lipitis , psoriasis type I, psoriasis type II, ideopathic leukopenia, autoimmune neutropenia, renal NOS diseases [renal NOS-disease], hl rollonephritis, microscopic renal vasculitis, discoid lupus erythematosus, ideopathic or male NOS fertility, [autoimmunity to sperm], all subtypes of multiple sclerosis, sympathetic ophthalmia, secondary pulmonary hypertension in connective tissue diseases, Goodpasturitis syndrome, pulmonary heart disease, rheumatic fever, pulmonary fibroid disease rheumatoid spondylitis, ankylosing spondylitis, Still's disease, systemic sclerosis, Schengen syndrome, Takayasu syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenia, autoimmune thyroiditis, hyperthyroidism, Hohimoto’s disease, autoimmune atrophic hypothyroidism, primary mexidema, phacogenic uveitis, primary vasculitis, vitiligo, acute liver disease, chronic liver disease, allergies, asthma, mental illnesses (including depression, Thyroid disease) type and Th1 type, conjunctivitis, allergic contact dermatitis, allergic rhinitis, alpha-1-antitrypsin deficiency, amyotrophic lateral sclerosis, anemia, cystic fibrosis, disease studies associated with cytokine therapy, demyelinating diseases, dermatitis, iridocyclitis / uveitis / optic neuritis, damage to ischemic reperfusion, ischemic stroke, juvenile rheumatoid arthritis, autoimmune immune loss, autoimmune immune doppler, and immune-dysfunctional lymphoma . An antibody can also treat any combination of the above disorders.

As used here, the term "violation in which TNFα activity is harmful", includes the inclusion of diseases and other disorders in which the presence of TNFα in a subject suffering from a violation, as shown or assumed, either determines the pathophysiology of the violation, or is a factor that exacerbates the violation. Accordingly, a disorder in which TNFα activity is harmful 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 increasing the concentration of TNFα in the biological fluid of a subject suffering from a violation (for example, increasing the concentration of TNFα in 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 harmful. The use of antibodies and antibody fragments corresponding to the violation 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, weakening of the myocardium, vascular leakage syndrome, organ necrosis, stimulation of toxic secondary neurotransmitter release and activation of the blood coagulation cascade (see, for example, Moeller A., et al. ( 1990), Cytokine 2: 162-169; U.S. Patent No. 5,231,024 to Moeller et at .; European Patent No. 2,60610 B1 to 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, septicemia caused by gram-negative bacteria, 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 reduce sepsis, such as an interleukin-1 inhibitor (described in PCT Publication No. WO 92/16221 and WO 92/17583), the cytokine interleukin-6 (see, for example, PCT publication No. WO 93/11793) or platelet activation factor antagonist (see, for example, European patent application No. EP 374510). Other methods of combination therapy for the treatment of sepsis are discussed below in subsection III.

In addition, in a preferred embodiment, an anti-TNFα antibody and antibody fragments of the invention are administered to a person from a subset of sepsis patients 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 found to play a role in the pathophysiology of a number of autoimmune diseases. For example, TNFα was involved in the activation of tissue inflammation and caused joint destruction in rheumatoid arthritis (see, e.g., Moeller A. et al. (1990), Cytokine 2: 162-169; U.S. Patent No. 5,231,024 Moeller et al .; European Patent No. 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 also participated in the stimulation of cell islet death and the formation of insulin resistance in diabetes (see, for example, Tracey and Cerami, supra; PCT publication No. WO 94/08609). TNFα also participated in the appearance of cytotoxicity against oligodendrocytes and the induction of inflammatory plaques in multiple sclerosis (see, for example, Tracey and Cerami, supra). Chimeric and humanized murine antibodies against TNFα have been clinically tested for the treatment of rheumatoid arthritis (see, for example, 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 others. Typically, an antibody or antibody fragment is administered systemically, although topical application of the antibody or antibody fragment in the area of inflammation (for example, local injection into joints in rheumatoid arthritis or external use in diabetic ulcers, alone or in combination with a cyclohexanilidene derivative, may be successful for some disorders). 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 used in the treatment of autoimmune diseases, as described further in subsection III.

C. Infectious diseases

Tumor necrosis factor was involved in the appearance of biological effects observed in a number of infectious diseases. For example, TNFα was involved in the development of cerebral inflammation and capillary thrombosis and heart attack in malaria. TNFα also participated in the development of inflammation of the brain, the induction of the destruction of the blood-brain barrier, the development of septic shock syndrome and activation of venous infarction with meningitis. TNFα also participated in the induction of cachexia, stimulation of the proliferation of viruses and the formation of damage to the central nervous system in acquired immunodeficiency syndrome (AIDS). Accordingly, antibodies and antibody fragments corresponding to 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, for example, European patent application No. EP 230574), as well as a secondary infection of 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 the symptoms associated with infectious diseases, including fever and myalgia due to infection (such as influenza), and secondary cachexia during infection (e.g. secondary to AIDS or αC).

D. Transplantation

Tumor necrosis factor was considered as a key mediator in transplant rejection and graft versus host disease (GVHD) and in the formation of an adverse reaction, which was noted when a rat OKTZ antibody directed against the T-cell receptor-CDR complex was used to inhibit renal transplant rejection (see ., for example, Eason JD et al. (1995) Transplantation 59: 300-305; Suthanthiran M. and Strom T. B. (1994) New Engl. J. Med. 331: 365-375). Accordingly, 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 may be used alone, it is more preferable to use them in combination with one or more agents that inhibit the immune response against an 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 surface molecules of a CD25 cell (receptor-β interleukin-2), 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 basic immunosuppressive agents, such as cyclosporin A or FK506.

E. Malignant neoplasms

Tumor necrosis factor was involved in the induction of cachexia, stimulation of tumor growth, increased metastatic potential and the appearance of cytotoxicity in malignant neoplasms. Accordingly, antibodies or antibody fragments of the invention are used in the treatment of malignant neoplasms to inhibit tumor growth or metastases and / or to facilitate secondary cachexia in malignant neoplasms. An antibody or antibody fragment may be administered systemically or topically to a tumor area.

F. Pulmonary disorders

Tumor necrosis factor was involved in the pathophysiology of adult respiratory distress syndrome (αDS), including stimulation of endothelial leukocyte activation, the orientation of cytotoxicity to pneumocytes and the induction of vascular leakage 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 used in the treatment of pulmonary disorders, as described in subsection III below.

G. Intestinal Disorders

Tumor necrosis factor was involved in the pathophysiology of inflammatory intestinal 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 murine antibodies against TNFα have been clinically tested to treat Crohn's disease (van Dullemen H.M. et al. (1995) Gastroenterology 109: 129-135). A human antibody or antibody fragment of the invention can also be used to treat intestinal disorders, such as idiopathic inflammatory bowel disease, which includes two syndromes - Crohn's disease and ulcerative colitis. An 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. Heart disorders

Antibodies or antibody fragments of the invention can also be used to treat a variety of cardiac disorders, including cardiac ischemia (see, for example, European patent application EP 453898) and heart failure (heart muscle weakness) (see, for example, PCT publication No WO 94/20139).

I. Others

Antibodies or antibody fragments of the invention can also be used to treat various disorders in which TNFα activity is harmful. Examples of other diseases and disorders in which TNFα activity is involved in 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. Ex p. 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 injuries (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, for example, 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 refers to a treatment that is not administered sequentially without interruption, but which is rather periodic in nature.

In the present description and in the following claims, unless the context provides otherwise, the words “have”, “include” and “contain” or their variations, such as “has”, “having”, “includes”, “including”, “ contains "or" containing ", it should be understood as including the indicated whole or group of whole, but not the exclusion of any other whole or group of whole.

DETAILED DESCRIPTION OF THE INVENTION

Water composition

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

Adalimumab and infliximab are examples of a recombinant monoclonal anti-TNFα antibody. 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 comprises 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 in 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, recombinant monoclonal anti-TNFα antibodies or antigen-binding portions thereof are typically provided in a therapeutic amount of 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.

A contemplated aqueous composition comprises a suitable buffer solution in combination with other pharmaceutically acceptable excipients that stabilize the pharmaceutical preparation. Suitable buffer solutions that can be used are selected from the group that is 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-based buffering agent (or buffer system). In a preferred embodiment, a suitable buffer comprises 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 mainly used in concentrations from about 1 mm to about 100 mm. In a preferred embodiment, the concentration of the buffer 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 in the range from 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 5.5. In a more preferred embodiment, the pH is maintained at about 5.0.

Said aqueous composition includes suitable surfactants which are pharmaceutically acceptable excipients used to protect the protein compositions from various stresses, such as stirring, shear, heat, freezing, etc. Suitable surfactants include, but are not limited to, polysorbate or poloxamer, 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 suitable surfactant is a combination of polysorbate and poloxamer. In a more preferred embodiment, the polysorbate is polysorbate 20 or polysorbate 80 (including those sold under the trademark 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 polysorbate 80 and up to 10 mg / ml 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 that are pharmaceutically acceptable excipients and which protect the pharmaceutically active ingredient from chemical and / or physical degradation during production, storage and use. In one embodiment, stabilizers include, but are not limited to, amino acids, oligosaccharides, or their suitable derivatives or mixtures. In another preferred embodiment, a suitable stabilizer is trehalose. In another preferred embodiment, a suitable stabilizer is proline. In another preferred embodiment, a suitable stabilizer is a combination of trehalose and proline. In another preferred embodiment, a suitable stabilizer is a combination of trehalose and arginine.

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

In some embodiments, 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 selected as the stabilizer. In some embodiments, trehalose is present in an amount up to 150 mg / ml and arginine in an amount up to 0.5 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 0.5 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, the amino acid that can be used as stabilizers is proline.

In some embodiments, proline is provided 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 provided 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 comprises from 50 to 200 mg / ml of recombinant monoclonal anti-TNFα 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 from 0.05 to 0.5 mg / ml arginine, acetic acid ice. to pH 5.0, from 0.1 mg / ml to 1 mg / ml of polysorbate 20. In a preferred embodiment of the aqueous composition, the recombinant monoclonal anti-TNFα antibody is adalimumab. In a more preferred embodiment, the composition comprises from 50 to 150 mg / ml adalimumab, 0.436 mg / ml sodium acetate trihydrate, 80 mg / ml trehalose dihydrate, acetic acid ice. up to pH 5.0, 0.5 mg / ml polysorbate 20. In a more preferred embodiment, 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 a pH of 5.0, 0.5 mg / ml polysorbate 20. In a more preferred embodiment, the composition comprises from 50 to 150 mg / ml adalimumab, 0.436 mg / ml sodium acetate trihydrate, 27 mg / ml proline, acetic acid ice. up to pH 5.0, 0.5 mg / ml polysorbate 20. In a more preferred embodiment, 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 5.0, 0.5 mg / ml polysorbate 20.

In some embodiments, the aqueous composition comprises from 50 to 200 mg / ml of recombinant monoclonal anti-TNFα 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 from 0.05 to 0.5 mg / ml arginine, acetic acid ice. to pH 5.0, from 0.1 mg / ml to 1 mg / ml of polysorbate 80. In a preferred embodiment of the aqueous composition, the recombinant monoclonal anti-TNFα antibody is adalimumab. In a more preferred embodiment, the composition comprises from 50 to 150 mg / ml adalimumab, 0.436 mg / ml sodium acetate trihydrate, 80 mg / ml trehalose dihydrate, acetic acid ice. up to pH 5.0, 0.5 mg / ml polysorbate 80. In a more preferred embodiment, 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. up to pH 5.0, 0.5 mg / ml polysorbate 80. In a more preferred embodiment, the composition comprises from 50 to 150 mg / ml adalimumab, 0.436 mg / ml sodium acetate trihydrate, 27 mg / ml proline, acetic acid ice. up to pH 5.0, 0.5 mg / ml polysorbate 80. In a more preferred embodiment, 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. up to pH 5.0, 0.5 mg / ml polysorbate 80.

In some embodiments, the implementation, the aqueous composition contains from 50 to 200 mg / ml of recombinant monoclonal antibodies to TNFα, 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 to 150 mg / ml trehalose and from 0.05 to 0.5 mg / ml arginine, acetic acid ice. to pH 5.0, from 0.1 mg / ml to 1 mg / ml of poloxamer 188. In a preferred embodiment, the recombinant monoclonal anti-TNFα antibody is adalimumab. In a more preferred embodiment, the composition comprises from 50 to 150 mg / ml adalimumab, 0.436 mg / ml sodium acetate trihydrate, 80 mg / ml trehalose dihydrate, acetic acid ice. to pH 5.0, 1 mg / ml poloxamer 188. In a more preferred embodiment, the composition comprises 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. up to pH 5.0, 1 mg / ml poloxamer 188. In a more preferred embodiment, the composition comprises from 50 to 150 mg / ml adalimumab, 0.436 mg / ml sodium acetate trihydrate, 27 mg / ml proline, acetic acid ice. up to pH 5.0, 1.0 mg / ml poloxamer 188. In a more preferred embodiment, the composition comprises 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 5.0, 1.0 mg / ml poloxamer 188.

The composition may further include one or more other suitable excipients that are well known to those skilled in the art.

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

In some embodiments, the implementation of the liquid composition remains stable during storage in the sense that it does not occur any further processes of aggregation of the protein or its modifications in comparison with the indicator of stability at zero time point.

Methods of treatment and use of aqueous composition

In another embodiment, the invention relates to a method for 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 and severe), active psoriatic arthritis, active ankylosing spondylitis, chronic plaque psoriasis (moderate or severe), ulcerative colitis (moderate and severe ), axial spondylitis, active purulent hydradenitis, juvenile idiopathic arthritis, Crohn’s disease (moderate or severe), uveitis, active enthesitis ovanny arthritis. Additional diseases or disorders that can be treated with the compositions of the present invention include the diseases described in US patent No. 6090382 and 8216583, the corresponding parts of which are incorporated herein by reference.

The provided pharmaceutical compositions can be administered to a subject 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 a site when the site is accessible by surgery; or through topical application.

In one embodiment, the invention relates to a method for 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 provided compositions depends on the condition to be treated, the severity of the condition, previous therapy and the patient’s medical history and response to the therapeutic agent. The appropriate dose can be adjusted according to the decision of the attending physician so that it can be administered to the patient once or through several administrations.

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

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

If the dose should be administered more than once a week, the illustrative dose range is the same as the above dose ranges or less, and it is preferably administered two or more times a week with a dose range of 25-100 mg / dose.

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

The dose can be administered once a week, once every two weeks or divided by several weeks (for example, from 2 to 8).

In one embodiment, adalimumab is administered at a dose of 40 mg through a single subcutaneous (s / c) injection.

In some cases, improvement in a 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. In order to achieve the desired degree of improvement, treatment for longer periods may be necessary. For incurable chronic conditions, the treatment regimen can be continued indefinitely. For pediatric patients (ages 4-17), a suitable treatment regimen may include administering a dose of adalimumab from 0.4 mg / kg to 4 mg / kg once or several times a week.

In another embodiment, the pharmaceutical compositions of the invention can be prepared in bulk form, and essentially the components of the pharmaceutical composition are present in amounts higher than may be required for administration, and are suitably diluted prior to administration.

The pharmaceutical compositions may be administered as a single therapeutic agent or in combination with additional therapeutic agents as necessary. 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. Another active agent can be administered before, during or after the administration of the pharmaceutical compositions of the present invention. Another active agent may be administered as part of the composition provided or, alternatively, as a separate composition.

The administration of the provided pharmaceutical compositions can be carried out in various ways, including parenteral, oral, buccal, nasal, rectal, intraperitoneal, intradermal, transdermal, subcutaneous, intravenous, intraarterial, intracardiac, intraventricular, intracranial, intramuscular, intramuscular introduction.

The pharmaceutical compositions of the present invention are particularly suitable for parenteral administration, i.e. subcutaneously, intramuscularly, intravenously, intraperitoneally, in the spinal cord, in the joints, intrasynovial and / or intrathecal. Parenteral administration may be via bolus injection or continuous infusion. Injectable pharmaceutical compositions may be in unit dosage form, for example, but not limited to, in ampoules, vials, prefilled syringes, or in 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 new methods, for example, 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 for not yet discovered methods of administration. Also see Langer, 1990, Science, 249: 1527-1533.

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

The pharmaceutical compositions, if desired, may be provided in a vial, package, or dispenser, which may contain one or more unit dosage forms containing the active ingredient. In one embodiment, the dispensing device may comprise a syringe containing a single dose of a liquid composition 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 can vary over a wide range, but typically within a range from about 1 to about 200 mg / ml of the aqueous composition. The kit may also be accompanied by instructions for use.

Production method

A method of obtaining the above compositions includes the addition of acetate buffering agents to the aqueous phase, followed by adding, in any sequence, the following components: an osmolytic and / or stabilizer selected from the group: trehalose, proline or a combination thereof, a recombinant monoclonal anti-TNFα antibody, surface-active a substance selected from the group: polysorbate 20, polysorbate 80, poloxamer 188, or a combination thereof.

Methodologies

1. Determination of protein concentration in the studied samples.

Protein concentration was determined using UV spectrophotometry at a wavelength of λ = 280 nm in UV spectrophotometry plates.

Each sample was diluted with a solution of the corresponding placebo 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 appropriate placebo solution was used as the comparison solution.

The concentration of protein (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 - total coefficient of dilution of the sample

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

2. Replacement of the buffer solution and concentration of samples

Diafiltration and concentration of samples was carried out in concentration cells of Stirred Cell (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 resulting solutions were filtered through a 0.45 μm Durapore filter.

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

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

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

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

• Analytical model: Protein analysis.

• Holding at a temperature before measuring for 30 seconds.

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

The point of aggregation of the studied proteins was determined on a Zetasizer Nano ZSP instrument. For this, the solution was placed in a quartz dedusted cuvette, which was gradually heated in the device with a constant measurement of the intensity of the scattered light in the Temperature trend measurement mode.

• Analytical model: Protein analysis.

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

• Holding at a temperature before measuring for 30 seconds.

• At each point, the average 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 method of "Thermostress 50 ° C."

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

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

The test samples were divided into 2 parts of 200 μl and placed in glass vials of 1 vial for each composition, placed in a refrigerator for storage at + 4 ° C, the rest were placed in a thermo shaker and shaken at a speed of 800 rpm at a temperature of 2 - 8 ° C for the specified time. After stress ended, the tubes were removed from the thermal shaker 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 stored in a refrigerator 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 stress ended, the tubes were removed from the freezer, kept at room temperature until the contents were completely thawed, the solutions were mixed with Vortex and transferred to the analysis according to the specification.

8. Determination of sample purity using gel filtration (HF) high performance liquid chromatography (HPLC) .

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

Column Temperature: 25ºС Mobile phase flow rate: 0.7 ml / min Volume of injection: 20 μl Sample Concentration: 5 mg / ml Detector Wavelength: 220 nm Elution Duration: 23 min Mobile phase: Used disodium hydrogen phosphate 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 samples on a Caliper LabChip GX II instrument .

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 solution of carboxypeptidase B (CpB) with a concentration of 5 mg / ml was added. Stirred and incubated for 2 hours at a temperature of 37 ° C. Test samples were dialyzed against three water changes in Amicon Ultra centrifuge tubes and concentrated to 2 mg / ml.

9.2. Preparation of working solutions.

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

9.3. Chip preparation.

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

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

10. Determination of sample purity on a Caliper Labchip GX II instrument under reducing and non-reducing conditions .

10.1. Preparation of analyzed samples.

To prepare the denaturing and reducing solutions, 700 μl of HT Protein Express Sample Buffer was used with the addition of 24.5 μl of 1M DTT for buffer under reducing conditions and 24.5 μl of 1M 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 to the other. Samples were denatured at 100 ° C for 5 minutes. Vortex tubes were mixed, 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.

Preparation of working solutions and preparation of the chip was carried out according to a standard method using the HT Protein Express Reagent Kit. Running an analysis is a standard operation. HT Protein Express 200 analysis method.

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

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

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

The electrophoresis chamber was assembled and installed in accordance with the instruction manual for the device for conducting vertical electrophoresis. Samples were prepared by diluting the 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 in a ratio of 3: 1 (volume / volume) were mixed with a 4-fold buffer solution for applying samples containing 2-mercaptoethanol (reducing conditions) and not containing 2-mercaptoethanol (non-reducing conditions) were mixed. The resulting solutions were incubated at a temperature of (99 ± 1) ° С for 3 min (samples containing 2-mercaptoethanol) and at a temperature of (99 ± 1) ° С for 1 min (samples containing 2-mercaptoethanol). The solutions were cooled to room temperature, mixed and applied to the PAG wells under a layer of electrode buffer solution.

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

After electrophoresis, the gels were separated from the glasses and the proteins were fixed in the fixing solution for 16-18 hours at room temperature. Then, staining of the gels (in a solution of acid blue 83) was carried out and washing to obtain a clear visualization of the bands. 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 WEHI-13 var culture (fibrosarcoma, Mus musculus), a culture variant that is highly sensitive to TNFα in the presence of Actinomycin D. Sample preparation was performed 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 CKO (quantitative determination medium).

The test antibody sample was diluted to 50 μg / ml with a dilution step of not more than 20 and placed in a robotic station. Using TecanEvo200 in culture plates, three independent dilutions of CO and AI were prepared in the range of 10,000 - 0.5 ng / ml, and 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 a temperature of (37 ± 1) ºС, 5% CO ₂ in humidified air for 20-24 hours.

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

For a better understanding of the invention, the following examples are provided. These examples are 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 referred to in this specification are incorporated herein by reference. Although the aforementioned invention has been described in some detail by way of illustration and example in order to avoid ambiguous interpretation, it will be readily understood by those skilled in the art that certain changes and modifications may be made without departing from the spirit and scope of the enclosed embodiments. the implementation of the invention.

Examples

Example 1. The choice of the nature of the buffer solution

The investigated compounds.

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

Determination of colloidal stability by PEG-aggregation.

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

Table 1. The average optical density of the solutions after preparation.

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

The analysis was performed according to procedure 4. The results are presented in table 2 and in FIG. 5 - 9.

Determination of thermal stability during prolonged exposure by thermostress method 50остС.

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

Results.

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

The conclusions of example 1.

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

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

Adalimumab in the composition of Humir 1 has less thermal stability compared to the recommended composition. Minimal colloidal and thermal stability was noted for the composition based on 5 mM citrate buffer solution, pH 5.0.

Example 2. Optimization of 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 prior art has shown, most patents and patent applications for pharmaceutical compositions containing adalimumab protect solutions with pH from 4.0 to 8.0. Thus, the aim of this section was to investigate the possibility of obtaining stable compositions containing acetate ions with a pH of less than 4.

The investigated compounds.

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. Data on 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 with thermostat method 50ºС.

The analysis was performed according to method 5.

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

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 shows the quality indicators of samples before and after thermostress, shake test and cryoconcentration.

2.6. The conclusions of example 2.

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

• The most stable sample among all studied is adalimumab in 5 mM acetate buffer solution with pH 5.0. He showed a minimal change in purity as well as acid-base profile during stress. However, to create an adalimumab composition, it is possible to use solutions with a larger buffer capacity.

Example 3. The choice of osmotic agent and stabilizers

According to the results of the first part of the study, adalimumab showed the best stability in acetate buffer solution with pH 5.0. Therefore, this composition was taken as the basis for further development.

The investigated compounds.

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 procedure 3. Data on the average optical density of the solutions are presented in Table 5. The results are also presented in FIG. 3 to 4.

Table 5. Average optical density of adalimumab solutions after preparation.

Table 6. The average optical density of the solutions after 24 hours of incubation at 25 ° C.

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 with thermostat method 50ºС.

The analysis was performed according to method 5.

3.5. The conclusions of example 3.

Based on the results of this study, promising auxiliary substances for adalimumab compositions are: trehalose dihydrate, glycine, proline, methionine and arginine hydrochloride. As surface-active substances for screening the final pharmaceutical composition are taken: polysorbate 80, polysorbate 20, poloxamer 188.

Adverse effects on the colloidal stability of adalimumab are: sodium chloride, arginine, EDTA, lysine, guanidine.

Adverse effects on the thermal stability of adalimumab are: sodium chloride, lysine, guanidine. The total cysteine content 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 excipient.

Example 4. The choice of the final pharmaceutical composition

According to the results of the third part of the study, the following promising auxiliary substances were selected: trehalose dihydrate, glycine, proline, methionine and arginine hydrochloride. As surface-active substances for screening the final pharmaceutical composition are taken: polysorbate 80, polysorbate 20, poloxamer 188.

4.1. Determination of thermal stability during prolonged exposure with thermostat method 50ºС.

The analysis was performed according to method 5.

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

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 table 8, which clearly shows the quality indicators of samples before and after thermostress, shake test and cryoconcentration.

Table 8

The conclusions of example 4.

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

The composition of Humir 2 has thermal stability comparable to alternative compositions (loss of purity during thermostress 0.38%). However, during freezing-thawing of adalimumab in this composition, a noticeable aggregation of protein occurs: the increase in aggregates on gel-filtration HPLC is more than 7%, which makes the use of the drug unsuitable for accidental freezing. A noticeable increase in impurities after freezing is also noted using DLS.

Alternative compositions based on acetate buffer solution with the addition of a number of auxiliary substances showed good thermal and colloidal stability during stress. Gel-filtration HPLC did not reveal a difference in the growth of impurities of these compositions. There was also 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 hydrochloride in arginine reduces the growth of impurities during the shake test, and the presence of methionine increases the accumulation of impurities during shaking.

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

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

To confirm the stability of the recommended pharmaceutical compositions, adalimumab at a concentration of 50 mg / ml, 100 mg / ml and 150 mg / ml was subjected to thermal stress at 50 ° C for 6 days.

The investigated compounds.

5.1. Determination of thermal stability during prolonged exposure with thermostat method 50ºС.

The analysis was performed according to method 5.

General conclusions

1. Screening of the 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 the buffer capacity of the solution, the choice of osmolytics and stabilizers, the choice of the final pharmaceutical composition and confirmation of the final pharmaceutical composition of highly concentrated adalimumab.

2. In the process, the thermal and colloidal stability of adalimumab in more than 85 formulations was studied using the following methods: PEG aggregation, shake test, freezing-defrosting, thermal stress followed by analysis of the degree of turbidity (UV spectrophotometry), purity (HPLC HPLC , DLS and gel electrophoresis, including using the LabChip, Caliper system), as well as the acid-base profile (LabChip, Caliper and IO HPLC).

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

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

5. Pharmaceutical compositions of adalimumab based on acetate buffer solution 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 with the original compositions and are the subject of the present invention.

6. During the process, when obtaining the 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 with surfactant, as well as when washing the ultrafiltration unit after concentration.

Claims (134)

1. An aqueous pharmaceutical composition for intravenous or subcutaneous administration, comprising: a) adalimumab in a concentration of from 50 to 200 mg / ml; b) an acetate ion-based buffering agent (or buffer system), wherein the concentration of the acetate buffer is from 1 to 100 mM; c) trehalose in a concentration of from 25 to 150 mg / ml, and / or Proline in a concentration of 5 to 40 mg / ml, or trehalose in a concentration of 25 to 150 mg / ml and arginine in a concentration of 0.05 to 0.5 mg / ml; d) polysorbate 20 in a concentration of from 0.05 mg / ml to 10 mg / ml, polysorbate 80 in a concentration of 0.05 mg / ml to 10 mg / ml, or poloxamer 188 in a concentration of from 0.05 mg / ml to 10 mg / ml, or a combination thereof. 2. The composition of claim 1, wherein the composition has a pH of 4 to 7. 3. The composition according to p. 1, containing: a) from 50 to 200 mg / ml adalimumab; b) from 0.4 to 1.2 mg / ml sodium acetate trihydrate; c) from 25 to 150 mg / ml trehalose, and / or from 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 5.0; e) from 0.1 mg / ml to 1 mg / ml of polysorbate 80. 4. The composition according to p. 3, containing: a) from 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 5.0; e) 0.5 mg / ml polysorbate 80. 5. The composition according to p. 3, containing: a) from 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 5.0; e) 0.5 mg / ml polysorbate 80. 6. The composition according to p. 3, containing: a) from 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 5.0; e) 0.5 mg / ml polysorbate 80. 7. The composition according to p. 3, containing: a) from 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 5.0; f) 0.5 mg / ml polysorbate 80. 8. The composition according to p. 1, containing: a) from 50 to 200 mg / ml adalimumab; b) from 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 5.0; e) from 0.1 mg / ml to 1 mg / ml of polysorbate 20. 9. The composition according to p. 8, containing: a) from 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 5.0; e) 0.5 mg / ml polysorbate 20. 10. The composition according to p. 8, containing: a) from 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 5.0; e) 0.5 mg / ml polysorbate 20. 11. The composition according to p. 8, containing: a) from 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 5.0; e) 0.5 mg / ml polysorbate 20. 12. The composition according to p. 8, containing: a) from 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 5.0; f) 0.5 mg / ml polysorbate 20. 13. The composition according to p. 1, containing: a) from 50 to 200 mg / ml adalimumab; b) from 0.4 to 1.2 mg / ml sodium acetate trihydrate; c) from 25 to 150 mg / ml trehalose, and / or 5 to 40 mg / ml proline, or from 25 to 150 mg / ml trehalose and from 0.05 to 0.5 mg / ml arginine; d) acetic acid ice. up to pH 5.0; e) from 0.1 mg / ml to 1 mg / ml of poloxamer 188. 14. The composition according to p. 13, containing: a) from 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 5.0; e) 1.0 mg / ml poloxamer 188. 15. The composition according to p. 13, containing: a) from 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 5.0; e) 1.0 mg / ml poloxamer 188. 16. The composition according to p. 13, containing: a) from 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 5.0; e) 1.0 mg / ml poloxamer 188. 17. The composition according to p. 13, containing: a) from 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 5.0; f) 1.0 mg / ml poloxamer 188. 18. A method for the treatment of diseases mediated by TNFα, comprising administering a composition according to claims. 1-17 in an effective amount. 19. The method of claim 18, wherein the disease mediated by TNFα is selected from the group: a) active rheumatoid arthritis (moderate and severe), b) active psoriatic arthritis, c) active ankylosing spondylitis, d) chronic plaque psoriasis (moderate or severe), e) ulcerative colitis (moderate to severe), f) axial spondylitis, g) active purulent hydradenitis, h) juvenile idiopathic arthritis, i) Crohn's disease (moderate or severe), j) uveitis k) active enthesitis-associated arthritis. 20. The use of the composition according to PP. 1-17 for the treatment of diseases mediated by TNFα. 21. The use of claim 20, wherein the disease is selected from the group: a) active rheumatoid arthritis (moderate and severe), b) active psoriatic arthritis, c) active ankylosing spondylitis, d) chronic plaque psoriasis (moderate or severe), e) ulcerative colitis (moderate to severe), f) axial spondylitis, g) active purulent hydradenitis, h) juvenile idiopathic arthritis, i) Crohn's disease (moderate or severe), j) uveitis k) active enthesitis-associated arthritis. 22. A method of obtaining a composition according to any one of paragraphs. 1-17, including the addition of acetate buffer agents to the aqueous phase, followed by the addition, in any sequence, of the following components: a) an osmolytic and / or stabilizer selected from the group: trehalose, proline, arginine, or a combination thereof; b) adalimumab; c) a surfactant selected from the group: polysorbate 20, polysorbate 80, poloxamer 188, or a combination thereof.

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