CN110536698A - Aqueous pharmaceutical composition of recombinant monoclonal anti-TNFα antibody - Google Patents

Abstract

The present invention relates to improved aqueous pharmaceutical compositions of recombinant monoclonal antibodies directed to TNF α and methods for their production. The invention also relates to the use of improved aqueous pharmaceutical compositions of recombinant monoclonal antibodies directed against TNF α for the treatment of TNF α -mediated diseases. The proposed invention makes it possible to prevent the physico-chemical instability indicated in the formation of aggregates and fragments of proteins or in the modification of proteins in solution, and also to prevent instability during freezing/thawing, agitation and shaking.

Description

Aqueous pharmaceutical composition of recombinant monoclonal anti-TNFα antibody

technical field of invention

The present invention relates to medical pharmacology, and in particular to aqueous pharmaceutical compositions and uses of human antibodies that specifically bind or neutralize TNFα.

Background of the Invention

Tumor necrosis factor alpha (TNFa) is a naturally occurring mammalian cytokine that is produced by various types of cells, including monocytes and macrophages, in response to endotoxin or other stimuli. TNFα is a major mediator of inflammatory, immune and pathophysiological responses (Grell, М., et аl. (1995) Cell, 83:793-802).

Soluble TNFα is formed by cleavage of precursor transmembrane proteins (Kriegler, et al. (1988) Cell 53:45-53), and the 17 kilodalton (kDa) secreted polypeptide aggregates to form soluble homotrimeric complexes (Smith, et al. (1987), J. Biol. Chem. 262:6951-6954; Butler, et al. (1986), Nature 320:584; Old (1986), Science 230:630). These complexes then bind to receptors found on a variety of cells. Binds to produce a number of pro-inflammatory effects, including (i) release of other pro-inflammatory cytokines, such as interleukin (IL)-6, IL-8, and IL-1, (ii) release of matrix metalloproteinases, and (iii) upregulation of endothelial Expression of cell adhesion molecules that further amplify the inflammatory and immune cascades by attracting leukocytes into extravascular tissues.

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

Physiologically, TNF[alpha] has also been implicated in the prevention of certain infections (Cerami. et al. (1988), Immunol. Today 9:28). TNFα is released by macrophages that have been activated by lipopolysaccharide of Gram-negative bacteria. Thus, TNFα appears to be a very important endogenous mediator involved in the development and pathogenesis of endotoxic shock associated with bacterial sepsis.

Adalimumab ( AbbVie, Inc.) is a human recombinant monoclonal IgG1 antibody specific for human TNF. This antibody is also known as D2E7. Adalimumab consists of 1330 amino acids and is described and patented in US 6090382 (WO9729131 ), the description of which is hereby incorporated by reference in its entirety. Adalimumab is typically produced in mammalian cell expression systems (such as Chinese hamster cells) by recombinant DNA technology. Adalimumab specifically binds to TNF and neutralizes TNF biological function by blocking its interaction with the TNF receptors p55 and p75 on the cell surface.

Various adalimumab formulations are known in the art (see eg WO2004016286 and WO2012065072).

WO2004016286 describes liquid compositions for stabilizing antibodies for the treatment of TNFα mediated diseases, comprising an antibody such as adalimumab, a buffer system, polysorbate and a solution isotonic agent such as mannitol and sodium chloride. The composition used citrate-phosphate buffer (Humira formulation 1). The above formulations have a number of disadvantages: 1) insufficient colloidal stability of high concentrations of antibody, 2) insufficient stability under heat stress conditions, and 3) aggregation during long-term storage.

WO2012065072 describes liquid compositions for stabilizing antibodies for the treatment of TNFα mediated diseases, comprising antibodies such as adalimumab and mannitol, polysorbate 80 (Humira formulation 2). The above formulations have a number of disadvantages: 1) significant freeze and thaw instability, and 2) low aggregation temperatures.

Therefore, there is a need to provide a new drug comprising an antibody that binds TNFα, such as adalimumab.

The present invention relates to stable aqueous compositions comprising adalimumab that enable long-term storage and also have good tolerance to freeze-thaw and heat 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, an acetate ion based buffer (or buffer system), trehalose or proline acid or a combination thereof or trehalose and arginine, polysorbate 20, polysorbate 80 or poloxamer 188 or a combination thereof.

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

In some embodiments of the composition, the monoclonal antibody concentration is 50-200 mg/mL.

In some embodiments of the composition, the acetate buffer solution is at a concentration of 1-100 mM.

In some embodiments, the pH of the composition is 4-7.

In some embodiments of the composition, the trehalose concentration is 25-150 mg/mL.

In some embodiments of the composition, the proline concentration is 5-40 mg/mL.

In some embodiments of the composition, the arginine concentration is 0.05-1.0 mg/mL.

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

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

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

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

In some embodiments, the composition comprises 50-150 mg/mL adalimumab, 0.436 mg/mL sodium acetate trihydrate, 80 mg/mL trehalose dihydrate, glacial acetic acid (up to pH 5.5), 0.5 mg/mL of polysorbate 80.

In some embodiments, the composition comprises 50-150 mg/mL adalimumab, 0.436 mg/mL sodium acetate trihydrate, 80 mg/mL trehalose dihydrate, 0.1 mg/mL arginine salt acid salt, glacial acetic acid (until pH 5.5), polysorbate 80 at 0.5 mg/mL.

In some embodiments, the composition comprises 50-150 mg/mL adalimumab, 0.436 mg/mL sodium acetate trihydrate, 27 mg/mL proline, glacial acetic acid (up to pH 5.5), 0.5 mg/mL mL of polysorbate 80.

In some embodiments, the composition comprises 50-150 mg/mL adalimumab, 0.436 mg/mL sodium acetate trihydrate, 40 mg/mL trehalose dihydrate, 13.5 mg/mL proline, Glacial acetic acid (until pH 5.5), 0.5 mg/mL polysorbate 80.

In some embodiments, the composition comprises 50-200 mg/mL recombinant monoclonal anti-TNFα antibody, 0.4-1.2 mg/mL sodium acetate trihydrate, 25-150 mg/mL trehalose and/or 5-40 mg/mL mL of proline or 25-150 mg/mL trehalose and 0.05-1.0 mg/mL arginine, glacial acetic acid (until pH 5.0-6.0), 0.1 mg/mL-1 mg/mL polysorbate 80 .

In some embodiments, the composition comprises 50-200 mg/mL recombinant monoclonal anti-TNFα antibody, 0.4-1.2 mg/mL sodium acetate trihydrate, 25-150 mg/mL trehalose and/or 5-40 mg/mL mL of proline or 25-150 mg/mL trehalose and 0.05-1.0 mg/mL arginine, glacial acetic acid (until pH 5.5), 0.1 mg/mL-1 mg/mL polysorbate 20.

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

In some embodiments, the composition comprises 50-150 mg/mL adalimumab, 0.436 mg/mL sodium acetate trihydrate, 80 mg/mL trehalose dihydrate, glacial acetic acid (up to pH 5.5), 0.5 mg/mL of polysorbate 20.

In some embodiments, the composition comprises 50-150 mg/mL adalimumab, 0.436 mg/mL sodium acetate trihydrate, 80 mg/mL trehalose dihydrate, 0.1 mg/mL arginine salt acid salt, glacial acetic acid (until pH 5.5), 0.5 mg/mL polysorbate 20.

In some embodiments, the composition comprises 50-150 mg/mL adalimumab, 0.436 mg/mL sodium acetate trihydrate, 27 mg/mL proline, glacial acetic acid (up to pH 5.5), 0.5 mg/mL mL of polysorbate 20.

In some embodiments, the composition comprises 50-150 mg/mL adalimumab, 0.436 mg/mL sodium acetate trihydrate, 40 mg/mL trehalose dihydrate, 13.5 mg/mL proline, ice Acetic acid (until pH 5.5), polysorbate 20 at 0.5 mg/mL.

In some embodiments, the composition comprises 50-200 mg/mL recombinant monoclonal anti-TNFα antibody, 0.4-1.2 mg/mL sodium acetate trihydrate, 25-150 mg/mL trehalose and/or 5-40 mg/mL mL of proline or 25-150 mg/mL trehalose and 0.05-1.0 mg/mL arginine, glacial acetic acid (until pH 5.0-6.0), 0.1 mg/mL-1 mg/mL poloxamer 188.

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

In some embodiments, the composition comprises 50-150 mg/mL adalimumab, 0.436 mg/mL sodium acetate trihydrate, 80 mg/mL trehalose dihydrate, glacial acetic acid (up to pH 5.5), 1 mg Poloxamer 188/mL.

In some embodiments, the composition comprises 50-150 mg/mL adalimumab, 0.436 mg/mL sodium acetate trihydrate, 80 mg/mL trehalose dihydrate, 0.1 mg/mL arginine salt acid salt, glacial acetic acid (until pH 5.5), Poloxamer 188 at 1 mg/mL.

In some embodiments, the composition comprises 50-150 mg/mL adalimumab, 0.436 mg/mL sodium acetate trihydrate, 27 mg/mL proline, glacial acetic acid (up to pH 5.5), 1 mg/mL Poloxamer 188.

In some embodiments, the composition comprises 50-150 mg/mL adalimumab, 0.436 mg/mL sodium acetate trihydrate, 40 mg/mL trehalose dihydrate, 13.5 mg/mL proline, Glacial acetic acid (until pH 5.5), Poloxamer 188 at 1 mg/mL.

In one aspect, the present invention relates to a method for treating a TNF[alpha]-mediated disease, the method comprising administering an effective amount of the above-described composition.

In some embodiments of the method of treatment, the TNFα-mediated disease is selected from active (moderate to severe) rheumatoid arthritis, active psoriatic arthritis, active ankylosing spondylitis, chronic (moderate to severe) to severe) plaque psoriasis, (moderate to severe) ulcerative colitis, axial spondyloarthritis, active hidradenitis suppurativa, juvenile idiopathic arthritis, (moderate or severe) gram Rhon's disease, uveitis, active enthesitis-related arthritis.

In one aspect, the present invention relates to the use of the above composition for the treatment of TNF[alpha] mediated diseases.

In some embodiments of the use, the disease is selected from active (moderate to severe) rheumatoid arthritis, active psoriatic arthritis, active ankylosing spondylitis, chronic (moderate to severe) plaque psoriasis, (moderate to severe) ulcerative colitis, axial spondyloarthritis, active hidradenitis suppurativa, juvenile idiopathic arthritis, (moderate or severe) Crohn's disease, grapevine Meningitis, active enthesitis-related arthritis.

In one aspect, the present invention relates to a method for producing the above composition, the method comprising adding an acetate buffer to an aqueous phase, followed by adding the following components in any order: an osmotic agent and/or a stabilizer (selected from trehalose, proline or a combination thereof), a recombinant monoclonal anti-TNFα antibody, a surfactant (selected from polysorbate 20, polysorbate 80, poloxamer 188 or a combination thereof).

Brief Description of Drawings

Figure 1. Dependence of optical density at 400 nm of solution on PEG 6000 concentration after preparation.

Figure 2. Dependence of optical density at 400 nm of solution on PEG 6000 concentration after preparation.

Figure 3. Dependence of optical density at 400 nm of solution on PEG 6000 concentration after preparation.

Figure 4. Adalimumab foci in acetate buffer (pH 5.0). _Tag 74.0°C. Dark grey: Z-mean, nm, light grey: intensity, kcps.

Figure 5. Adalimumab foci in citrate buffer (pH 5.0). _Tag 69.5°C. Dark grey: Z-mean, nm, light grey: intensity, kcps.

Figure 6. Adalimumab foci in histidine buffered solution (pH 5.0). _Tag 69.5°C. Dark grey: Z-mean, nm, light grey: intensity, kcps.

Figure 7. Adalimumab aggregation sites in phosphate buffered solution (pH 5.0). _Tag 71.0°C. Dark grey: Z-mean, nm, light grey: intensity, kcps.

Figure 8. Adalimumab foci in Humira Formulation 1 (pH 5.0). _Tag 69.5°C. Dark grey: Z-mean, nm, light grey: intensity, kcps.

Disclosure of the Invention

Definition and General Approach

Terms used in this specification generally have their ordinary meaning in the art in the context of the present invention and in the specific context in which each term is used. Below or elsewhere herein, certain terms used to describe the invention are defined to provide practitioners with additional guidance for describing the invention. Provide synonyms for some terms. The recitation of one or more synonyms does not preclude the use of other synonyms. The use of examples anywhere in this specification, including examples of any terms described herein, is illustrative only and is not intended to limit the scope and meaning of the invention or any exemplary subject matter. The present invention is not limited to the various embodiments presented in this specification.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, this document, including definitions, will control.

The term "adalimumab" is associated with The active pharmaceutical ingredient in is synonymous with a protein considered or intended to be a biosimilar or biomodified variant thereof. Adalimumab is a human recombinant monoclonal IgG1 antibody specific for human TNF. Adalimumab is also known as D2E7. Adalimumab contains two light chains (each with a molecular weight of about 24 kDa) and two IgGl heavy chains (each with a molecular weight of about 49 kDa). Each light chain consists of 214 amino acid residues and each heavy chain consists of 451 amino acid residues. Therefore, adalimumab consists of 1330 amino acids. The term adalimumab is also intended to include commercially available The so-called biosimilar or biomodified adalimumab protein variants used in For example, when commercially available A variant with commercially available substantially the same pharmacological action, even though it may exhibit a Similar, if not identical, certain physical properties, such as glycosylation profile and acid-base profile, may also be acceptable to the FDA.

For the purposes of this application, the term "adalimumab" also includes minor modifications in amino acid structure (including deletions, additions and/or substitutions of amino acids) or in glycosylation properties and acid-base distribution (which are not significant Adalimumab that affects the function of the polypeptide). The term "adalimumab" includes All forms and formulations of , including (but not limited to) concentrated formulations; injectable ready-to-use formulations; formulations reconstituted with water, alcohol and/or other ingredients, etc.

The term "human TNFα", also known as hTNFα or hTNF or TNFα, means a human cytokine in a 17kDa secreted form and a 26kDa membrane-associated form whose biologically active form consists of trimers of non-covalently bound 17kDa molecules . 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 rhTNFα is intended to include recombinant human TNFα, which can be prepared by standard recombinant expression methods or commercially available (R&D Systems, catalog. No. 210-TA, Minneapolis, Minn.).

The term "antibody" as used herein refers to an immunoglobulin-type molecule consisting of four polypeptide chains, two heavy (H) and two light (L) chains interconnected by internal disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region consists of 3 domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL consists of 3 CDRs and 4 FRs, arranged from N-terminus to C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In one embodiment of the invention, the formulations comprise antibodies having CDRl, CDR2 and CDR3 sequences similar to those described in US Pat. Nos. 6,090,382, 6,258,562 and 8,216,583.

An antibody or antigen-binding portion thereof may be part of a larger immunoadhesion molecule formed by covalent or non-covalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of streptavidin core regions to prepare tetrameric scFv molecules (Kipriyanov, S.M., et al. (1995) Human Antibodies and Hybridomas 6:93-101) and the use of semi- Cystine residues, marker peptides and C-terminal polyhistidine tags to prepare bivalent and biotinylated scFv molecules (Kipriyanov, S.M., et al. (1994) Mol. Immunol. 31: 1047-1058). Antibody portions (such as Fab and F(ab') ₂ fragments of whole antibodies) can be prepared from whole antibodies using conventional methods, such as papain or pepsin digestion, respectively. Further, antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques described herein.

As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificities (eg, an isolated antibody that specifically binds TNFα is substantially free of antigens other than TNFα that specifically binds bound antibody). However, isolated antibodies that specifically bind TNFα may be cross-reactive with other antigens, such as TNFα molecules from other species. Furthermore, the isolated antibody can be substantially free of other cellular material and/or chemicals.

The term "anti-TNFa antibody" refers to the human anti-TNFa antibodies described herein and in US Pat. Nos. 6,090,382, 6,258,562, 6,509,015, 7,223,394, and 6,509,015. The term anti-TNFα antibody used in the present invention is an anti-TNFα antibody or fragment thereof, including infliximab ( Johnson and Johnson, described in US Pat. No. 5,656,272), CDP571 (humanized monoclonal anti-TNFα IgG4 antibody), CDP870 (humanized monoclonal anti-TNFα antibody fragment), anti-TNF dAb (Peptech), CNTO148 (Goley Limumab; Centocor, see WO 02/12502 and US521206 and US7250165) and adalimumab ( Abbott Laboratories, a human anti-TNF mAb, described in US 6090382 as D2E7). Additional anti-TNF antibodies useful in the present invention are described in US Pat. Nos. 6,593,458, 6,498,237, 6,451,983, and 6,448,380. In another embodiment, the TNFα inhibitor is a TNF fusion protein, such as etanercept ( Amgen; described in WO 91/03553 and WO 09/406476). In another embodiment, the TNFα inhibitor is recombinant TNF binding protein (r-TBP-I) (Serono).

The term "long-term storage" or "long-term stability" is to be understood to mean that the pharmaceutical composition can be stored for 3 months or longer, 6 months or longer, and preferably one year or longer, most A minimum stable shelf life of at least two years is preferred. In general, the terms "long-term storage" and "long-term stability" further include stable storage durations that are at least comparable or better than the stable shelf-life typically required for currently available commercial formulations of adalimumab, without loss of Stability, this loss would render the formulation unsuitable for its intended pharmaceutical application. Long term storage is also understood to mean that the pharmaceutical composition is stored at 2-8°C liquid or frozen (eg at -18°C or lower). It is also contemplated that the composition can be frozen and thawed more than once.

The term "steady state" with respect to long-term storage is understood to mean that the adalimumab contained in the pharmaceutical composition does not lose more than 20%, or more preferably 15%, of the activity relative to the composition at the start of storage, or Even more preferably 10%, and most preferably 5% of its activity.

The terms "excipient" or "additive" are used herein to describe any ingredient other than those previously described herein.

The term "sugar" refers to monosaccharides, disaccharides and polysaccharides or mixtures thereof. Examples of sugars include, but are not limited to, sucrose, trehalose, glucose, dextrose, and the like.

The term "polyol" as used herein refers to an excipient having multiple hydroxyl groups and includes sugar alcohols and sugar acids. Examples of polyols include, but are not limited to, mannitol, sorbitol, and the like.

The terms "buffer", "buffer composition", "buffer agent" as used herein refer to an added composition that enables a liquid antibody formulation, generally through the action of its acid-base conjugated component to resist changes in pH. When referring to the concentration of a buffer, it is intended that the concentration represents the total molar concentration of the free acid or free base form of the buffer. Examples of buffers known in the art and found in the literature include, but are not limited to, histidine, citrate, succinate, acetate, phosphate, phosphate buffered saline, citrate-phosphoric acid Salt buffers and tromethamine based buffers and the like or suitable mixtures thereof.

As used herein, the terms "tonicity agent" or "tonicity adjusting agent" and "osmolyte" or "osmotic agent" refer to excipients that can adjust the osmotic pressure of a liquid antibody formulation. In certain embodiments, the tonicity agent can adjust the osmotic pressure of the liquid antibody formulation to isotonic, making the antibody formulation physiologically compatible with the cells of the subject's body tissue. In yet another embodiment, a "tonicity agent" may help to improve the stability of the antibodies described herein. An "isotonic" formulation is one that has an osmotic pressure comparable to that of human blood. Isotonic formulations typically have an osmolarity of about 250-350 mOsm. The term "hypotonic" describes a formulation with an osmotic pressure lower than that of human blood. Therefore, the term "hypertonic" is used to describe formulations with an osmotic pressure higher than that of human blood. Isotonicity can be measured using, for example, vapor pressure or a frozen-type osmometer. Tonicity agents can be in enantiomeric (eg L- or D-enantiomer) or racemic form, in isomeric form (such as alpha or beta, including alpha, alpha; or beta, beta; or alpha, beta; or β,α), in free acid or free base form, in salt form, in hydrated form (eg monohydrate) or in anhydrous form.

The term "surfactant" (also known as surfactant or detergent or SAS) as used herein refers to an excipient that can alter the surface tension of a liquid antibody formulation. In certain embodiments, the surfactant reduces the surface tension of the liquid antibody formulation. In other embodiments, "surface active substances" may help improve the colloidal stability or solubility of any antibody in the formulation. Surfactants can reduce aggregation of the resulting antibody formulation and/or minimize particle formation in the formulation and/or reduce adsorption. Surfactants can also improve antibody stability during and after freeze/thaw cycles and upon shaking. Surface-active substances can be ionic or non-ionic. Exemplary nonionic surfactants that can be included in the formulations of the present invention include, for example, alkyl poly(ethylene oxide), alkyl polyglucosides (eg, octyl glucoside and decyl maltoside), fatty alcohols such as cetyl Waxy and Oleyl Alcohols, Cocamide MEA, Cocamide DEA and Cocamide TEA. Particular nonionic surfactants that may be included in the formulations of the present invention include, for example, polysorbates such as polysorbate 20 (Tween 20), polysorbate 28, polysorbate 40, polysorbate 60, polysorbate Sorbitan 65, Polysorbate 80 (Tween 80), Polysorbate 81 and Polysorbate 85; Poloxamers such as Poloxamer 188 (KolliphorP188), Poloxamer 407; Polyethylene glycol Alcohol-polypropylene glycol; or polyethylene glycol (PEG), ethylene glycol-propylene glycol copolymer (eg, pluronics PF68, etc.).

The term "lyophilized" as used herein refers to a formulation that has undergone a process known in the art as freeze-drying, which involves freezing the formulation and subsequent removal of ice from the frozen contents.

The term "amino acid" as used herein refers to amino acids (free amino acids, ie not in peptide or protein sequences). 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 amino acid, tryptophan, serine, cysteine, methionine and proline.

"Pharmaceutical composition" means comprising an antibody of the invention and at least one selected from the group consisting of pharmaceutically acceptable and pharmacologically compatible excipients, solvents, diluents, carriers, additives, dispersants, delivery agents, The composition of components such as preservatives, stabilizers, emulsifying agents, suspending agents, thickening agents, prolonged delivery modifiers, the selection and ratio of which will depend on the nature and method of administration and administration. Exemplary suspending agents include ethoxylated isostearyl alcohols, polyoxyethylenes, sorbitol and sorbitol esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth, and mixtures thereof. Protection from the action of microorganisms can be provided by a variety of antibacterial and antifungal agents such as parabens, chlorobutanol, sorbic acid, and the like. The compositions may also contain isotonic agents such as sugars, sodium chloride, and the like. Prolonged action of the compositions can be brought about by agents which delay absorption of the active agent, such as aluminum monostearate and gelatin. Exemplary suitable carriers, solvents, diluents and delivery agents include water, ethanol, polyols and mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Exemplary excipients include lactose, milk sugar, sodium citrate, calcium carbonate, calcium phosphate, and the like. Pharmaceutical compositions of an active agent (alone or in combination with another active agent) for oral, sublingual, transdermal, intramuscular, intravenous, subcutaneous, topical or rectal administration, as a combination with conventional pharmaceutical carriers The mixtures are administered to animals and humans in unit dosage form. Suitable unit dosage forms include parenteral forms, implants and transdermal systems.

"Drugs (preparations)" are those in the form of tablets, capsules, powders, lyophilisates, injections, infusions, ointments and others intended to restore, correct or alter the physiological functions of humans and animals, and provide disease treatment and Substances (or mixtures of substances in the form of pharmaceutical compositions) present in finished dosage forms for prophylaxis, diagnosis, anesthesia, contraception, cosmetic, etc.

"Treat," "treating," and "treatment" refer to methods for reducing or eliminating a biological disorder and/or at least one of its accompanying symptoms. As used herein, "reducing" a disease, disorder or condition means reducing the severity and/or frequency of symptoms of the disease, disorder or condition. Additionally, references herein to "treatment" include references to curative, palliative and prophylactic treatment.

In one aspect, the subject or patient to be treated is a mammal, preferably a human subject. The above subjects can be male or female of any age.

The term "disorder" refers to any condition that can be ameliorated by the treatment of the present invention. The term is defined to include both chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to the disorder. Non-limiting examples of diseases to be treated include benign and malignant tumors; leukemia and lymphoid malignancies, especially breast, ovarian, gastric, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreas Carcinoma, prostate or bladder cancer; neural, glial, astrocyte, hypothalamic and other glandular, macrophage, epithelial, stromal and blastocoel disorders; inflammatory, angiogenic and immune disorders. Preferred disorders to be treated according to the present invention are autoimmune diseases.

The terms "immune response", "autoimmune response", "autoimmune inflammation" refer to, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules (including those produced by selective Injury, destruction or elimination of human invasive pathogens, pathogen-infected cells or tissues, cancer cells, or normal human cells or tissues in the case of autoimmunity or pathological inflammation The role of antibodies, cytokines and complement produced by .

The term "autoimmune disease" as used herein means a non-malignant disease or disorder that occurs and is directed against self (self) antigens and/or tissues in an individual.

This definition includes, but is not limited to, rheumatoid arthritis, arthropathy, juvenile chronic arthritis, septic arthritis, Lyme joint disease, psoriatic arthritis, reactive arthritis, spondyloarthropathy, Systemic lupus erythematosus, Crohn's disease, ulcerative colitis, inflammatory bowel disease, diabetes, thyroiditis, asthma, allergic disease, psoriasis, atopic dermatitis, scleroderma, graft-versus-host reaction , transplant rejection, acute or chronic immune disease associated with transplantation, sarcoidosis, Kawasaki disease, Grave's disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Hen-Scherer's purpura, microscopic Renal vasculitis, chronic active hepatitis, uvenita, septic shock, toxic shock syndrome, septic syndrome, cachexia, acquired immunodeficiency syndrome, acute transverse myelitis, Huntington's disease, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia, adult (acute) respiratory distress syndrome, alopecia, focal alopecia, seronegative arthropathy, arthropathy, Reiter's disease, Psoriatic arthropathy, arthropathy associated with ulcerative colitis, atopic allergy, autoimmune bullous disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid disease, linear IgA , autoimmune hemolytic anemia, Coombs positive hemolytic anemia, pernicious anemia, juvenile pernicious anemia, arthritis, primary sclerosing hepatitis A, cryptogenic autoimmune hepatitis, pulmonary fibrosis, cryptogenic Fibroalveolitis, post-inflammatory interstitial lung disease, interstitial pneumonia, chronic eosinophilic pneumonia, post-infectious interstitial lung disease, gouty arthritis, autoimmune hepatitis, autoimmune hepatitis type 1 (classic autoimmune hepatitis or lipids), autoimmune hepatitis type 2, osteoarthritis, primary sclerosing cholangitis, psoriasis type 1, psoriasis type 2, idiopathic leukopenia, autoimmune neutropenia Cytopenia, renal NOS disease, glomerulonephritis, microscopic renal vasculitis, discoid lupus erythematosus, idiopathic or male NOS-fertility, [sperm autoimmunity], multiple sclerosis of all subtypes, sexual intercourse Sensory ophthalmia, secondary pulmonary hypertension associated with connective tissue disease, Goodpasture syndrome, pulmonary manifestations of polyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis, ankylosing spondylitis, Still's disease, systemic sclerosis, Sjogren's syndrome, Kaohsiung's disease, autoimmune thrombocytopenia, essential thrombocythemia, autoimmune thyroiditis, hyperthyroidism, Hashimoto's disease , autoimmune atrophic hypothyroidism, primary myxedema, lens-derived uveitis, primary vasculitis, vitiligo, acute liver disease, chronic liver disease, allergy, asthma, psychiatric disorders (including depressive syndromes) and schizophrenia), Th2- and Th1-mediated diseases, conjunctivitis, allergic contact dermatitis, allergic rhinitis, alpha-I antitrypsin deficiency, amyotrophic lateral sclerosis, anemia, Cystic fibrosis, diseases associated with cytokine therapy, demyelinating disease, dermatitis , iridocyclitis/uveitis/ophthalmic neuritis, ischemia-reperfusion injury, ischemic stroke, juvenile rheumatoid arthritis, autoimmune enteropathy, autoimmune hearing loss, autoimmune lymphoid Proliferative syndrome, autoimmune myocarditis, autoimmune premature ovarian failure, and blepharitis. Antibodies can also treat any combination of the disorders listed above.

As used herein, the term "disorder in which TNFα activity is detrimental" includes diseases and other disorders in which the presence of TNFα in a subject with the disorder has been shown or suspected to be responsible for the pathophysiology of the disorder or a factor contributing to the exacerbation of the disorder . Thus, a disorder in which TNFα activity is detrimental is one in which inhibition of TNFα activity is expected to reduce the symptoms and/or progression of the disorder. For example, the disorder can be detected by an increase in the concentration of TNFα in the biological fluid of the subject with the disorder (eg, in the subject's serum, plasma, synovial fluid, etc.) An increase can be detected, for example, using an anti-TNFa antibody as described above. There are many examples of disorders in which TNFα activity is detrimental. The use of disorders-appropriate antibodies and antibody fragments in the treatment of specific disorders is discussed further below:

A. Sepsis

Tumor necrosis factor plays an established role in the pathophysiology of sepsis, and its biological effects include hypotension, myocardial depression, vascular leak syndrome, organ necrosis, stimulation of the release of toxic secondary mediators, and activation of the coagulation cascade (see For example Moeller A., et al. (1990), Cytokine 2:162-169; U.S. Patent No. 5231024 Moeller etat.; European Patent No. 260610B1 Moeller A.; Tracey K.J. and Cerami A. (1994) Annu. Rev. Med 45:491-503; Russel D. and Thompson R.C. (1993) Curr. Opin. Biotech. 4:714-721). Accordingly, the 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, sepsis caused by Gram-negative bacteria, and toxic shock syndrome.

Additionally, for sepsis treatment, the anti-TNFα antibodies and antibody fragments of the invention can be co-administered with one or more therapeutic agents that further alleviate sepsis, such as interleukin-1 inhibitors (described in PCT Publication No. WO 92/16221 and WO 92/17583), the cytokine interleukin-6 (see eg PCT Publication No. WO 93/11793) or platelet activating factor antagonists (see eg European Patent Application No. EP 374 510). Other combination therapies for sepsis treatment are discussed in subsection III below.

Additionally, in a preferred embodiment, the anti-TNFα antibodies and antibody fragments of the invention are administered to sub-patients from sepsis with serum or plasma IL-6 concentrations above 500 pg/mL and more preferably 1000 pg/mL during treatment group (see PCT Publication No. WO 95/20978 Daum, L., et al.).

B. Autoimmune diseases

Tumor necrosis factor has been shown to play a role in the pathophysiology of various autoimmune diseases. For example, TNFα is also involved in activating tissue inflammation and causing joint destruction in rheumatoid arthritis (see eg, Moeller A. et al. (1990), Cytokine 2:162-169; US Patent No. 5231024 Moeller et al.; European Patent No. 260610B1 Moeller A.; Tracey K.J. and Cerami A., supra; end W.P. and Dayer J.M. (1995) ath. Rheum. 38:151-160; Fava R.A. et al. (1993) Clin.Exp.Immunol. ). TNFα is also involved in promoting islet cell death and the development of insulin resistance in diabetes (see, eg, Tracey and Cerami, supra; PCT publication No. WO 94/08609). TNFα is also involved in cytotoxic formation of oligodendrocytes and induction of inflammatory plaques in multiple sclerosis (see, eg, Tracey and Cerami, supra). Chimeric and humanized murine anti-hTNFα antibodies have been clinically tested for the treatment of rheumatoid arthritis (see, eg, Elliot M.J. et al. (1994); Lancet 344:1125-1127; Elliot M.J. et al. (1994) Lancet 344 : 1105-1110; Rankin E.C. et al. (1995) Br. J. Rheumatol. 34:334-342).

The human antibodies and antibody fragments of the invention are useful in the treatment of autoimmune diseases, particularly those associated with inflammation, including rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis and gouty arthritis, allergies, Multiple sclerosis, autoimmune diabetes, autoimmune uveitis, and renal syndrome. Typically, the antibody or antibody fragment is administered systemically, although for certain disorders, local administration of the antibody or antibody fragment at the site of inflammation may be beneficial (eg, topical administration to the joint in rheumatoid arthritis or topical administration to diabetic ulcers, alone or in combination with the cyclohexane-subunit derivatives described in PCT Publication No. WO 93/19751). The antibodies and antibody fragments of the invention may also be used with one or more additional therapeutic agents for the treatment of autoimmune diseases, as further described in subsection III.

c. Infectious diseases

Tumor necrosis factor is involved in the production of biological effects observed in a variety of infectious diseases. For example, TNFα is involved in the development of brain inflammation and capillary thrombosis and infarction in malaria. TNFα is also involved in the development of brain inflammation, induction of blood-brain barrier rupture, development of septic shock syndrome, and activation of venous infarction in meningitis. TNFα is also involved in the induction of cachexia, stimulation of viral proliferation and the development of central nervous system damage in acquired immunodeficiency syndrome (AIDS). Accordingly, the antibodies and antibody fragments of the invention are useful in the treatment of infectious diseases, including bacterial meningitis (see, eg, European Patent Application No. EP 585705), cerebral malaria, AIDS and the AIDS-related complex (αC) (see, eg, European Patent Application No. EP 585705) Patent Application No. EP 230574) and cytomegalovirus infection secondary to transplantation (see eg Fietze, E., et al. (1994) Transplantation 58:675-680). The antibodies and antibody fragments of the invention can also be used to reduce symptoms associated with infectious diseases, including fever and myalgia due to infection (eg, influenza) and cachexia secondary to infection (eg, secondary to AIDS or alphaC).

D. to transplant

Tumor necrosis factor is thought to play a role in allograft rejection and graft-versus-host disease (GVHD) and adverse effects observed when rejection of kidney transplants is inhibited using the rat antibody OKT3 directed against the T-cell receptor CD3 complex. Critical media (see eg Eason J.D. et al. (1995) Transplantation 59:300-305; Suthanthiran M. and Strom T.В. (1994) New Engl. J. Med. 331:365-375). Accordingly, the antibodies and antibody fragments of the present invention can be used to inhibit transplant rejection (including allograft and xenograft rejection) and inhibit GVHD. Although antibodies or antibody fragments can be used alone, more preferably, they are used in combination with one or more other agents that inhibit the immune response to allografts or inhibit GVHD. For example, in one embodiment, the antibodies or antibody fragments of the invention are combined with one or more other targets involved in the modulation of immune responses (such as the cell surface molecules CD25 (interleukin-2 receptor-beta), CD11a (LFA) -1), CD54 (ICAM-1), CD4, CD45, CD28/CTLA4, CD80 (В7-1) and/or CD86 (B7-2)) antibodies were used in combination. In yet another embodiment, the antibodies or antibody fragments of the invention are used in combination with one or more general immunosuppressive agents, such as cyclosporin A or FK506.

E. Malignant tumor

Tumor necrosis factor is involved in the induction of cachexia, stimulation of tumor growth, enhancement of metastatic potential and the development of cytotoxicity in malignant tumors. Accordingly, the antibodies and antibody fragments of the present invention are useful for treating malignancies, inhibiting tumor growth or metastasis, and/or alleviating cachexia secondary to malignancies. Antibodies or antibody fragments can be administered systemically or locally to the tumor site.

F. Lung disorders

Tumor necrosis factor is involved in the pathophysiology of adult respiratory distress syndrome (αDS), including stimulation of endothelial leukocyte activation, directing cytotoxicity to lung cells, and induction of vascular leaky syndrome. Accordingly, the antibodies and antibody fragments of the invention can be used to treat various pulmonary disorders, including adult respiratory distress syndrome (see eg, PCT Publication No. WO 91/04054), shock lung, chronic pulmonary inflammatory diseases, pulmonary nodules disease, pulmonary fibrosis and silicosis. The antibodies and antibody fragments of the invention can be administered with one or more additional therapeutic agents for the treatment of pulmonary disorders, as described in subsection III below.

G. Intestinal disorders

Tumor necrosis factor is involved in the pathophysiology of inflammatory bowel disease (see, eg, Tracy K.J., et al. (1986) Science 234:470-474; Sun X-M., et al. (1988) J.Clin.Invest.81:1328- 1331; MacDonald T.T., et al. (1990) Clin. Exp. Immunol. 81:301-305). Chimeric murine anti-TNFa antibodies have been clinically tested for the treatment of Crohn's disease (van Dullemen H.M. et al. (1995) Gastroenterology 109:129-135). The human antibodies or antibody fragments 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). The antibodies and antibody fragments of the invention may also be administered with one or more additional therapeutic agents for the treatment of intestinal disorders, as described in subsection III below.

H. Heart disorders

The antibodies or antibody fragments of the invention are also useful in the treatment of various cardiac disorders, including cardiac ischemia (see eg European Patent Application No. EP 453898) and heart failure (myocardial weakness) (see eg PCT Publication No. WO 94/20139).

I. Other

The antibodies or antibody fragments of the invention can also be used to treat various disorders in which TNFa activity is detrimental. Examples of other diseases and disorders in which TNFα activity is involved in pathophysiology and thus can be treated using the antibodies or antibody fragments of the invention include inflammatory bone diseases and bone resorption diseases (see, e.g., Bertolini D.R., et al. (1986) Nature 319: 516-518; Konig A., et al. (1988) J. Bone Miner. Res. 3:621-627; Lerner U.H. and Ohiin A. (1993) J. Bone Miner. Res. 8:147-155; and Shanka G. and Stern P.H. (1993) Bone 14:871-876), hepatitis (including alcoholic hepatitis) (see eg McClain C.J. and Cohen D.A. (1989) Hepatology 9:349-351; Felver M.E. et al. (1990) Alcohol Clin. Exp. Res. 14: 255-259; and Hansen J. et al. (1994) Hepatology 20: 461-474), viral hepatitis (Sheron N. et al. (1991) J. Hepatol. 12:241 -245; and Hussain M.J. et al. (1994) J. Clin. Pathol. 47: 1112-1115) and fulminant hepatitis; coagulopathy (see eg van der PollТ. et al. (1990) N. Engl. J. Med. 322: 1622-1627; van der PollТ. et al. (1991) Prog. Clin. Biol. Res. 367: 55-60; H118-124; Liu X.S. et al. (1994) Burns 20:40-44), reperfusion injury (see eg Scales W.E. et al. (1994) Am.J.Physiol. 26:1122-1127; SerrickС.etal. (1994) Transplantation 58:1158-1162; Yao Y.M. et al. (1995) Resuscitation 29:157-168), keloid formation (see eg 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 considered to be the amount of a therapeutic agent administered during treatment that will alleviate to some extent one or more symptoms of the disease being treated.

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

"Intermittent" administration refers to treatment that is not continuous without interruption, but is cyclic in nature.

In this specification and the claims that follow, unless the context requires otherwise, the words "having", "including" and "comprising" or variations thereof, such as "has", "having", "including" "includes", "including", "comprises" or "comprising" should be understood to include the stated integer or group of integers, but not to exclude any other integer or group of integers.

Detailed description of the invention

Aqueous composition

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

Adalimumab and infliximab are examples of recombinant monoclonal anti-TNFa antibodies. Infliximab is an example of a chimeric antibody. Adalimumab is an example of a human antibody. In a preferred embodiment, the antibody used in the composition is a human antibody. In a more preferred embodiment, the antibody used in the composition is a human anti-human TNFα antibody. In yet another embodiment, the composition comprises D2E7 and D2E7 in combination with other antibodies. The D2E7 antibody, commonly referred to as adalimumab, binds TNFα with high affinity, low dissociation kinetics, and high neutralizing capacity. Adalimumab can be trademarked Obtained in the pharmaceutical market. The names adalimumab and D2E7 used throughout this specification represent the same human monoclonal antibody. In a preferred embodiment, the monoclonal antibody is adalimumab or an antigen-binding portion thereof.

In some embodiments, the recombinant monoclonal anti-TNFa antibody or antigen-binding portion thereof is typically present 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.

The aqueous compositions in question comprise suitable buffer solutions in combination with other pharmaceutically acceptable excipients that stabilize pharmaceutical formulations. Suitable buffer solutions that can be used are selected from groups known in the art and can be found in the literature. In one embodiment, suitable buffer solutions include, but are not limited to, acetate ion-based buffers (or buffer systems). In a preferred embodiment, a suitable buffer comprises acetate buffer. In an even more preferred embodiment, the acetate buffer is selected from a combination of sodium acetate trihydrate and acetic acid.

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

In some embodiments, sodium acetate trihydrate is present in an amount of 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 of 4.0 to about 7.0, depending on the monoclonal antibody used. In a preferred embodiment, the buffer used maintains the pH of the composition in the range of about 4.5-6.0. In a more preferred embodiment, the pH is maintained at about 5.0-6.0. In a more preferred embodiment, the pH is maintained at about 5.5.

The aqueous composition contains suitable surface active substances, which are pharmaceutically acceptable excipients, for protecting the protein composition from various stressful effects, such as stirring, shearing, high temperature, freezing, and the like. Suitable surface active substances include, but are not limited to, polysorbates or poloxamers or combinations of polysorbates and poloxamers. In a preferred embodiment, suitable surface-active substances are polysorbates. In a preferred embodiment, a suitable surface-active substance is a poloxamer. In a preferred embodiment, a suitable surface-active substance is a combination of polysorbate and poloxamer. In a more preferred embodiment, the polysorbate is polysorbate 20 or polysorbate 80 (also known as the trademark 20 or 80 distribution). In a more preferred embodiment, the poloxamer is Poloxamer 188 (also known under the trademark Kolliphor distribution). In another preferred embodiment, a suitable surface-active substance is a combination of polysorbate 20/polysorbate 80 and poloxamer 188.

In some embodiments, polysorbate 20 is present in an amount of 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 of 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 of 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 0.5 mg/mL.

In some embodiments, the combination of polysorbate 20/polysorbate 80 and poloxamer 188 is polysorbate 80 present in an amount up to 10 mg/mL and poloxamer present in an amount up to 10 mg/mL Losham 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 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.

Aqueous compositions contain one or more suitable stabilizers which are pharmaceutically acceptable excipients and which protect the pharmaceutically active ingredient from chemical and/or physical degradation during manufacture, storage and use. In one embodiment, stabilizers include, but are not limited to, amino acids, oligosaccharides, or suitable derivatives or mixtures thereof. In another preferred embodiment, a suitable stabilizer is trehalose. In another preferred embodiment, 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, an oligosaccharide that can also be used as a stabilizer includes trehalose.

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

In another such embodiment, the amino acid useful as a stabilizer is proline.

In some embodiments, proline is present in an amount of 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 trehalose present in an amount up to 150 mg/mL and proline present in an amount up to 40 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 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 50-200 mg/mL recombinant monoclonal anti-TNFα antibody, 0.4-1.2 mg/mL sodium acetate trihydrate, 25-150 mg/mL trehalose, and/or 5-40 mg Proline/mL or 25-150mg/mL trehalose and 0.05-0.5mg/mL arginine, glacial acetic acid (to pH 5.0-6.0), 0.1mg/mL-1mg/mL polysorbate 20. In a preferred embodiment of the aqueous composition, the recombinant monoclonal anti-TNFa antibody is adalimumab. In a more preferred embodiment, the composition comprises 50-150 mg/mL adalimumab, 0.436 mg/mL sodium acetate trihydrate, 80 mg/mL trehalose dihydrate, glacial acetic acid (up to pH 5.5 ), 0.5 mg/mL of polysorbate 20. In a more preferred embodiment, the composition comprises 50-150 mg/mL adalimumab, 0.436 mg/mL sodium acetate trihydrate, 80 mg/mL trehalose dihydrate, 0.1 mg/mL essence Amino acid hydrochloride, glacial acetic acid (until pH 5.5), polysorbate 20 at 0.5 mg/mL. In a more preferred embodiment, the composition comprises 50-150 mg/mL adalimumab, 0.436 mg/mL sodium acetate trihydrate, 27 mg/mL proline, glacial acetic acid (up to pH 5.5), Polysorbate 20 at 0.5 mg/mL. In a more preferred embodiment, the composition comprises 50-150 mg/mL adalimumab, 0.436 mg/mL sodium acetate trihydrate, 40 mg/mL trehalose dihydrate, 13.5 mg/mL acetic acid, glacial acetic acid (until pH 5.5), polysorbate 20 at 0.5 mg/mL.

In some embodiments, the aqueous composition comprises 50-200 mg/mL recombinant monoclonal anti-TNFα antibody, 0.4-1.2 mg/mL sodium acetate trihydrate, 25-150 mg/mL trehalose, and/or 5-40 mg Proline/mL or 25-150 mg/mL trehalose and 0.05-0.5 mg/mL arginine, glacial acetic acid (until pH 5.0-6.0), 0.1 mg/mL-1 mg/mL polysorbate 80 . In a preferred embodiment of the aqueous composition, the recombinant monoclonal anti-TNFa antibody is adalimumab. In a more preferred embodiment, the composition comprises 50-150 mg/mL adalimumab, 0.436 mg/mL sodium acetate trihydrate, 80 mg/mL trehalose dihydrate, glacial acetic acid (up to pH 5.5 ), 0.5 mg/mL of polysorbate 80. In a more preferred embodiment, the composition comprises 50-150 mg/mL adalimumab, 0.436 mg/mL sodium acetate trihydrate, 80 mg/mL trehalose dihydrate, 0.1 mg/mL essence Amino acid hydrochloride, glacial acetic acid (until pH 5.5), polysorbate 80 at 0.5 mg/mL. In a more preferred embodiment, the composition comprises 50-150 mg/mL adalimumab, 0.436 mg/mL sodium acetate trihydrate, 27 mg/mL proline, glacial acetic acid (up to pH 5.5), Polysorbate 80 at 0.5 mg/mL. In a more preferred embodiment, the composition comprises 50-150 mg/mL adalimumab, 0.436 mg/mL sodium acetate trihydrate, 40 mg/mL trehalose dihydrate, 13.5 mg/mL acetic acid, glacial acetic acid (until pH 5.5), polysorbate 80 at 0.5 mg/mL.

In some embodiments, the aqueous composition comprises 50-200 mg/mL recombinant monoclonal anti-TNFα antibody, 0.4-1.2 mg/mL sodium acetate trihydrate, 25-150 mg/mL trehalose, and/or 5-40 mg Proline/mL or 25-150 mg/mL trehalose and 0.05-0.5 mg/mL arginine, glacial acetic acid (until pH 5.0-6.0), 0.1 mg/mL-1 mg/mL poloxamer 188 . In a preferred embodiment, the recombinant monoclonal anti-TNFa antibody is adalimumab. In a more preferred embodiment, the composition comprises 50-150 mg/mL adalimumab, 0.436 mg/mL sodium acetate trihydrate, 80 mg/mL trehalose dihydrate, glacial acetic acid (up to pH 5.5 ), Poloxamer 188 at 1 mg/mL. In a more preferred embodiment, the composition comprises 50-150 mg/mL adalimumab, 0.436 mg/mL sodium acetate trihydrate, 80 mg/mL trehalose dihydrate, 0.1 mg/mL essence Acid hydrochloride, glacial acetic acid (until pH 5.5), Poloxamer 188 at 1 mg/mL. In a more preferred embodiment, the composition comprises 50-150 mg/mL adalimumab, 0.436 mg/mL sodium acetate trihydrate, 27 mg/mL proline, glacial acetic acid (up to pH 5.5), Poloxamer 188 at 1.0 mg/mL. In a more preferred embodiment, the composition comprises 50-150 mg/mL adalimumab, 0.436 mg/mL sodium acetate trihydrate, 40 mg/mL trehalose dihydrate, 13.5 mg/mL proline acid, glacial acetic acid (until pH 5.5), Poloxamer 188 at 1.0 mg/mL.

Further, the composition may additionally comprise one or more other suitable excipients well known to those skilled in the art.

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

In some embodiments, the liquid composition retains storage stability to the effect that there is no further protein aggregation process or modification compared to the stability measurement at time zero.

Methods of treatment and use of aqueous compositions

In another embodiment, the present invention relates to a method for treating a mammal, the method comprising administering to the mammal a therapeutically effective amount of a pharmaceutical composition of the present invention, wherein the mammal is afflicted with a disease that is effectively treatable with adalimumab or obstacles.

In a preferred embodiment, the mammal is a human.

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

The provided pharmaceutical compositions can be injected systemically, such as intravenously or subcutaneously, or by intramuscular injection; or by injection or applied to the site of interest, such as by direct injection or direct application to the site when the site is exposed during surgery; Or by topical application, to a subject in need of treatment.

In one embodiment, the present 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.

A therapeutically effective amount of adalimumab in the provided compositions depends on the condition being treated, the severity of the condition, previous treatments and the patient's medical history and response to the therapeutic agent. Appropriate doses can be adjusted according to the judgment of the attending physician so that they can be administered to the patient in one or more administrations.

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

Alternatively, a fixed dose may be administered, which may be in the range of 2-500 mg/dose, 2-100 mg/dose, or about 10-80 mg/dose.

Exemplary dosage ranges are the same or lower than those described above if the dosage is administered more than once a week, and preferably are administered in a dosage range of 25-100 mg/dose twice or more per week.

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

Dosages may be administered weekly, biweekly, or separated over several weeks (eg, from 2 to 8).

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

In some instances, improvement in the patient's condition can be achieved by administering a dose of up to about 100 mg of the pharmaceutical composition 1-3 times per week for a period of at least 3 weeks. A longer period of treatment may be required to achieve the desired level of improvement. For incurable chronic conditions, the treatment regimen can continue indefinitely. For pediatric patients (ages 4-17 years), a suitable treatment regimen may include administration of adalimumab at doses of 0.4 mg/kg to 4 mg/kg one or more times per week.

In another embodiment, the pharmaceutical formulations of the present invention may be prepared as bulk formulations, and thus, the components of the pharmaceutical composition are present in higher amounts than are required for administration, and are appropriately diluted prior to administration.

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

Administration of the provided pharmaceutical compositions can be performed in a variety of ways, including parenteral, oral, buccal, nasal, rectal, intraperitoneal, intradermal, transdermal, subcutaneous, intravenous, intraarterial, intracardiac, cardiac Intraventricular, intracranial, intratracheal, intrathecal, intramuscular, intravitreal and topical.

The pharmaceutical compositions of the present invention are particularly useful for parenteral administration, ie subcutaneous, intramuscular, intravenous, intraperitoneal, intramedullary, intraarticular, intrasynovial and/or intrathecal. Parenteral administration can be by bolus injection or continuous infusion. Pharmaceutical compositions for injection may be presented in unit dosage forms such as, but not limited to, ampoules, vials, prefilled syringes, or multi-dose containers with an added preservative. In addition, many recent drug delivery methods have been developed and the pharmaceutical compositions of the present invention are suitable for administration by these new methods, such as Inject- Syringe such as and needle-free devices such as and The pharmaceutical compositions of the present invention are also suitable for yet-to-be-discovered modes of administration. See also Langer, 1990, Science, 249:1527-1533.

The provided pharmaceutical compositions can also be formulated as depots. Such long-acting formulations can be administered by implantation (eg, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the formulations can be modified with 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).

If desired, the pharmaceutical compositions can be presented in vials, packs or dispenser devices which can contain one or more unit dosage forms containing the active ingredient. In one embodiment, the dispenser device may comprise a syringe containing a single dose of a ready-to-use injectable liquid formulation. Instructions may be attached to the syringe.

In another embodiment, the present invention provides a kit or container containing an aqueous pharmaceutical composition of the present invention. The polypeptide concentration in the aqueous pharmaceutical composition can vary widely, but is generally in the range of about 1 to about 200 mg/mL aqueous formulation. The kit can also be accompanied by instructions for use.

production method

The method for producing the above composition comprises adding an acetate buffer to the aqueous phase, followed by adding the following components in any order: osmotic and/or stabilizer selected from trehalose, proline or combinations thereof; reconstitution Monoclonal anti-TNFa antibody; a surfactant selected from polysorbate 20, polysorbate 80, poloxamer 188, or a combination thereof.

technology

1. Determination of Protein Concentration in Test Samples

Protein concentrations were determined using UV spectrophotometry at a wavelength of 280 nm in UV spectrophotometric plates.

Dilute each sample with the appropriate blank solution to a concentration of ~0.5 mg/mL. Place 150 μL of the diluted sample into the wells of a UV spectrophotometric plate. The optical density of the plate solution was measured at a wavelength of 280 nm using a plate reader. Use an appropriate blank solution as the reference solution.

Calculate the protein concentration (C) in mg/mL by the following formula:

А ₂₈₀ is the optical density value at the wavelength of 280 nm;

ε is the extinction value of the studied protein;

b is the total dilution ratio of the sample;

l is the layer thickness per plate well, for 150 μL, l = 0.42 cm.

2. Buffer Exchange and Sample Concentration

Diafiltration and concentration of the samples were performed under pressure in a Stirred Cell (Millipore) concentration cell.

3. "PEG Aggregation"

Preparation of PEG 6000 solution

A solution of PEG 6000 was prepared at a concentration of 20-25% by mass in the study additive formulation. The resulting solution was filtered through a 0.45 μm Durapore filter.

Transfer the calculated amounts of sample, additive solution, and 20-25% PEG 6000 solution into a 96-well UV spectrophotometric plate such that the PEG 6000 concentration per well is 0-18% and the protein concentration per well is 1 mg/mL . All the resulting well solutions were mixed well by pipetting.

Next, the degree of turbidity of the solution was visually evaluated, and the optical density of the solution was measured at a wavelength of 400 nm. The more unstable the sample, the lower the PEG 6000 concentration will form visible aggregates (opalescence). The degree of turbidity of the solutions was also assessed one or more days after solution preparation.

4. Determination of Protein Homogeneity and Aggregation Points Using Dynamic Light Scattering (DLS)

Study sample homogeneity determinations were performed on a Zetasizer Nano ZSP in size measurement mode. For this purpose, 0.05 mL of the solution was placed in a dust-free disposable plastic cuvette.

· Analysis model: protein analysis.

• Hold the temperature for 30 seconds before measurement.

• At each point, 13 measurements were averaged in 3 replicates.

Assays to study protein foci were performed on a Zetasizer Nano ZSP. For this purpose, the solution is placed in a quartz dust-free cuvette and heated gradually in the instrument while the intensity of the scattered light is continuously measured in the temperature trend measurement mode.

· Analysis model: protein analysis.

Temperature trend, mod: protein aggregation point. Heat from 50°C to 85°C in 1.5°C steps.

• Hold the temperature for 30 seconds before measurement.

• At each point, average 15 measurements with 1 repetition.

Temperature trends are plotted using the instrument software, which automatically calculates protein aggregation points (aggregates).

5. Determination of thermal stability by the "heat stress 50°C" technique

The test samples were divided into 2 and placed in separate tubes: 1 tube of each formulation was stored in a freezer at +4°C, the rest were placed in an incubator and incubated at 50°C for the indicated time. After completion of the heating period, the tubes were removed from the incubator, allowed to stand at room temperature for about 15 minutes, and analyzed according to the instructions.

6. Determination of colloidal stability by the technique of "oscillation test"

Divide the test samples into 2 aliquots of 200 μL each and place in glass vials, store 1 vial of each formulation in a freezer at +4°C, and place the rest in a thermal shaker and refrigerate at 2-8°C. Shake at 800 rpm for the indicated time. Upon completion of the stress, the tubes were removed from the thermal shaker and analyzed according to the instructions.

7. Determination of colloidal stability by low temperature concentration technique

The test samples were divided into 2 and placed in polymer tubes: 1 tube of each formulation was stored in the freezer at +4°C, the rest were placed in the freezer and stored at -16°C to 20°C for the indicated time. Upon completion of the stress, tubes were removed from the freezer, allowed to stand at room temperature until the contents were completely thawed, the solutions were mixed using a Vortex and analyzed according to the instructions.

8. Determination of sample purity by size exclusion high performance liquid chromatography (SE-HPLC) technique

Column: Tosoh TSK-Gel G3000 SW _XL 7.8mm ID x 30cm, cat. #08541.

Column temperature: 25°C.

Mobile phase flow rate: 0.7 mL/min.

Injection volume: 20 μL.

Sample concentration: 5 mg/mL.

Detector wavelength: 220nm.

Elution time: 23 minutes.

Mobile phase: anhydrous 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 orthophosphoric acid.

9. Determination of sample acid-base distribution using the Caliper LabChip GX II instrument

9.1. Sample Preparation

Samples were diluted to a concentration of 1 mg/mL. 2 μL of 5 mg/mL carboxypeptidase B (CpB) solution was added to 200 μL of the resulting solution. It was stirred and incubated at 37°C for 2 hours. Test samples were dialyzed against 3 water changes in Amicon Ultra centrifuge tubes and concentrated to 2 mg/mL.

9.2. Working solution preparation

Using the HT Protein Charge Variant Labeling Kit, prepare working solutions and plates with assay samples according to the manufacturer's procedure.

9.3. Chip Preparation

Chips and tubes with buffer were prepared according to the manufacturer's procedure using buffer solutions from the Protein Charge Variant Buffer Kit.

Assay initiation is standard operation. Protein Charge Variant 68s assay.

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

10.1. Assay sample preparation

700 μL of HT protein expression sample buffer supplemented with 24.5 μL of 1 M DTT for buffer under reducing conditions and 700 μL of HT protein expression sample buffer supplemented with 24.5 μL of 1 M IAM for buffer under non-reducing conditions were used to prepare denaturing and reducing solutions. The samples were diluted to a concentration of 2 mg/mL. Prepare two microtubes for each sample - add 35 µL of denaturing buffer to one and 35 µL of reducing buffer to the other. The samples were denatured at 100°C for 5 minutes. Vortex the tubes, then add 70 μL of water to each tube and mix. 44 μL of each sample was transferred to a 96-well plate for analysis.

10.2. Working solution preparation and chip loading

Working solution and chip preparation were performed according to standard procedures using the HT Protein Express Reagent kit. Assay initiation is standard operation. HT Protein Express 200 assay.

11. Determination of sample acid-base distribution using ion exchange (IE) HPLC

12. Purity determination by polyacrylamide gel electrophoresis (PAGE) under reducing and non-reducing conditions

PAGs were prepared in glass plates in the presence of sodium dodecyl sulfate, consisting of a concentrated layer of 4% PAGE and a separated layer of 12.5% PAG for reducing conditions and 8% PAG for non-reducing conditions.

Assemble and install the electrophoresis chamber according to the operating manual of the vertical electrophoresis device. Samples were prepared by diluting the samples with purified water to a final concentration of 1 mg/mL. Take a volume equivalent to 40 μg and mix the prepared test samples with samples containing 2-mercaptoethanol (reducing conditions) and without 2-mercaptoethanol (non-reducing conditions) in a ratio of 3:1 (v/v) Apply buffer solution mix, mix. The resulting solution was incubated at a temperature of (99±1)°C for 3 minutes (samples containing 2-mercaptoethanol) and 1 minute at a temperature of (99±1)°C (samples without 2-mercaptoethanol). sample). The solution was allowed to cool to room temperature, mixed and transferred to the PAG wells under the running buffer layer.

Run electrophoresis in DC mode using a water cooling system. The power supply parameters were set as follows: the voltage was 110V when the dye front was passed through the laminated gel. Once the dye front entered the lower running gel 5-7mm, the voltage was increased to 180V. When the dye front reaches the lower limit of the gel, turn off the power.

Once electrophoresis is over, separate the gel from the glass and fix the protein with fixative for 16-18 hours at room temperature. Further, the gel was stained (in acid blue 83 solution) and washed until a clear view of the bands. Scan the gel. Purity and impurities in the test samples were assessed using GelPro software.

13. Specific activity assay

Sample specific activity was assessed by the ability to specifically bind and neutralize TNFα, which in turn was cytotoxic and resulted in the death of WEHI-13var cultures (fibrosarcoma, Mus musculus), a Variants of cultures with increased sensitivity to TNFα in the presence of actinomycin D. Sample preparation was performed using a Tecan Evo 200 robotic workstation, RPMI1640, 2 mM Gln, 10% FBS, 2.5 μg/mL actinomycin D, 5 μg/mL gentamicin were used as QM (quantitative medium).

Test antibody samples were diluted to 50 μg/mL with no more than 20 dilution steps and placed in the robotic workstation. Using a Tecan Evo 200, 3 separate dilutions of RS and IS were prepared in culture plates ranging from 10000-0.5 ng/mL, and 500 pg/mL of TNFα solution was added to the prepared dilutions. The resulting mixture was stirred and incubated at room temperature for 1 hour.

After incubation, (0.5±0.1) x 106 cells/mL of WEHI- ^13var cell suspension was added. The plates were placed in a CO ₂ incubator and incubated at a temperature of (37±1)° C. in humidified air with 5% CO ₂ for 20-24 hours.

At the end of the incubation period, the vital dye alamar blue was added to the plates and the plates were incubated under the same conditions until color development. Fluorescence intensity was evaluated at excitation/emission wavelengths of 544/590 nm using an Infinite M200 Pro reader. Fluorescence intensity versus protein concentration curves were plotted using Magellan 7.2 software and the parallel alignment of the resulting curves was assessed. The relative specific activity of the test sample was determined as the ratio of the ED50 of the reference sample to the ED50 of the test sample, expressed as a percentage.

The following examples are provided for the best understanding of the present invention. These examples are provided for illustrative purposes only, and should not be construed to limit the scope of the invention in any way.

All publications, patents and patent applications cited in this specification are incorporated herein by reference. Although the above invention has been described in some detail by way of illustration and example for the sake of clarity, as those skilled in the art will appreciate based on the teachings disclosed herein, the invention may be practiced without departing from the spirit and scope of the appended embodiments of the invention. subject to certain changes and modifications.

Example

Example 1. Selection of buffer solution properties.

research preparation

Four buffer solutions were selected for this study and the initial Humira formulation (for formulations with protein content of 50 mg/mL and 100 mg/mL) was used as a control.

1.1. Determination of colloidal stability by PEG aggregation.

For each sample, the "PEG aggregation" test was performed in triplicate. Analysis was performed according to procedure 3. The data on the average optical density of the solutions are shown in Table 1. The results are also shown in Figure 1.

Table 1. Average optical density of solutions after preparation

Visible aggregation is present.

1.2. Determination of thermal stability by protein foci using dynamic light scattering (DLS) techniques.

Analysis was performed according to procedure 4. The results are shown in Table 2 and Figures 4-8.

1.3. Determination of thermal stability under long-term exposure using the heat stress 50°C method.

Analysis was performed according to procedure 5. The results are shown in Table 2.

1.4. Results.

Table 2. Overview of buffer solution property selection

positive result negative result Average result

1.5. Conclusion of Example 1.

Based on the results of this study, the recommended storage formulation (excluding additional additives) is:

This formulation showed the best stability performance of all samples tested: the least increase in impurities during heat stress (0.02% on par with the Humira 2 formulation), the absence of aggregation at 18% PEG 6000 and the highest aggregation temperature (74 °C).

Adalimumab in the Humira 1 formulation has lower thermal stability than the recommended formulation. Minimal colloidal and thermal stability was noted for formulations based on 5 mM citrate buffer at pH 5.0.

Example 2. Composition pH and buffer capacity optimization.

Based on the results of the first part of the study, adalimumab showed the best stability in acetate buffer at pH 5.0. According to the prior art, most patents and patent applications for pharmaceutical compositions comprising adalimumab protect solutions at pH 4.0-8.0. Therefore, the purpose of this section was to investigate the possibility of obtaining stable compositions containing acetate ions with a pH of less than 4.

research preparation

abbreviation Concentration, mM рН Acet 5mM pH 6 5 6.0 Acet 5mM pH 5.5 5 5.5 Acet 5mM pH 5 5 5.0 Acet 5mM pH 4 5 4.0 Acet 5mM pH 3.75 5 3.75 Acet 5mM pH 3.5 5 3.5 Acet 10mM pH 6 10 6.0 Acet 10mM pH 5.5 10 5.5 Acet 10mM pH 5 10 5.0 Acet 10mM pH 4 10 4.0 Acet 10mM pH 3.75 10 3.75 Acet 10mM pH 3.5 10 3.5 Acet 20mM pH 6 20 6.0 Acet 20mM pH 5.5 20 5.5 Acet 20mM pH 5 20 5.0 Acet 20mM pH 4 20 4.0 Acet 20mM pH 3.75 20 3.75 Acet 20mM pH 3.5 20 3.5

2.1. Determination of colloidal stability by PEG aggregation.

For each sample, the "PEG aggregation" test was performed in triplicate. Analysis was performed according to procedure 3. The data on the average optical density of the solutions are shown in Table 3. The results are also shown in Figure 2.

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

2.2. Determination of thermal stability under long-term exposure using the heat stress 50°C method.

Analysis was performed according to procedure 5.

2.3. Determination of colloidal stability by the "shake test" technique.

Analysis was performed according to procedure 6.

2.4. Determination of colloidal stability by low temperature concentration technique.

Analysis was performed according to procedure 7.

An overview is provided in Table 4, where sample quality measurements before and after heat stress, shaking tests, and cryoconcentration are demonstrated.

2.5. Results.

Table 4. Overview of sample quality measures before and after stress

positive result negative result

Average Results/Baseline Data

2.6. Conclusion of Example 2.

· Studies have shown that the presence of adalimumab in an acidic solution of placebo results in a significant increase in pH levels. In addition, compositions with pH less than 5.0 exhibited poor stability during dialysis (low purity on Labchip Caliper under reducing conditions) and heat stress (low purity after stress for all assays used).

• The most stable sample in all studies was adalimumab in 5 mM acetate buffer pH 5.0-6.0. They demonstrated minimal changes in purity and acid-base distribution during stress. However, to generate adalimumab compositions, solutions with greater buffer capacity may be used.

Example 3. Penetrant and Stabilizer Selection.

Based on the results of the first part of the study, adalimumab showed the best stability in acetate buffer solution at pH 5.0-6.0. A pH 5.5 formulation was used as the basis for osmotic and stabilizer selection.

research preparation

3.1. Determination of colloidal stability by PEG aggregation.

For each sample, the "PEG aggregation" test was performed in triplicate. Analysis was performed according to procedure 3. The data on the average optical density of the solutions are shown in Table 6. The results are also shown in Figure 3.

Table 6. Average Optical Densities of Adalimumab Solutions After Preparation

Visible aggregation is present.

3.2. Determination of thermal stability by protein foci using dynamic light scattering (DLS) techniques.

Analysis was performed according to procedure 4.

3.3. Determination of thermal stability under long-term exposure using the heat stress 50°C method.

Analysis was performed according to procedure 5.

3.4. Results.

3.5. Conclusion of Example 3.

Based on the results of this study, promising additives for adalimumab compositions are trehalose dihydrate, glycine, proline, methionine, and arginine hydrochloride. Polysorbate 80, polysorbate 20, and poloxamer 188 were used as surface active substances for final pharmaceutical composition screening.

Sodium chloride, arginine, EDTA, lysine, guanidine have adverse effects on the colloidal stability of adalimumab.

Sodium chloride, lysine, guanidine have adverse effects on the thermal stability of adalimumab. Cysteine contained in the formulation resulted in complete fragmentation of the antibody. When the EDTA additive was added to the formulation, a change in the acid-base distribution of the protein was noted.

Example 4. Final Pharmaceutical Composition Selection.

Based on the results of the third part of the study, trehalose dihydrate, glycine, proline, methionine and arginine hydrochloride were selected as promising additives. Polysorbate 80, polysorbate 20, and poloxamer 188 were used as surface active substances for final pharmaceutical composition screening.

Study Formulation (mg/mL)

4.1. Determination of thermal stability under long-term exposure using the heat stress 50°C method.

Analysis was performed according to procedure 5.

4.2. Determination of colloidal stability by the "shake test" technique.

Analysis was performed according to procedure 6.

4.3. Determination of colloidal stability by low temperature concentration technique.

Analysis was performed according to procedure 7.

An overview is provided in Tables 8 and 9, where sample quality measurements before and after heat stress, shaking tests, and cryoconcentration are demonstrated.

4.4. Results.

4.4. Conclusion of Example 4

The study showed that adalimumab in Humira Formulation 1 was the least thermolabile of all samples tested. Heat stress at 50°C for 120 hours resulted in the largest increase in impurities (1.2%) and the largest change in acid-base profile based on SE-HPLC (over 33% total absolute change for all fractions on the LabChip Caliper instrument). The colloidal stability of the samples in Humira Formulation 1 was comparable to the alternative formulation.

Humira formulation 2 had comparable thermal stability to one of the alternative formulations (0.38% loss of purity during heat stress). However, after freeze-thaw of adalimumab in the formulation of the invention, there was significant aggregation of the protein: the increase in aggregates in size exclusion HPLC was over 7%, making the formulation unusable if it was accidentally frozen. A significant increase in impurities was also noted after freezing using DLS.

Alternative formulations based on acetate buffer solutions with the addition of many additives showed good thermal and colloidal stability during stress. Size exclusion HPLC showed no difference in impurity increase in the formulations of the invention. Furthermore, the surfactant did not significantly affect the stability of adalimumab at a concentration of 15 mg/mL.

Analysis of stressed samples using DLS showed that the presence of arginine hydrochloride in the formulation reduced the increase in impurities during the shaking test, and the presence of methionine increased the accumulation of impurities during shaking.

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

Example 5. Final highly concentrated adalimumab pharmaceutical composition validation.

To confirm the stability of the recommended pharmaceutical composition, adalimumab at concentrations of 50 mg/mL, 100 mg/mL and 150 mg/mL were exposed to heat stress at 50°C for 6 days.

research preparation

5.1. Determination of thermal stability under long-term exposure using the heat stress 50°C method.

Analysis was performed according to procedure 5.

5.2. Results.

General conclusions

1. The screening of stable adalimumab pharmaceutical compositions is carried out in several stages: selection of buffer solution properties, selection of pH and buffer capacity of the solution, selection of osmotic agents and stabilizers, selection of final pharmaceutical composition and confirmation of the final highly concentrated adalimumab pharmaceutical composition.

2. During the study period, the thermal and colloidal stability of adalimumab in more than 90 formulations were investigated using the following methods: PEG aggregation, shaking test, freeze-thaw, heat stress followed by analysis of the degree of turbidity ( UV spectrophotometry), purity (size exclusion HPLC, dynamic light scattering and gel electrophoresis including use of LabChip, Caliper systems) and acid-base distribution (LabChip, Caliper and ion exchange HPLC).

3. The study showed that the thermal stability of the original Humira formulation (Humira 1 ) used in a commercial formulation of adalimumab at a concentration of 50 mg/mL was low compared to the formulation of the present invention.

4. Experiments showed that in the original Humira formulation (Humira 2) used in the commercial formulation of Adalimumab 100 mg/mL, the freezing and thawing process had a significant effect on Adalimumab aggregation compared to the formulation of the present invention. influences.

5. Acetate based buffer solution supplemented with trehalose dihydrate, arginine hydrochloride, proline and a surfactant selected from polysorbate 20, polysorbate 80 and poloxamer 188 The adalimumab pharmaceutical composition (pH 5.0-6.0) showed higher thermal stability and colloidal stability at a concentration of 50-150 mg/mL compared with the original formulation, and was the theme.

The optimal additive formulation of adalimumab was selected for the solution at pH 5.5. The stability of Adalimumab in formulations in the pH range of 5.0-6.0 was demonstrated based on samples with protein concentrations of 50, 100 and 150 mg/mL.

6. The preparation method of the pharmaceutical composition of the present invention with an adalimumab concentration of 150 mg/mL enables concentration up to 200 mg/mL without loss of protein mass for subsequent dilution when adding SAS and washing the ultrafiltration device after concentration .

Claims (34)

1. a kind of aqueous pharmaceutical composition for intravenously or subcutaneously giving, it includes:

A) a kind of recombinant monoclonal anti-TNF alpha antibodies；

B) a kind of buffer (or buffer system) based on acetate ion；

C) trehalose and/or proline, or

Trehalose and arginine；

D) polysorbate20, polysorbate80 or PLURONICS F87 or combinations thereof.

2. the composition of claim 1, wherein the recombinant monoclonal anti-TNF alpha antibodies are adalimumab.

3. the composition of claim 1-2, wherein the MAb concentration is 50-200 mg/mL.

4. the composition of claim 1, wherein the acetate buffer solution concentration is 1-100 mM.

5. the composition of claim 1, wherein the pH of the composition is 4-7.

6. the composition of claim 1, wherein the trehalose concentration is 25-150 mg/mL.

7. the composition of claim 1, wherein the concentration of proline is 5-40 mg/mL.

8. the composition of claim 1, wherein the trehalose concentration is 25-150 mg/mL and the arginine concentrations are 0.05-0.5 mg/mL。

9. the composition of claim 1, wherein the polysorbate20 concentration is 0.05 mg/mL-10 mg/mL.

10. the composition of claim 1, wherein the polysorbate80 concentration is 0.05 mg/mL-10 mg/mL.

11. the composition of claim 1, wherein the PLURONICS F87 concentration is 0.05 mg/mL-10 mg/mL.

12. the composition of claim 1, it includes:

A) the recombinant monoclonal anti-TNF alpha antibodies of 50-200 mg/mL；

B) sodium acetate trihydrate of 0.4-1.2 mg/mL；

C) proline of the trehalose of 25-150 mg/mL and/or 5-40 mg/mL, or

The trehalose of 25-150 mg/mL and the arginine of 0.05-0.5 mg/mL；

D) glacial acetic acid, until pH 5.0-6.0；

E) polysorbate80 of 0.1 mg/mL-1 mg/mL.

13. the composition of claim 12, wherein the recombinant monoclonal anti-TNF alpha antibodies are adalimumab.

14. the composition of claim 12, it includes:

A) adalimumab of 50-150 mg/mL；

B) sodium acetate trihydrate of 0.436 mg/mL；

C) trehalose dihydrate of 80 mg/mL closes object；

D) glacial acetic acid, until pH 5.5；

E) polysorbate80 of 0.5 mg/mL.

15. the composition of claim 12, it includes:

A) adalimumab of 50-150 mg/mL；

B) sodium acetate trihydrate of 0.436 mg/mL；

C) trehalose dihydrate of 80 mg/mL closes the arginine monohydrochloride of object and 0.1 mg/mL；

D) glacial acetic acid, until pH 5.5；

E) polysorbate80 of 0.5 mg/mL.

16. the composition of claim 12, it includes:

A) adalimumab of 50-150 mg/mL；

B) sodium acetate trihydrate of 0.436 mg/mL；

C) proline of 27 mg/mL；

D) glacial acetic acid, until pH 5.5；

E) polysorbate80 of 0.5 mg/mL.

17. the composition of claim 12, it includes:

A) adalimumab of 50-150 mg/mL；

B) sodium acetate trihydrate of 0.436 mg/mL；

C) 40 mg/mL trehalose dihydrates close object；

D) proline of 13.5 mg/mL；

E) glacial acetic acid, until pH 5.5；

F) polysorbate80 of 0.5 mg/mL.

18. the composition of claim 1, it includes:

A) the recombinant monoclonal anti-TNF alpha antibodies of 50-200 mg/mL；

B) sodium acetate trihydrate of 0.4-1.2 mg/mL；

C) proline of the trehalose of 25-150 mg/mL and/or 5-40 mg/mL, or

The trehalose of 25-150 mg/mL and the arginine of 0.05-0.5 mg/mL；

D) glacial acetic acid, until pH 5.0-6.0；

E) polysorbate20 of 0.1 mg/mL-1 mg/mL.

19. the composition of claim 18, wherein the recombinant monoclonal anti-TNF alpha antibodies are adalimumab.

20. the composition of claim 18, it includes:

A) adalimumab of 50-150 mg/mL；

B) sodium acetate trihydrate of 0.436 mg/mL；

C) trehalose dihydrate of 80 mg/mL closes object；

D) glacial acetic acid, until pH 5.5；

E) polysorbate20 of 0.5 mg/mL.

21. the composition of claim 18, it includes:

A) adalimumab of 50-150 mg/mL；

B) sodium acetate trihydrate of 0.436 mg/mL；

C) trehalose dihydrate of 80 mg/mL closes the arginine monohydrochloride of object and 0.1 mg/mL；

D) glacial acetic acid, until pH 5.5；

E) polysorbate20 of 0.5 mg/mL.

22. the composition of claim 18, it includes:

A) adalimumab of 50-150 mg/mL；

B) sodium acetate trihydrate of 0.436 mg/mL；

C) proline of 27 mg/mL；

D) glacial acetic acid, until pH 5.5；

E) polysorbate20 of 0.5 mg/mL.

23. the composition of claim 18, it includes:

A) adalimumab of 50-150 mg/mL；

B) sodium acetate trihydrate of 0.436 mg/mL；

C) 40 mg/mL trehalose dihydrates close object；

D) proline of 13.5 mg/mL；

E) glacial acetic acid, until pH 5.5；

F) polysorbate20 of 0.5 mg/mL.

24. the composition of claim 1, it includes:

A) the recombinant monoclonal anti-TNF alpha antibodies of 50-200 mg/mL；

B) sodium acetate trihydrate of 0.4-1.2 mg/mL；

C) proline of the trehalose of 25-150 mg/mL and/or 5-40 mg/mL, or

The trehalose of 25-150 mg/mL and the arginine of 0.05-0.5 mg/mL；

D) glacial acetic acid, until pH 5.0-6.0；

E) PLURONICS F87 of 0.1 mg/mL-1 mg/mL.

25. the composition of claim 24, wherein the recombinant monoclonal anti-TNF alpha antibodies are adalimumab.

26. the composition of claim 24, it includes:

A) adalimumab of 50-150 mg/mL；

B) sodium acetate trihydrate of 0.436 mg/mL；

C) trehalose dihydrate of 80 mg/mL closes object；

D) glacial acetic acid, until pH 5.5；

E) PLURONICS F87 of 1.0 mg/mL.

27. the composition of claim 24, it includes:

A) adalimumab of 50-150 mg/mL；

B) sodium acetate trihydrate of 0.436 mg/mL；

C) trehalose dihydrate of 80 mg/mL closes the arginine monohydrochloride of object and 0.1 mg/mL；

D) glacial acetic acid, until pH 5.5；

E) PLURONICS F87 of 1.0 mg/mL.

28. the composition of claim 24, it includes:

A) adalimumab of 50-150 mg/mL；

B) sodium acetate trihydrate of 0.436 mg/mL；

C) proline of 27 mg/mL；

D) glacial acetic acid, until pH 5.5；

E) PLURONICS F87 of 1.0 mg/mL.

29. the composition of claim 24, it includes:

A) adalimumab of 50-150 mg/mL；

B) sodium acetate trihydrate of 0.436 mg/mL；

C) 40 mg/mL trehalose dihydrates close object；

D) proline of 13.5 mg/mL；

E) glacial acetic acid, until pH 5.5；

F) PLURONICS F87 of 1.0 mg/mL.

30. a kind of method for treating the disease of TNF α mediation, the method includes giving a effective amount of claim 1-29 Composition.

31. the method for claim 30, wherein the disease that the TNF α mediates is selected from:

A) activity (moderate to severe) rheumatoid arthritis,

B) activity psoriasis arthropathica,

C) activity ankylosing spondylitis,

D) chronic (moderate to severe) psoriasis in plaques,

E) (moderate to severe) ulcerative colitis,

F) mesinae joint of vertebral column is scorching,

G) activity suppurative hidradenitis,

H) juvenile idiopathic arthritis,

I) (moderate or severe) Crohn disease,

J) uveitis,

K) activity Enthesopathy correlation arthritis.

32. the purposes that the composition of claim 1-29 is used to treat the disease of TNF α mediation.

33. the purposes of claim 32, wherein the disease is selected from:

A) activity (moderate to severe) rheumatoid arthritis,

B) activity psoriasis arthropathica,

C) activity ankylosing spondylitis,

D) chronic (moderate to severe) psoriasis in plaques,

E) (moderate to severe) ulcerative colitis,

F) mesinae joint of vertebral column is scorching,

G) activity suppurative hidradenitis,

H) juvenile idiopathic arthritis,

I) (moderate or severe) Crohn disease,

J) uveitis,

K) activity Enthesopathy correlation arthritis.

34. a kind of method for generating the composition of any one of claim 1-29, the method includes into water phase Acetate buffer is added, following components is then added in any order:

A) bleeding agent and/or stabilizer of trehalose, proline, arginine or combinations thereof are selected from；

B) recombinant monoclonal anti-TNF alpha antibodies；

C) it is selected from the surface reactive material of polysorbate20, polysorbate80, PLURONICS F87 or combinations thereof.