CN110536698B

CN110536698B - Aqueous pharmaceutical compositions of recombinant monoclonal anti-TNFα antibodies

Info

Publication number: CN110536698B
Application number: CN201780087767.4A
Authority: CN
Inventors: E.A.隆科娃; A.O.艾克弗勒夫; V.O.希缇科娃; A.M.莱科弗斯卡亚; D.V.莫洛佐夫
Original assignee: Biocard Jsc
Current assignee: Biocard Jsc
Priority date: 2016-12-30
Filing date: 2017-12-29
Publication date: 2024-02-13
Anticipated expiration: 2037-12-29
Also published as: RU2016152691A3; CN110536698A; RU2016152691A; MA46169B2; RU2665966C2; MA46169A1

Abstract

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

Description

Aqueous pharmaceutical compositions of recombinant monoclonal anti-TNFα antibodies

Field of the invention

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

Background

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

Soluble TNFα is formed by cleavage of precursor transmembrane proteins (Kriegler, et al (1988) Cell 53:45-53), and secreted polypeptides of 17 kilodaltons (kDa) aggregate 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. Binding produces a number of pro-inflammatory effects including (i) the release of other pro-inflammatory cytokines such as Interleukins (IL) -6, IL-8 and IL-1, (ii) the release of matrix metalloproteinases, and (iii) up-regulation of endothelial cell adhesion molecule expression, further expanding 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, TNFα is also involved in the prevention of specific infections (Cerami et al (1988), immunol. Today 9:28). Tnfα is released by macrophages which have been activated by lipopolysaccharides of gram-negative bacteria. Tnfα thus 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, described and patented in US 6090382 (WO 9729131), 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 techniques. Adalimumab binds specifically to TNF The combination neutralizes TNF biological functions by blocking its interaction with TNF receptors p55 and p75 on the cell surface.

A variety of adalimumab formulations are known in the art (see, e.g., WO2004016286 and WO 2012065072).

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

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

Accordingly, there is a need to provide a new drug comprising antibodies that bind tnfα, such as adalimumab.

The present invention relates to stable aqueous compositions comprising adalimumab, enabling long-term storage and also having good tolerance to freeze-thawing and heat stress.

Summary of The Invention

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

In one aspect, the invention relates to a method for treating a tnfα -mediated disease, 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 the group consisting of active (moderate to severe) rheumatoid arthritis, active psoriatic arthritis, active ankylosing spondylitis, chronic (moderate to severe) plaque psoriasis, (moderate to severe) ulcerative colitis, central axis type spondylitis, active suppurative sweat gland inflammation, juvenile idiopathic arthritis, (moderate or severe) crohn's disease, uveitis, active attachment point inflammation-associated arthritis.

In one aspect, the invention relates to the use of the above composition for the treatment of tnfα mediated diseases.

In some embodiments of 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, central axis type spondyloarthritis, active suppurative sweat gland inflammation, juvenile idiopathic arthritis, (moderate or severe) crohn's disease, uveitis, active attachment point inflammation-related arthritis.

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

Brief Description of Drawings

FIG. 1 dependence of the optical density of the solution at 400nm on the concentration of PEG 6000 after preparation.

FIG. 2 dependence of the optical density of the solution at 400nm on the concentration of PEG 6000 after preparation.

FIG. 3 dependence of the optical density of the solution at 400nm on the concentration of PEG 6000 after preparation.

Fig. 4. Adalimumab aggregation sites in acetate buffer (pH 5.0). T (T) _ag 74.0 ℃. Dark grey: z-average, nm, light grey: intensity, kcps.

Fig. 5. Adalimumab aggregation points in citrate buffer (pH 5.0). T (T) _ag 69.5 ℃. Dark grey: z-average, nm, light grey: intensity, kcps.

Fig. 6. Adalimumab aggregation point in histidine buffer solution (pH 5.0). T (T) _ag 69.5 ℃. Dark grey: z-average, nm, light grey: intensity, kcps.

Fig. 7. Adalimumab aggregation sites in phosphate buffered solution (pH 5.0). T (T) _ag 71.0 ℃. Dark grey: z-average, nm, light grey: intensity, kcps.

Fig. 8. Adalimumab aggregation points in humira formulation 1 (pH 5.0). T (T) _ag 69.5 ℃. Dark grey: z-average, nm, light grey: intensity, kcps.

Disclosure of the invention

Definition and general method

The terms used in the present 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. In the following or elsewhere herein, certain terms used to describe the invention are defined to provide additional guidance to the practitioner for describing the invention. Synonyms for some of the terms are provided. The recitation of one or more synonyms does not exclude 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 invention is not limited to the various embodiments presented in this specification.

Unless defined otherwise, 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 the event of a conflict, the present document, including definitions, will control.

The term "adalimumab" andis considered synonymous with or intended as a biologically similar or biologically modified variant thereof. Adalimumab is a human recombinant monoclonal IgG1 antibody specific for human TNF. Adalimumab is also known as D2E7. Adalimumab comprises two light chains (each of about 24kDa molecular weight) and two IgG1 heavy chains (each of about 49kDa molecular weight). Each light chain consists of 214 amino acid residues and each heavy chain consists of 451 amino acid residues. Thus, adalimumab consists of 1330 amino acids. The term adalimumab is also intended to include commercially available +.>So-called biosimilar or biologically modified variants of adalimumab protein used in (a) are provided. For example, when commercially available->Variants of (2) have the same meaning asCommercially available->Substantially the same pharmacological action, even though it may exhibit a pharmacological effect which may be as good as +.>Similar (if not identical) certain physical properties (such as glycosylation profile and acid-base profile) may also be FDA acceptable.

For the purposes of this application, the term "adalimumab" also includes adalimumab having minor modifications in the amino acid structure (including deletions, additions and/or substitutions of amino acids) or in glycosylation characteristics and acid-base profile (which do not significantly affect the function of the polypeptide). The term "adalimumab" includes Including, but not limited to, concentrated formulations; an injectable ready-to-use formulation; formulations reconstituted with water, alcohols, and/or other ingredients, and the like.

The term "human TNFα", also known as hTNFα or TNFα, means a human cytokine which exists in a 17kDa secreted form and in a 26kDa membrane-associated form, the biologically active form of which consists of a trimer 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, category No.210-TA, minneapolis, minn.).

The term "antibody" as used herein refers to an immunoglobulin-type molecule consisting of 4 polypeptide chains, two heavy chains (H) and two light chains (L) being interconnected by an internal disulfide bond. 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 (CH 1, CH2 and CH 3). 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. VH and VL regions can be further subdivided into regions of higher variability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FR). 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 formulation comprises antibodies having CDR1, CDR2 and CDR3 sequences similar to those described in U.S. patent nos. 6090382, 6258562 and 8216583.

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

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

The term "anti-tnfα antibody" refers to human anti-tnfα antibodies described herein and in U.S. patent nos. 6090382, 6258562, 6509015, 7223394 and 6509015. The term anti-TNFα antibody as used herein is an anti-TNFα antibody or a combination thereofFragments, including infliximabJohnson and Johnson, described in U.S. patent No. 5656272), CDP571 (humanized monoclonal anti-tnfa IgG4 antibody), CDP870 (humanized monoclonal anti-tnfa antibody fragment), anti-TNF dAb (Peptech), CNTO148 (golimumab; centocor, see WO 02/12502 and U.S.521206 and U.S. 7250165) and adalimumab (>Abbott Laboratories, a human anti-TNF mAb, described in US 6090382 as D2E 7). Additional anti-TNF antibodies useful in the present invention are described in U.S. patent nos. 6593458, 6498237, 6451983 and 6448380. In another embodiment, the TNFα inhibitor is a TNFfusion protein, e.g., etanercept (/ -A)>Amgen; described in WO 91/03553 and WO 09/406476). In another embodiment, the TNFα inhibitor is a recombinant TNFbinding protein (r-TBP-I) (Serono).

The term "long term storage" or "long term stability" is understood to mean that the pharmaceutical composition can be stored for 3 months or more, 6 months or more, and preferably for one year or more, most preferably for a minimum stable shelf life of at least two years. In general, the terms "long-term storage" and "long-term stability" further include a stable storage duration that is at least comparable to or better than the stable shelf-life typically required for currently available commercial formulations of adalimumab without losing stability, which would render the formulation unsuitable for its intended pharmaceutical use. Long term storage is also understood to mean that the pharmaceutical composition is stored as a liquid at 2-8 ℃ or frozen (e.g. at-18 ℃ or lower). It is also contemplated that the composition may be frozen and thawed more than once.

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

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

The term "sugar" refers to mono-, di-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 excipients 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 to resist changes in pH, typically by the action of its acid-base conjugated components. When referring to the concentration of 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 buffered saline, citrate-phosphate buffers, tromethamine-based buffers, and the like or suitable mixtures thereof.

The terms "tonicity agent" or "tonicity agent" and "osmotic agent" or "osmotic agent" as used herein refer to excipients that can regulate the osmotic pressure of a liquid antibody formulation. In certain embodiments, the tonicity agent can adjust the osmolality of the liquid antibody formulation to be isotonic, such that the antibody formulation is 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 human blood. Isotonic formulations typically have an osmotic pressure of about 250-350 mOsm. The term "hypotonic" describes a formulation with an osmotic pressure lower than human blood. Thus, the term "hypertonic" is used to describe a formulation with a higher osmotic pressure than human blood. Isotonicity can be measured using, for example, a vapor pressure or ice-cold type osmometer. The tonicity agent may exist as enantiomers (e.g., L-or D-enantiomers) or racemates, as isomers (such as alpha or beta, including alpha, or beta, or alpha, beta, or beta, alpha), as free acid or free base, as salts, as hydrates (e.g., monohydrate), or as anhydrous forms.

The term "surfactant" (also referred to as a 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 surface active substance reduces the surface tension of the liquid antibody formulation. In other embodiments, a "surface active agent" may help improve the colloidal stability or solubility of any antibody in the formulation. The surface-active substance may reduce aggregation of the resulting antibody preparation and/or minimize particle formation and/or reduce adsorption in the preparation. The surface active substances may also improve the stability of the antibody during and after freeze/thaw cycles and upon shaking. The surface active material may be ionic or nonionic. Exemplary nonionic surfactants that may be included in the formulations of the present invention include, for example, alkyl poly (ethylene oxide), alkyl polyglucosides (e.g., octyl glucoside and decyl maltoside), fatty alcohols such as cetyl alcohol and oleyl alcohol, cocamide MEA, cocamide DEA, and cocamide TEA. Specific nonionic surface active substances 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 65, polysorbate 80 (Tween 80), polysorbate 81, and polysorbate 85; poloxamers such as poloxamer 188 (Kolliphor P188), poloxamer 407; polyethylene glycol-polypropylene glycol; or polyethylene glycol (PEG), ethylene glycol-propylene glycol copolymers (e.g., pluronics PF68, etc.).

The term "lyophilized" as used herein refers to a formulation that has undergone a process known in the art as lyophilization, which includes freezing the formulation and then removing ice from the frozen contents.

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

"pharmaceutical composition" means a composition comprising an antibody of the invention and at least one component selected from pharmaceutically acceptable and pharmacologically compatible excipients, solvents, diluents, carriers, additives, dispersants, delivery agents, such as preservatives, stabilizers, emulsifiers, suspending agents, thickeners, and extended delivery modifiers, the choice and ratio of which depend on the nature and method of administration and administration. Exemplary suspending agents include ethoxylated isostearyl alcohols, polyoxyethylene, sorbitol and sorbitol esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof. Various antibacterial and antifungal agents, such as nipagins, chlorobutanol, sorbic acid, and the like, can be used to provide protection from microbial action. The composition may also contain isotonic agents, for example, sugars, sodium chloride, and the like. Agents that delay the absorption of the active agent (e.g., aluminum monostearate and gelatin) may be used to provide a prolonged action of the composition. Exemplary suitable carriers, solvents, diluents, and vehicles 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, sodium citrate, calcium carbonate, calcium phosphate, and the like. Pharmaceutical compositions of the active agent (alone or in combination with another active agent) for oral, sublingual, transdermal, intramuscular, intravenous, subcutaneous, topical or rectal administration can be administered to animals and humans in unit dosage forms as a mixture with conventional pharmaceutical carriers. Suitable unit dosage forms include parenteral forms, implants and transdermal systems.

"drug (preparation)" is a substance (or mixture of substances in the form of pharmaceutical compositions) in the form of tablets, capsules, powders, lyophilized products, injections, infusions, ointments and other finished dosage forms intended to restore, correct or alter physiological functions in humans and animals and to provide disease treatment and prevention, diagnosis, anesthesia, contraception, cosmetology, etc.

"treatment" and "treatment" refer to methods for alleviating or eliminating a biological disorder and/or at least one of its concomitant symptoms. As used herein, "alleviating" a disease, disorder or condition means reducing the severity and/or frequency of symptoms of the disease, disorder or condition. In addition, 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 may be male or female of any age.

The term "disorder" means any condition that can be ameliorated by the treatment of the present invention. The definition of the term includes chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to said disorders. Non-limiting examples of diseases to be treated include benign and malignant tumors; leukemia and lymphoid malignancies, in particular breast, ovarian, gastric, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic, prostate or bladder cancer; neurological, glial, astrocyte, hypothalamic and other glandular, macrophage, epithelial, stromal and blastula cavity disorders; inflammatory, angiogenic and immune disorders. A preferred disorder to be treated according to the invention is an autoimmune disease.

The terms "immune response", "autoimmune inflammation" refer to the action of, for example, lymphocytes, antigen presenting cells, phagocytes, granulocytes, and soluble macromolecules produced by the above cells or hepatocytes, including antibodies, cytokines, and complements produced by selectively damaging, destroying, or eliminating invasive pathogens, pathogen-infected cells or tissues, cancer cells, or normal human cells or tissues in the case of autoimmune or pathological inflammation.

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.

The definition includes, but is not limited to, rheumatoid arthritis, arthropathy, juvenile chronic arthritis, septic arthritis, lyme's arthropathy, psoriatic arthritis, reactive arthritis, spondyloarthropathies, systemic lupus erythematosus, crohn's disease, ulcerative colitis, inflammatory bowel disease, diabetes, thyroiditis, asthma, allergic diseases, psoriasis, atopic dermatitis, scleroderma, graft versus host reaction, transplant rejection, acute or chronic immune diseases associated with transplantation, sarcoidosis, kawasaki disease, grave's disease, nephrotic syndrome, chronic fatigue syndrome, wegener's granulomatosis, henry-sjogren's purpura, microscopic renal vasculitis, chronic active hepatitis, uvnita, septic shock, toxic shock syndrome, septic syndrome, cachexia, acquired immunodeficiency syndrome, acute transverse myelitis, huntington's chorea, parkinson's disease, alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia, adult (acute) respiratory distress syndrome, alopecia, focal alopecia, seronegative arthropathy, leptospirosis, psoriasis arthropathy, arthropathy associated with ulcerative colitis, atopic allergy, autoimmune bullous disease, pemphigus vulgaris, pemphigus, pemphigoid, linear IgA, autoimmune hemolytic anemia, coombs positive hemolytic anemia, pernicious anemia, juvenile pernicious anemia, arthritis, primary sclerotic hepatitis a, cryptogenic autoimmune hepatitis, pulmonary fibrosis, cryptogenic fibroalveolar inflammation, post inflammatory interstitial lung disease, interstitial pneumonia, chronic eosinophilic pneumonia, post-infection interstitial lung disease, gouty arthritis, autoimmune hepatitis type 1 (classical autoimmune hepatitis or lipid), autoimmune hepatitis type 2, osteoarthritis, primary sclerosing cholangitis, psoriasis type 1, psoriasis type 2, idiopathic leukopenia, autoimmune neutropenia, renal NOS disease, glomerulonephritis, microscopic renal vasculitis, discoid lupus erythematosus, idiopathic or male NOS-fertility, [ sperm autoimmunity ], multiple sclerosis of all subtypes, sympathetic ophthalmitis, secondary pulmonary arterial hypertension associated with connective tissue disease, goodescher syndrome, pulmonary manifestations of nodular polyarteritis, acute rheumatic fever, rheumatoid spondylitis, ankylosing spondylitis, szechwan's disease, systemic sclerosis Sjogren's syndrome, gaoan's disease, autoimmune thrombocytopenia, primary thrombocythemia, autoimmune thyroiditis, hyperthyroidism, hashimoto's disease, autoimmune atrophic hypothyroidism, primary myxoedema, uveitis of lenticular origin, primary vasculitis, vitiligo, acute liver disease, chronic liver disease, allergies, asthma, psychotic disorders (including depressive syndrome and schizophrenia), th 2-and Th 1-mediated diseases, conjunctivitis, allergic contact dermatitis, allergic rhinitis, alpha-I antitrypsin deficiency, amyotrophic lateral sclerosis, anemia, cystic fibrosis, diseases associated with cytokine therapy, demyelinating diseases, dermatitis, iridocyclitis/uveitis/ophthalmic neuritis, ischemic reperfusion injury, ischemic stroke, juvenile rheumatoid arthritis, autoimmune enteropathy, autoimmune hearing loss, autoimmune lymphoproliferative syndrome, autoimmune myocarditis, autoimmune premature ovarian failure, and blepharitis. Antibodies may also treat any combination of the disorders listed above.

The term "disorder in which tnfα activity is detrimental" as used herein includes diseases and other disorders in which the presence of tnfα in a subject suffering from the disorder has been shown or suspected to be the cause of the pathophysiology of the disorder or to contribute 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 alleviate symptoms and/or progression of the disorder. Such disorders can be detected, for example, by an increase in the concentration of tnfα in a biological fluid of a subject having the disorder (e.g., an increase in the concentration of tnfα in the serum, plasma, synovial fluid, etc., of the subject), which increase can be detected, for example, using an anti-tnfα antibody as described above. There are many examples of disorders in which tnfα activity is detrimental. The use of antibodies and antibody fragments suitable for the treatment of a particular disorder is discussed further below:

A. sepsis of the body

Tumor necrosis factor plays a definite role in the pathophysiology of sepsis, and its biological effects include hypotension, myocardial inhibition, vascular leakage syndrome, organ necrosis, stimulation of toxic secondary mediator release and activation of the coagulation cascade (see, e.g., moeller A., et al (1990), cytokine 2:162-169;U.S.Patent No.5231024Moeller et at; 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 are useful in the treatment of sepsis present in any clinical manifestation thereof, including septic shock, endotoxic shock, sepsis caused by gram negative bacteria, and toxic shock syndrome.

In addition, for sepsis treatment, the anti-tnfα antibodies and antibody fragments of the present invention may be co-administered with one or more therapeutic agents that may further reduce sepsis, such as an interleukin-1 inhibitor (described in PCT publication nos. WO 92/16221 and WO 92/17583), cytokine interleukin-6 (see, e.g., PCT publication No. WO 93/11793), or a platelet-activating factor antagonist (see, e.g., european patent application No. EP 374 510). Other combination therapy methods for sepsis treatment are discussed in section III below.

In addition, in a preferred embodiment, the anti-TNFα antibodies and antibody fragments of the invention are administered to humans from a subset of sepsis patients who have serum or plasma IL-6 concentrations above 500pg/mL and more preferably 1000pg/mL during treatment (see PCT publication No. WO 95/209788 Daum, L., et al).

B. Autoimmune diseases

Tumor necrosis factors have been shown to play a role in the pathophysiology of a variety of autoimmune diseases. For example, TNF alpha is also involved in activating tissue inflammation and causing joint destruction in rheumatoid arthritis (see, e.g., moeller A.et al (1990), cytokine 2:162-169; U.S. Pat. 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.94:261-266). TNFα is also involved in promoting islet cell death and the development of insulin resistance in diabetes (see, e.g., tracey and Cerami, supra; PCT publication No. WO 94/08609). Tnfα is also involved in the cytotoxic formation of oligodendrocytes and induction of inflammatory plaques in multiple sclerosis (see, e.g., tracey and Cerami, supra). Chimeric and humanized murine anti-hTNFα antibodies have been clinically tested for the treatment of rheumatoid arthritis (see, e.g., 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. Rheumatoid.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 it may be beneficial to administer the antibody or antibody fragment locally at the site of inflammation (e.g., locally to the joint in rheumatoid arthritis or to administer the diabetic ulcer locally, alone or in combination with a cyclohexane-subunit derivative as 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 section III.

C. Infectious diseases

Tumor necrosis factor is involved in the generation of biological effects observed in a variety of infectious diseases. For example, tnfα is involved in the development of cerebral inflammation and capillary thrombosis and infarction in malaria. Tnfα is also involved in the development of brain inflammation, in the induction of blood brain barrier rupture, in the development of septic shock syndrome, and in the activation of venous infarction in meningitis. Tnfα is also involved in inducing cachexia, stimulating viral proliferation and the formation of central nervous system lesions 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, e.g., european patent application No. EP 585705), cerebral malaria, AIDS and AIDS related complex (αc) (see, e.g., european patent application No. EP 230574), and cytomegalovirus infection secondary to Transplantation (see, e.g., fietze, e., et al (1994) translation 58:675-680). The antibodies and antibody fragments of the invention are also useful for alleviating symptoms associated with infectious diseases, including fever and myalgia due to infection (such as influenza) and cachexia secondary to infection (e.g., secondary to AIDS or oc).

D. Transplantation

Tumor necrosis factor is believed to be a critical mediator in allograft rejection and Graft Versus Host Disease (GVHD) and in the development of adverse reactions observed when using the rat antibody OKT3 against the T cell receptor CD3 complex to inhibit rejection of kidney transplants (see, e.g., easton J.D.et al. (1995) transformation 59:300-305;Suthanthiran M. And boom T.B. (1994) New Engl.J.Med.331:365-375). Accordingly, the antibodies and antibody fragments of the invention are useful for inhibiting graft rejection (including allograft and xenograft rejection) and inhibiting GVHD. Although the antibodies or antibody fragments may be used alone, more preferably they are used in combination with one or more other agents that inhibit immune responses against allografts or inhibit GVHD. For example, in one embodiment, the antibodies or antibody fragments of the invention are used in combination with one or more antibodies directed against other targets involved in the modulation of an immune response, such as the cell surface molecules CD25 (interleukin-2 receptor-beta), CD11a (LFA-1), CD54 (ICAM-1), CD4, CD45, CD28/CTLA4, CD80 (B7-1) and/or CD86 (B7-2). In yet another embodiment, the antibodies or antibody fragments of the invention are used in combination with one or more general immunosuppressants (such as cyclosporin a or FK 506).

E. Malignant tumor

Tumor necrosis factor is involved in inducing cachexia, stimulating tumor growth, enhancing metastatic potential and development of cytotoxicity in malignant tumors. Accordingly, the antibodies and antibody fragments of the invention are useful for treating malignancy, inhibiting tumor growth or metastasis and/or alleviating cachexia secondary to malignancy. The antibody or antibody fragment may be administered to the tumor site either systemically or locally.

F. Pulmonary disorders

Tumor necrosis factor is involved in the pathophysiology of adult respiratory distress syndrome (αds), including stimulation of endothelial leukocyte activation, guidance of cytotoxicity to lung cells, and induction of vascular leakage syndrome. Accordingly, the antibodies and antibody fragments of the invention are useful in the treatment of a variety of pulmonary disorders, including adult respiratory distress syndrome (see, e.g., PCT publication No. WO 91/04054), shock lung, chronic pulmonary inflammatory disease, pulmonary sarcoidosis, pulmonary fibrosis, and silicosis. The antibodies and antibody fragments of the invention may be administered with one or more additional therapeutic agents for treating pulmonary disorders, as described in section III below.

G. Intestinal disorders

Tumor necrosis factor is involved in the pathophysiology of inflammatory bowel disease (see, e.g., 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-TNFα 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 are also useful in the treatment of intestinal disorders, such as idiopathic inflammatory bowel disease, which includes both 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 section III below.

H. Heart disorders

The antibodies or antibody fragments of the invention may also be used to treat a variety of cardiac disorders, including cardiac ischemia (see, e.g., european patent application No. EP 453898) and heart failure (myocardial weakness) (see, e.g., PCT publication No. WO 94/20139).

I. Others

The antibodies or antibody fragments of the invention may also be used to treat various disorders in which tnfα activity is detrimental. Examples of other diseases and disorders in which TNFα activity is involved in pathophysiology and thus can be treated using 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. (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, e.g., clalin C.J. and Cohen D.A. (1989) Hepatogy 9:349-351;Felver M.E.et al (1990) Alcohol Clin.Exp.Res.14:255-259; and Hansen J.J.4:241 and Shain P.H. (1993) Bone 14:871-876), hepatitis (see, e.g., clalin C.J. and Cohen D.A. (1989) Hectol.M.9:349.349. (1990) and Hansen.P.E.1:474); coagulation disorders (see, e.g., van der Poll. Et al (1990) N.Engl. J.Med.322:1622-1627;van der Poll T.et al (1991) prog. Clin. Biol. Res.367:55-60; burns (see, e.g., giroir B.P.et al (1994) am. J.Physiol.267:H118-124;Liu X.S.et al (1994) Burns 20:40-44), reperfusion injury (see, e.g., scales W.E.et al (1994) am. J.Physiol.26:1122-1127; serrick. Et al (1994) transplatin 58:1158-1162;Yao Y.M.et al) (1995) Resuscitation 29:157-168), keloid formation (see, e.g., mcCaul R.et al et al (1992) J.Immunol. 12:300), scar formation and formation of toxic heat radiation, periodontal disease and periodontal disease.

A "therapeutically effective amount" is considered to be the amount of 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 the continuous (long-term) administration of an agent as opposed to an acute (short-term) administration, so as to maintain an initial therapeutic effect (activity) for an extended period of time.

By "intermittent" administration is meant that the treatment is not carried out uninterrupted continuously, but rather is cyclical in nature.

In this specification and the claims which follow, unless the context requires otherwise, the words "comprise", "comprising" and "include" or variations thereof such as "have", "include", "comprise" or "include" will be understood to include the stated integer or group of integers but not to exclude any other integer or group of integers.

Detailed Description

Aqueous composition

The present invention relates to new and improved aqueous compositions, which may optionally be lyophilized, comprising an appropriate amount of recombinant monoclonal anti-tnfα antibody in a suitable buffer, one or more suitable stabilizers and other excipients selected from suitable surfactants and isotonic agents. 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 a combination of D2E7 with other antibodies. The D2E7 antibody, commonly referred to as adalimumab, has high affinity for binding tnfα, low dissociation kinetics and high neutralization capacity. Adalimumab can be trademarkedAre available in the pharmaceutical market. The designations adalimumab and D2E7 used throughout this specification represent the same human monoclonal antibody. In a preferred embodiment, the monoclonal antibody is adalimumab or antigen binding portion thereof.

In some embodiments, the recombinant monoclonal anti-tnfα 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 1mg/mL to about 150mg/mL. In a more preferred embodiment, the therapeutic amount is from about 1mg/mL to about 100mg/mL. In a more preferred embodiment, the therapeutic amount is from about 50mg/mL to about 100mg/mL. In an even more preferred embodiment, the therapeutic amount is about 100mg/mL.

The aqueous compositions in question comprise a suitable buffer solution in combination with pharmaceutically acceptable excipients of other stable pharmaceutical formulations. Suitable buffer solutions that may be used are selected from the group 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, the suitable buffer comprises an acetate buffer. In an even more preferred embodiment, the acetate buffer is selected from the group consisting of sodium acetate trihydrate in combination with acetic acid.

The buffer solution is typically used at a concentration of about 1mM to about 100 mM. In a preferred embodiment, the buffer concentration is from about 1mM to about 50mM. In a more preferred embodiment, the buffer concentration is from about 1mM to about 20mM. In an even more preferred embodiment, the buffer concentration is about 5mM.

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

In one embodiment, the liquid composition maintains the 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 to about 6.0. In a more preferred embodiment, the pH is maintained at about 5.5.

The aqueous composition comprises a suitable surface active substance, which is a pharmaceutically acceptable excipient, for protecting the protein composition from various stress effects, such as stirring, shearing, high temperature, freezing, etc. Suitable surface-active substances include, but are not limited to, polysorbates or poloxamers or a combination of polysorbates and poloxamers. In a preferred embodiment, a suitable surface active substance is polysorbate. In a preferred embodiment, a suitable surfaceThe active substance is 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 under the trademark polysorbate 80 20 or->80 distribution). In a more preferred embodiment, the poloxamer is poloxamer 188 (also 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 10mg/mL. In a preferred embodiment, the amount of polysorbate 20 is about 0.05mg/mL to about 10mg/mL. In a more preferred embodiment, the amount of polysorbate 20 is about 0.1mg/mL to about 1mg/mL. In a more preferred embodiment, the amount of polysorbate 20 is about 0.5mg/mL.

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

In some embodiments, poloxamer 188 is present in an amount of up to 10mg/mL. In a preferred embodiment, the amount of poloxamer 188 is between about 0.05mg/mL and about 10mg/mL. In a more preferred embodiment, the amount of poloxamer 188 is between about 0.1mg/mL and about 1mg/mL. In a more preferred embodiment, the amount of poloxamer 188 is about 0.5mg/mL.

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

The aqueous composition comprises one or more suitable stabilizers which are pharmaceutically acceptable excipients and protect the pharmaceutically active ingredient from chemical and/or physical degradation during manufacture, storage and use. In one embodiment, the stabilizing agent includes, but is not limited to, an amino acid, an oligosaccharide, or a suitable derivative or mixture 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, oligosaccharides that may also be used as stabilizers include trehalose.

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

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

In another such embodiment, the amino acid that can be used as a stabilizer is proline.

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

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

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

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

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

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

The above composition is suitable for intravenous, subcutaneous or intramuscular administration.

In some embodiments, the liquid composition maintains storage stability to the effect of: there was no further protein aggregation process or modification compared to the stability measurement at zero time.

Methods of treatment and use of aqueous compositions

In another embodiment, the 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 invention, wherein the mammal may have a disease or disorder that is effectively treatable with adalimumab.

In a preferred embodiment, the mammal is a human.

Diseases or disorders treatable with the provided compositions include, as non-limiting examples, active (moderate to severe) rheumatoid arthritis, active psoriatic arthritis, active ankylosing spondylitis, chronic (moderate to severe) plaque psoriasis, (moderate to severe) ulcerative colitis, central axis spondylitis, active suppurative sweat gland inflammation, juvenile idiopathic arthritis, (moderate or severe) crohn's disease, uveitis, active attachment point inflammation-related arthritis. Additional diseases or disorders that may be treated with the compositions of the present invention include those described in U.S. patent nos. 6090382 and 8216583, the relevant portions of which are incorporated herein by reference.

The pharmaceutical compositions provided may be injected systemically, e.g., intravenously or subcutaneously, or intramuscularly; or by injection or application to the relevant site, for example 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, the method comprising administering to a mammal in need thereof a therapeutically effective amount of one of the provided adalimumab compositions.

The therapeutically effective amount of adalimumab in the provided compositions depends on the condition to be treated, the severity of the condition, the prior treatment and the patient's medical history, and the response to the therapeutic agent. The appropriate dosage may be adjusted at the discretion of the attendant physician so that it may be administered to the patient in one or more administrations.

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

Alternatively, a fixed dose may be administered in an amount ranging from 2 to 500 mg/dose, from 2 to 100 mg/dose, or from about 10 to 80 mg/dose.

If the dose is administered more than once a week, exemplary dose ranges are the same as or lower than the above-described dose ranges, and are preferably administered twice or more weekly in a dose range of 25-100 mg/dose.

In another embodiment, the acceptable dose administered by injection comprises 80-100 mg/dose or 80mg per dose.

The dose may be administered once a week, or several weeks apart (e.g., from 2 to 8).

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

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

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

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

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

The pharmaceutical compositions of the invention are particularly usefulFor parenteral administration, i.e., subcutaneous, intramuscular, intravenous, intraperitoneal, intramedullary, intra-articular, intrasynovial and/or intrathecal. Parenteral administration may be by bolus injection or continuous infusion. The pharmaceutical composition for injection may be presented in unit dosage form such as, but not limited to, an ampoule, a vial, a prefilled syringe, or a multi-dose container containing 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 novel methods, e.g., project- Syringes such as +.>And needleless devices such as +.>And->The pharmaceutical compositions of the present invention are also suitable for use in an as yet undiscovered mode of administration. See also Langer,1990, science,249:1527-1533.

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

If desired, the pharmaceutical compositions may be provided in vials, packages, or dispenser devices, which may contain one or more unit dosage forms containing the active ingredient. In one embodiment, the dispenser device may comprise a syringe containing a single dose of a ready-to-use injectable liquid formulation. The syringe may be accompanied by instructions for administration.

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

Method of production

The method for producing the above composition comprises adding an acetate buffer to the aqueous phase followed by the following components in any order: an osmotic agent and/or a stabilizing agent selected from trehalose, proline, or a combination thereof; recombinant monoclonal anti-tnfα antibodies; a surfactant selected from polysorbate 20, polysorbate 80, poloxamer 188, or a combination thereof.

Techniques for

1. Protein concentration determination in test samples

Protein concentration was determined in a UV spectrophotometry plate using UV spectrophotometry at a wavelength of 280 nm.

Each sample was diluted with the appropriate placebo solution to a concentration of 0.5 mg/mL. 150 μl of the diluted sample was placed in the wells of a UV spectrophotometry plate. The optical density of the plate solution was measured at a wavelength of 280nm using a plate reader. A suitable placebo solution was used as a reference solution.

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

А ₂₈₀ an optical density value at a wavelength of 280 nm;

epsilon is the extinction value of the research protein;

b is the total dilution ratio of the sample;

l is the layer thickness of each plate well, l=0.42 cm for 150 μl.

2. Buffer exchange and sample concentration

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

"PEG aggregation"

Preparation of PEG 6000 solution

PEG 6000 solution with mass concentration of 20-25% in the research additive preparation is prepared. The resulting solution was filtered through a 0.45 μm Durapore filter.

Calculated amounts of sample, additive solution and 20-25% PEG 6000 solution were transferred to a 96-well UV spectrophotometry plate such that the PEG 6000 concentration per well was 0-18% and the protein concentration per well was 1mg/mL. All resulting solutions in the wells were thoroughly mixed by pipetting.

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

4. Determination of protein homogeneity and aggregation point using Dynamic Light Scattering (DLS) techniques

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

Analytical model: and (5) protein analysis.

The temperature was maintained for 30 seconds prior to measurement.

At each point, an average of 13 measurements was made in 3 replicates.

The assay to investigate the protein aggregation point was performed on Zetasizer Nano ZSP. For this purpose, the solution is placed in a quartz dust-free cuvette and gradually heated in the instrument while the intensity of the scattered light is continuously measured in a temperature trend measurement mode.

Analytical model: and (5) protein analysis.

Trend of temperature, mod: protein aggregation sites. Heating from 50 ℃ to 85 ℃ at a step distance of 1.5 ℃.

The temperature was maintained for 30 seconds prior to measurement.

At each point, 15 measurements were taken on average in 1 repeat.

Temperature trends were plotted using instrument software, which automatically calculated protein aggregation points (aggregation points).

5. Determination of thermal stability by the "Heat stress 50 ℃ technique

Test samples were divided into 2 portions and placed in separate tubes: 1 tube of each formulation was stored in a refrigerator at +4℃, the remainder was placed in an incubator and incubated at 50℃for the indicated time. After the heating period was completed, the tube was 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 "Oscillating test" technique

The test samples were divided into 2 parts, 200 μl each, and placed in glass vials, 1 vial of each formulation was stored in a refrigerator at +4℃, the remainder was placed in a hot shaker, and the shaker was run at 800rpm for the indicated time at 2-8 ℃. Upon completion of the stress, the tube was removed from the thermal shaker and analyzed according to the instructions.

7. Determination of colloidal stability by cryoconcentration technique

The test samples were divided into 2 portions and placed in a polymer tube: 1 tube of each formulation was stored in a +4℃ refrigerator, the remainder was placed in a freezer and stored at-16 ℃ to 20 ℃ for a specified period of time. Upon completion of the stress, the tube was removed from the freezer, allowed to stand at room temperature until the contents were completely thawed, the solution was mixed using 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×30cm,cat.#08541。

Column temperature: 25 ℃.

Mobile phase flow rate: 0.7mL/min.

Injection volume: 20. Mu.L.

Sample concentration: 5mg/mL.

Detector wavelength: 220nm.

Elution time: 23 minutes.

Mobile phase: anhydrous disodium hydrogen phosphate: 7.1mg/mL.

Sodium chloride: 17.54mg/mL.

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

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

9.1. Sample preparation

The sample was diluted to a concentration of 1mg/mL. mu.L of 5mg/mL carboxypeptidase B (CpB) solution was added to 200. Mu.L of the resulting solution. It was stirred and incubated at 37℃for 2 hours. The test samples were dialyzed against 3 changes of water in an Amicon Ultra centrifuge tube and concentrated to 2mg/mL.

9.2. Working solution preparation

Using the HT Protein Charge Variant labeling kit, working solutions and plates with assay samples were prepared 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.

The assay starts with standard operation. Protein Charge Variant 68 s.

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

10.1. Assay sample preparation

A700. Mu.L HT protein expression sample buffer supplemented with 24.5. Mu.L 1M DTT for buffer under reducing conditions and 24.5. Mu.L 1M IAM for buffer under non-reducing conditions was used to prepare denaturing and reducing solutions. The sample was diluted to a concentration of 2 mg/mL. Two microtubes were prepared for each sample-35. Mu.L of denaturation buffer was added to one and 35. Mu.L of reduction buffer to the other. The sample was denatured at 100℃for 5 minutes. Vortex tubes and 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 solutions and chip preparation were performed according to standard procedures using HT Protein Express Reagent kit. The assay starts with standard operation. HT Protein Express 200 measurement.

11. Determination of sample acid-base distribution using Ion Exchange (IE) HPLC

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

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

The electrophoresis chamber is assembled and installed according to an operation manual of the vertical electrophoresis apparatus. Samples were prepared by diluting the samples with purified water to a final concentration of 1 mg/mL. A volume corresponding to 40. Mu.g was taken out, and the samples of the prepared test samples were mixed with a sample containing 2-mercaptoethanol (reducing conditions) and no 2-mercaptoethanol (non-reducing conditions) at a ratio of 3:1 (v/v) with application of a buffer solution, and mixed. The resulting solution was incubated at a temperature of (99.+ -. 1) ℃ for 3 minutes (sample containing 2-mercaptoethanol) and at a temperature of (99.+ -. 1) ℃ for 1 minute (sample containing no 2-mercaptoethanol). The solution was allowed to cool to room temperature, mixed and transferred into the PAG wells under the running buffer solution layer.

Electrophoresis was run in direct current mode using a water cooling system. The power supply parameters were set as follows: the voltage was 110V when the dye front passed through the laminated gel. Once the dye front enters the lower run gel 5-7mm, the voltage is increased to 180V. When the dye front reaches the lower limit of the gel, the power supply is turned off.

Once electrophoresis was completed, the gel was separated from the glass and the protein was fixed with a fixing solution at room temperature for 16-18 hours. Further, the gel was stained (in acid blue 83 solution) and washed until a clear view of the band. The gel was scanned. Purity and impurities in the test samples were evaluated using GelPro software.

13. Determination of specific Activity

The specific activity of the samples was assessed by the ability to specifically bind and neutralize tnfα, which in turn has a cytotoxic effect and leads to death of WEHI-13var cultures (fibrosarcoma, mice), a variant 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, 2mM gin, 10% fbs, 2.5 μg/mL actinomycin D, 5 μg/mL gentamicin was used as QM (quantitative medium).

The test antibody samples were diluted to 50 μg/mL with no more than 20 dilution steps and placed in a robotic workstation. Using Tecan Evo 200, 3 independent dilutions of RS and IS were prepared in culture plates ranging from 10000-0.5ng/mL, and 500pg/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). Times.10 is added ⁶ WEHI-13var cell suspensions per cell/mL. Placing the plate in CO ₂ In an incubator and at a temperature of (37.+ -. 1) ℃ and 5% CO ₂ Is incubated in humidified air for 20-24 hours.

At the end of the incubation period, the vital dye alamar blue was added to the culture plates and the plates were incubated under the same conditions until color developed. Fluorescence intensity was assessed using an Infinite M200 Pro reader at excitation/emission wavelengths of 544/590 nm. Fluorescence intensity versus protein concentration curves were plotted using the magollan 7.2 software and the parallel arrangement of the resulting curves was assessed. The relative specific activity of the test samples was determined as the ratio of the reference sample ED50 to the test sample ED50, 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 as limiting the scope of the invention in any way.

All publications, patents and patent applications cited in this specification are herein incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity, as will be appreciated by one of ordinary skill in the art based on the teachings disclosed herein, certain changes and modifications may be made without departing from the spirit and scope of the appended embodiments of the invention.

Examples

Example 1. Buffer solution Properties selection.

Study preparation

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

1.1. Colloidal stability was determined 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 FIG. 1.

TABLE 1 average optical Density of the solutions after preparation

There is visible aggregation.

1.2. Thermal stability was determined by protein aggregation point using Dynamic Light Scattering (DLS) technique.

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

1.3. The thermal stability under prolonged exposure was determined using a heat stress 50 ℃ method.

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

1.4. As a result.

TABLE 2 overview of buffer property selections

Positive results->Negative result->Average result

1.5. Conclusion of example 1.

Based on the results of this study, the recommended stock formulation (excluding further additives) was:

the present formulation showed the best stability performance among all test samples: the least increase in impurities during heat stress (0.02% level equivalent to the Humira 2 formulation), absence of aggregation at 18% PEG 6000 and highest aggregation temperature (74 ℃).

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 5mM citrate buffer solution at pH 5.0.

Example 2 composition pH and buffer capacity optimisation.

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

Study preparation

Abbreviations (abbreviations)	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. Colloidal stability was determined 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 FIG. 2.

TABLE 3 average optical Density of the solutions after preparation (400 nm)

2.2. The thermal stability under prolonged exposure was determined using a heat stress 50 ℃ method.

Analysis was performed according to procedure 5.

2.3. Colloidal stability was determined by the "shake test" technique.

Analysis was performed according to procedure 6.

2.4. Colloidal stability was determined by low temperature concentration techniques.

Analysis was performed according to procedure 7.

An overview is provided in table 4, where sample mass measurements before and after heat stress, shaking test and low temperature concentration are demonstrated.

2.5. As a result.

TABLE 4 overview of sample mass measurements before and after stress

Positive results->Negative results

Mean results/baseline data->

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2.6. Conclusion of example 2.

Studies have shown that the presence of adalimumab in the acidic solution of the placebo results in a significant increase in pH levels. In addition, compositions with a pH of less than 5.0 show 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 5mM acetate buffer at pH 5.0-6.0. They demonstrate minimal changes in purity and acid-base distribution during stress. However, in order to produce 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 solutions at pH 5.0-6.0. Formulations with pH 5.5 were used as the basis for the selection of penetrants and stabilizers.

Study preparation

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3.1. Colloidal stability was determined 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 FIG. 3.

TABLE 6 average optical Density of adalimumab solution after preparation

There is visible aggregation.

3.2. Thermal stability was determined by protein aggregation point using Dynamic Light Scattering (DLS) technique.

Analysis was performed according to procedure 4.

3.3. The thermal stability under prolonged exposure was determined using a heat stress 50 ℃ method.

Analysis was performed according to procedure 5.

3.4. As a result.

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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, poloxamer 188 were used as surface active substances for the final pharmaceutical composition screening.

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

Sodium chloride, lysine, guanidine have an adverse effect on the thermal stability of adalimumab. The cysteine contained in the formulation resulted in complete fragmentation of the antibody. When EDTA additives were added to the formulation, a change in the acid-base profile of the protein was noted.

Example 4. Selection of the final pharmaceutical composition.

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, poloxamer 188 were used as surface active substances for the final pharmaceutical composition screening.

Research preparation (mg/mL)

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4.1. The thermal stability under prolonged exposure was determined using a heat stress 50 ℃ method.

Analysis was performed according to procedure 5.

4.2. Colloidal stability was determined by the "shake test" technique.

Analysis was performed according to procedure 6.

4.3. Colloidal stability was determined by low temperature concentration techniques.

Analysis was performed according to procedure 7.

An overview is provided in tables 8 and 9, where sample mass measurements before and after heat stress, shaking test and low temperature concentration are demonstrated.

4.4. As a result.

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4.4. Conclusion of example 4

Studies have shown that adalimumab in Humira formulation 1 is the least heat resistant of all the test samples. Heat stress at 50 ℃ for 120 hours resulted in the greatest increase in SE-HPLC based impurities (1.2%) and the greatest change in acid-base profile (total absolute change of all fractions was over 33% on LabChip Caliper instrument). The colloidal stability of the sample in Humira formulation 1 was comparable to the alternative formulation.

Humira formulation 2 had a thermal stability comparable to one of the alternative formulations (loss of purity during heat stress 0.38%). However, there is significant aggregation of the protein following freeze-thawing of adalimumab in the formulation of the invention: the increase in aggregates in size exclusion HPLC was over 7%, which made the formulation unusable in the event of its accidental freezing. The use of DLS also noted a significant increase in impurities after freezing.

Alternative formulations based on acetate buffer solutions and with the addition of a number of additives show good thermal and colloidal stability during stress. Size exclusion HPLC showed no difference in impurity increase in the formulations of the present invention. Furthermore, the surfactant material had no significant effect on the stability of adalimumab at a concentration of 15 mg/mL.

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

Note that the change in acid-base distribution was minimal 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 were:

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

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

Study preparation

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5.1. The thermal stability under prolonged exposure was determined using a heat stress 50 ℃ method.

Analysis was performed according to procedure 5.

5.2. As a result.

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General conclusion

1. The screening of stable adalimumab pharmaceutical compositions is performed in several stages: the choice of buffer solution properties, the choice of pH and buffer capacity of the solution, the choice of osmotic agent and stabilizer, the choice of final pharmaceutical composition and the confirmation of final highly concentrated adalimumab pharmaceutical composition.

2. During the study, the thermal and colloidal stability of adalimumab in more than 90 formulations was studied using the following method: PEG aggregation, shaking testing, freeze-thawing, heat stress followed by analysis of turbidity (UV spectrophotometry), purity (exclusion HPLC, dynamic light scattering and gel electrophoresis, including using LabChip, caliper system) and acid-base distribution (LabChip, caliper and ion exchange HPLC).

3. Studies have shown that the original Humira formulation (Humira 1) used in commercial formulations with a concentration of adalimumab of 50mg/mL has low thermal stability compared to the formulation of the present invention.

4. Experiments have shown that in the original Humira formulation (Humira 2) in a commercially available formulation for adalimumab 100mg/mL, the freezing and thawing process has a significant effect on adalimumab aggregation compared to the formulation of the invention.

5. Adalimumab pharmaceutical compositions (pH 5.0-6.0) based on acetate buffer supplemented with trehalose dihydrate, arginine hydrochloride, proline and a surface active substance selected from polysorbate 20, polysorbate 80 and poloxamer 188 exhibit higher thermal and colloidal stability at concentrations of 50-150mg/mL compared to the original formulation and are the subject of the present invention.

The best additive formulation of adalimumab was chosen for the solution at ph 5.5. The stability of adalimumab in formulations with a pH range of 5.0-6.0 was confirmed 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 having a concentration of adalimumab of 150mg/mL enables concentration up to 200mg/mL without losing the protein quality for subsequent dilution after concentration when adding SAS and washing the ultrafiltration device.

Claims

1. An aqueous pharmaceutical composition for intravenous or subcutaneous administration comprising:

a) Adalimumab with the concentration of 50-150 mg/mL;

b) Acetate buffer at a concentration of 1-100 mM;

c) Proline, wherein the proline concentration is 27 mg/mL;

d) Polysorbate 20, polysorbate 80, or poloxamer 188, or combinations thereof; wherein the polysorbate 20 concentration is 0.05 mg/mL to 10 mg/mL, wherein the polysorbate 80 concentration is 0.05 mg/mL to 10 mg/mL, and wherein the poloxamer 188 concentration is 0.05 mg/mL to 10 mg/mL;

wherein the pH of the composition is from 5.0 to 6.0.

2. The composition of claim 1, comprising:

a) Adalimumab 50-150 mg/mL;

b) Sodium acetate trihydrate at 0.4-1.2 mg/mL;

c) 27 mg/mL proline;

d) Glacial acetic acid until the pH is 5.0-6.0;

e) Polysorbate 80 at 0.1 mg/mL-1 mg/mL.

3. The composition of claim 2 comprising:

a) Adalimumab 50-150 mg/mL;

b) Sodium acetate trihydrate at 0.436 mg/mL;

c) 27 mg/mL proline;

d) Glacial acetic acid, up to pH 5.5;

e) Polysorbate 80 at 0.5 mg/mL.

4. The composition of claim 1, comprising:

a) Adalimumab 50-150 mg/mL;

b) Sodium acetate trihydrate at 0.4-1.2 mg/mL;

c) 27 mg/mL proline;

d) Glacial acetic acid until the pH is 5.0-6.0;

e) 0.1. 0.1 mg/mL-1. 1 mg/mL polysorbate 20.

5. The composition of claim 4 comprising:

a) Adalimumab 50-150 mg/mL;

b) Sodium acetate trihydrate at 0.436 mg/mL;

c) 27 mg/mL proline;

d) Glacial acetic acid, up to pH 5.5;

e) Polysorbate 20 at 0.5 mg/mL.

6. The composition of claim 1, comprising:

a) Adalimumab 50-150 mg/mL;

b) Sodium acetate trihydrate at 0.4-1.2 mg/mL;

c) 27 mg/mL proline;

d) Glacial acetic acid until the pH is 5.0-6.0;

e) Poloxamer 188 at 0.1 mg/mL to 1 mg/mL.

7. The composition of claim 6 comprising:

a) Adalimumab 50-150 mg/mL;

b) Sodium acetate trihydrate at 0.436 mg/mL;

c) 27 mg/mL proline;

d) Glacial acetic acid, up to pH 5.5;

e) Poloxamer 188 at 1.0 mg/mL.

8. Use of a composition according to any one of claims 1-7 in the manufacture of a medicament for the treatment of a tnfα -mediated disease, wherein the tnfα -mediated disease is selected from the group consisting of:

a) Active moderate to severe rheumatoid arthritis,

b) An active psoriatic arthritis, which is characterized by the presence of a compound of formula (i),

c) The active ankylosing spondylitis is treated by the method,

d) Chronic moderate to severe plaque psoriasis,

e) Moderate to severe ulcerative colitis is indicated to be a condition of moderate to severe ulcerative colitis,

f) A central axis type of the vertebral column arthritis,

g) Active hidradenitis suppurativa,

h) Juvenile idiopathic arthritis is treated by a combination of a compound of the formula,

i) Moderate or severe crohn's disease,

j) The preparation method comprises the steps of (1) treating uveitis,

k) Active attachment point inflammation-associated arthritis.

9. A process for producing the composition of any one of claims 1-7, the process comprising adding an acetate buffer to the aqueous phase followed by the following components in any order:

a) Proline as an osmotic agent and/or stabilizer;

b) Adalimumab;

c) A surfactant selected from polysorbate 20, polysorbate 80, poloxamer 188, or a combination thereof.