OA19427A - Aqueous pharmaceutical composition of a recombinant monoclonal antibody to FNOα. - Google Patents

Aqueous pharmaceutical composition of a recombinant monoclonal antibody to FNOα. Download PDF

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OA19427A
OA19427A OA1201900260 OA19427A OA 19427 A OA19427 A OA 19427A OA 1201900260 OA1201900260 OA 1201900260 OA 19427 A OA19427 A OA 19427A
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composition
polysorbate
adalimumab
trehalose
sodium acetate
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OA1201900260
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Dmitry Valentinovich MOROZOV
Ekaterina Aleksandrovna LOMKOVA
Aleksandr Olegovich IAKOVLEV
Viktoriya Olegovna SHITIKOVA
Anastasiya Mikhajlovna RYAKHOVSKAYA
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Joint Stock Company "Biocad"
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Abstract

The invention relates to improved aqueous pharmaceutical compositions of a recombinant monoclonal antibody to TNFα, and to a method for producing same. The present invention also relates to the use of improved aqueous pharmaceutical compositions of a recombinant monoclonal antibody to TNFα to treat TNFα-mediated diseases. The proposed invention allows prevention of physical-chemical instability expressed in the formation of aggregates and fragments of proteins or in the modification of proteins in a solution, and also prevents instability during freezing/thawing, agitating and shaking.

Description

The terms used in this spécification generally hâve their ordinary meanings in the art, within the context of the invention, and in the spécifie context where each term is used. Below, or elsewhere herein, certain terms used to describe the invention are defined to provide a practitioner with an additional guidance for describing the invention. Synonyms for some terms are provided. A récital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this spécification, including examples of any term described herein, is illustrative only and is not intended to limit the scope and meaning of the invention or of any exemplified subject matter. The invention is not limited to various embodiments given in this spécification.
Unless otherwise defined, ail technical and scientific terms used herein hâve the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the case of conflict, the présent document, including définitions, will control.
The term adalimumab is synonymous with the active pharmaceutical ingrédient in Humira® as well as protein considered or intended as bio-similar or bio-improved variants thereof. Adalimumab is a human recombinant monoclonal IgGl antibody spécifie for human TNF. Adalimumab is also known as D2E7 . Adalimumab comprises two light chains each with a molecular weight of approximately 24 kDa and two IgGl heavy chains each with a molecular weight of approximately 49 kDa. 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 so-called bio-similar or bioimproved variants of the adalimumab protein used in commercially available Humira®. For example, a variant of commercial Humira® may be acceptable to the FDA when it has essentially the same pharmacological effect as commercially available Humira®, even though it may exhibit certain physical properties, such as a glycosylation profile and an acid-base profile, that may be similar if not identical to Humira®.
For the purposes of the présent application, the term adalimumab also includes adalimumab with minor modifications in the amino acid structure (including délétions, additions, and/or substitutions of amino acids) or in the glycosylation properties and the acid-base profile, which do not significantly affect the function of the polypeptide. The term adalimumab includes ail forms and formulations of Humira®, including but not limited to concentrated formulations, injectable ready-to-use formulations; formulations reconstituted with water, alcohol, and/or other ingrédients, and others.
The term human TNFa, which also known as hTNFa or hTNF, or TNFa, is intended to refer to a human cytokine that exists as a 17 kDa secreted form and a 26 kDa membrane-associated form, the biologically active form of which is composed of a trimer of noncovalently bound 17 kDa molécules. The structure of TNFa is described further in, for example, Pennica, D., et al. (1984) Nature 312:724-729; Davis, J. M., et al. (1987) Biochemistry 26:1322-1326; and Jones, E. Y., et al. (1989) Nature 338:225-228. The term human rhTNFa is intended to include recombinant human TNFa, which can be prepared by standard recombinant expression methods or purchased commercially ( R & D Systems, catalog. No. 210-TA, Minneapolis, Minn.).
As used herein, the term antibody refers to immunoglobulin-type molécules comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by internai disulfide bonds. Each heavy chain consists of a heavy chain variable région (abbreviated herein as HCVR or VH) and a heavy chain constant région. The heavy chain constant région consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable région (abbreviated herein as LCVR or VL) and a light chain constant région. The light chain constant région consists of one domain, CL. The VH and VL régions can be further subdivided into régions of hypervariability, referred to as complementarity determining régions (CDRs), interspersed with régions that are more conserved, termed framework régions (FRs) . Each VH and VL consists of three CDRs and four FRs, arranged from N-terminus to C-terminus in the following order: FRI, CDR1, FR2, CDR2, FR3, CDR3, FR4. In one embodiment of the invention, the formulation comprises an antibody with CDR1, CDR2, and CDR3 sequences similar to those described in U.S. Pat. Nos. 6090382; 6258562, and 8216583.
An antibody or antigen-binding portion thereof may be part of a larger immunoadhesion molécule, formed by the covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molécules include use of the streptavidin core région to make a tetrameric scFv molécule (Kipriyanov, S. M., et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molécules (Kipriyanov, S. M., et al. (1994) Mol. Immunol. 31:1047-1058). From whole antibodies, antibody portions, such as Fab and F(ab' ) 2 fragments of whole antibodies, can be prepared using conventional methods, such as papain or pepsin digestion, respectively. Further, antibodies, antibody portions, and immunoadhesion molécules can be obtained using standard recombinant DNA techniques, as described herein.
As used herein, the term isolated antibody refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to TNFa is substantially free of antibodies that specifically bind to antigens other than TNFa) . However, an isolated antibody that specifically binds to TNFa may hâve cross-reactivity to other antigens, such as TNFa molécules from other species. Furthermore, an isolated antibody may be substantially free of other cellular materials and/or Chemicals.
The term anti-TNFa antibody refers to a human anti-TNFa antibody as described herein, as well as described in US Pat. Nos. 6090382; 6258562; 6509015; 7223394 and 6509015. The term anti-TNFa antibody used in the invention is an anti-TNFa antibody or a fragment thereof, including infliximab (Remicade®, Johnson and Johnson, described in US Patent No. 5656272); CDP571 (a humanized monoclonal anti-TNF-alpha IgG4 antibody); CDP870 (a humanized monoclonal anti-TNF-alpha antibody fragment); an anti-TNF dAb (Peptech); CNTO148 (golimumab; Centocor, see WO 02/12502 and U.S. 521,206 and U.S. 7250165); and adalimumab (HUMIRA® Abbott Laboratories, a human anti-TNF mAb, described in US 6090382 as D2E7). Additional anti-TNF antibodies that may be used in the invention are described in U.S. Patent Nos. 6593458; 6498237; 6451983; and 6448380. In another embodiment, the TNFa inhibitor is a TNF fusion protein, for example, etanercept (Enbrel®, Amgen; described in WO 91/03553 and WO 09/406476). In another embodiment, the TNFa inhibitor is a recombinant TNF binding protein (r-TBP-I) (Serono).
The term long-term storage or long-term stability is to be understood to mean that a pharmaceutical composition can be stored for three months or more, for six months or more, and preferably for one year or more, most preferably a minimum stable shelf life of at least two years. In general, the terms long-term storage and long-term stability further include stable storage durations that are at least comparable to or better than the stable shelf life typically required for currently available commercial formulations of adalimumab, without losses in stability that would render the formulation unsuitable for its intended pharmaceutical application. Longterm storage is also understood to mean that the pharmaceutical composition is stored either as a liquid at 2 to 8°C., or frozen, e.g., at -18°C., or lower. It is also contemplated that the composition can be frozen and thawed more than once.
The term stable State with respect to long-term storage is understood to mean that adalimumab contained in the pharmaceutical compositions does not lose more than 20%, or more preferably 15%, or even more preferably 10%, and most preferably 5% of its activity relative to activity of the composition at the start of storage.
The term excipient or additive is used herein to describe any ingrédient other than those previously described in the présent invention.
The term sugar refers to monosaccharides, disaccharides and polysaccharides or mixtures thereof. Examples of sugars include, but are not limited to, sucrose, trehalose, glucose, dextrose, and others.
The term polyol as used herein refers to an excipient having multiple hydroxyl groups and includes sugar alcohols and sugar acids. Examples of polyols include, but are not limited to, mannitol, sorbitol, and others.
The term buffer, buffer composition, buffer agent, as used herein, refers to an added composition that allows a liquid antibody formulation to resist changes in pH, typically by the action of its acid-base conjugate components. When reference is made to a concentration of a buffer, it is intended that said concentration represents the total molar concentration of the free acid or free base form of the buffer. Examples of buffers that are known in the art and can be found in the literature include, but are not limited to, histidine, citrate, succinate, acetate, phosphate, phosphatebuffered saline, citrate-phosphate buffers, and tromethaminebased buffers and the like, or suitable mixtures thereof.
The terms tonicity agent or tonicifier, as well as osmolyte or osmotic agent as used herein refer to an excipient that can adjust the osmotic pressure of a liquid antibody préparation. In certain embodiments, the tonicity agent can adjust the osmotic pressure of the liquid antibody préparation to isotonie so that the antibody préparation is physiologically compatible with the cells of the body tissue of the subject. In yet another embodiment, the tonicity agent may contribute to an improvement in stability of the antibodies described herein. An isotonie préparation is a préparation that has the osmotic pressure équivalent to human blood. Isotonie préparations generally hâve an osmotic pressure of about 250 to 350 mOsm. The term hypotonie describes a préparation having an osmotic pressure below that of human blood. Accordingly, the term hypertonie is used to describe a préparation having an osmotic pressure above that of human blood. Isotonicity can be measured using, for example, a vapor pressure or ice-freezing type osmometer. The tonicity agent may be in an enantiomeric (e.g., L- or Denantiomer) or racemic form; in the form of isomers, such as alpha or beta, including alpha, alpha; or beta, beta; or alpha, beta; or beta, alpha; in a free acid or free base form; in a sait form; in a hydrated form (for example, monohydrate) or in an anhydrous form.
The term surface-active substance (also referred to as surfactant or detergent, or SAS) as used herein refers to an excipient that can alter the surface tension of a liquid antibody préparation. In certain embodiments, the surfaceactive substance reduces the surface tension of the liquid antibody préparation. In other embodiments, the surfaceactive substance may contribute to an improvement in colloïdal stability or solubility of any antibody in the préparation. The surface-active substance may reduce aggregation of the resulting antibody préparation and/or minimize the formation of particulates in the préparation and/or reduce adsorption. The surface-active substance may also improve the stability of the antibody during and after a freeze/thaw cycle and while shaking. The surface-active substances may be ionic or non-ionic. Exemplary non-ionic surface-active substances that can be included in the formulations of the présent invention include, e.g., 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. Spécifie non-ionic surface-active substances that can be included in the formulations of the présent invention include, e.g., Polysorbates such as Polysorbate 20 (Tween 20), Polysorbate 28, Polysorbate 40, Polysorbate 60, Polysorbate 65, Polysorbate 80 (Tween 80), Polysorbate 81, and Polysorbate 85; Poloxamers such as Poloxamer 188 (Kolliphor P188), Poloxamer 407; polyethylene-polypropylene glycol; or polyethylene glycol (PEG), ethylene- and propylene glycol copolymers {e.g., pluronics PF68, etc.) .
The term lyophilized as used herein refers to a préparation that has undergone a process known in the art as freeze-drying, involving freezing the préparation and subsequently removing the ice from the frozen content.
The term amino acid as used herein dénotés an amino acid (a free amino acid, i.e. not an amino acid in a peptide or protein sequence). Amino acids as used in the présent invention comprise, but are not limited to, e.g. arginine, glycine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tryptophan, serine, cysteine, méthionine, and proline.
Pharmaceutical composition means a composition comprising an antibody of the invention and at least one of components selected from the group consisting of pharmaceutically acceptable and pharmacologically compatible excipients, solvents, diluents, carriers, additive, distributing agents, delivery agents, such as preservatives, stabilizing agents, emulsifiers, suspending agents, thickeners, prolonged delivery regulators, the choice and ratio of which dépends on the nature and method for administering and dosing. Exemplary suspending agents include ethoxylated isostearyl alcohol, polyoxyethylene, sorbitol and sorbitol ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof. Protection from the microorganism action can be provided with multiple antibacterial and antifungal agents, e.g., such as parabens, chlorobutanol, sorbic acid and the like. The composition may also comprise isotonie agents, e.g., sugars, sodium chloride and the like. The prolonged action of the composition may be provided with agents that delay the absorption of the active agent, e.g. aluminum monostearate and gelatin. Exemplary appropriate carriers, solvents, diluents and delivery agents include water, éthanol, polyalcohols, and mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). Exemplary excipients include lactose, milk sugar, sodium citrate, calcium carbonate, calcium phosphate and the like. A pharmaceutical composition for oral, sublingual, transdermal, intramuscular, intravenous, subcutaneous, topical or rectal administration of the active agent, alone or in combination with another active agent, can be administered to animais and humans in a unit dosage form as a mixture with conventional pharmaceutical carriers. Appropriate unit dosage forms include parentéral forms, implants and transdermal Systems.
A drug (préparation) is a substance (or a substance mixture in a form of a pharmaceutical composition) in the form of tablets, capsules, powders, lyophilizates, injections, infusions, ointments and other finished dosage forms intended to restore, correct or alter physiological functions in humans and animais, and to provide disease treatment and prévention, diagnostics, anesthésia, contraception, cosmetology and others.
Treat, treating, and treatment refer to a method for alleviating or eliminating a biological disorder and/or at least one of its attendant symptoms. As used herein, to alleviate a disease, disorder or condition means to reduce the severity and/or frequency of the disease, disorder or condition symptoms. In addition, the references to treatment herein include references to curative, palliative and préventive thérapies.
In one aspect, the subject to be treated or the patient is a mammal, preferably a human subject. The above subject may be male or female of any âge.
The term disorder dénotés any condition that can be improved by the treatment according to the invention. The définition of this term includes chronic and acute disorders or diseases including pathological conditions, which prédisposé the mammal to said disorder. Non-limiting examples of the diseases to be treated include benign and malignant tumors; leukemias and lymphoid malignancies, in particular, breast, ovarian, stomach, endometrium, salivary gland, lung, kidney, colon, thyroid, pancréas, prostate or bladder cancer; neural, glial, astrocytic, hypothalamus and other glandular, macrophage, épithélial, stromal and blastocoelic disorders; inflammatory, angiogenic and immunological disorders. The preferred disorder to be treated according to the invention is autoimmune diseases.
The terms immune response, autoimmune response, autoimmune inflammation refer, e.g. to the action of lymphocytes, antigen-presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by above cells or liver cells (including antibodies, cytokines, and complément that resuit from the sélective damage, destruction or élimination from the human body of invasive pathogens, cells or tissues infected with pathogens, cancer cells or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues).
The term autoimmune disease as used herein means nonmalignant disease or disorder that occurs and is directed against the self (auto) antigens and/or tissues in an individual.
This définition encompasses but is not limited to rheumatoid arthritis, arthrosis, juvénile chronic arthritis, septic arthritis, Lyme's arthrosis, psoriatic arthritis, reactive arthritis, spondyloarthropathy, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, inflammatory bowel disease, diabètes mellitus, thyroiditis, asthma, allergie diseases, psoriasis, atopie dermatitis, scleroderma, graft versus host reaction, transplant rejection, acute or chronic immune diseases associated with transplantation, sarcoidosis, Kawasaki'’s disease, Graves' disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Henoch-Schonlein's purpura, microscopie rénal vasculitis, chronic active hepatitis, uvenita, septic shock, toxic shock syndrome, septic syndrome, cachexia, acquired immunodeficiency syndrome, acute transverse myelitis, Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anémia, adult (acute) respiratory distress syndrome, alopecia, focal alopecia, séronégative arthropathia, arthropathy, Reiter's disease, psoriatic arthropathy, arthropathy associated with ulcerative colitis, atopie allergies, autoimmune bullous diseases, pemphigus vulgaris, pemphigus foliaceus, pemphigoid disease, linear IgAs, autoimmune hemolytic anémia, Coombs-positive hemolytic anémia, malignant anémia, juvénile malignant anémia, arthritis, primary sclerosing hepatitis A, cryptogenic autoimmune heppatitis, pulmonar fibrosing disease, cryptogenic fibrous alveolitis, post-inflammatory interstitial lung diseases, interstitial pneumonitis, chronic éosinophilie pneumonia, postinfectious interstitial lung diseases, urarthritis, autoimmune hepatitis, autoimmune hepatitis type 1 (classic autoimmune hepatitis or lipoid), autoimmune hepatitis type 2, osteoarthritis, primary sclerosing cholangitis, psoriasis type 1, psoriasis type 2, idiopathic leukopenia, autoimmune neutropenia, rénal NOS-diseases, glomerulonephritis, microscopie rénal vasculitis, discoid lupus erythematosus, idiopathic or male NOS-fertility, [autoimmunity to sperm], ail subtypes of multiple sclerosis, sympathetic ophthalmia, secondary pulmonary hypertension associated with connective tissue disease, Goodpasture's syndrome, pulmonary manifestation of polyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis, ankylosing spondylitis, Still's disease, systemic sclerosis, Sjogren's syndrome, Takayasu's disease, autoimmune thrombocytopenia, essential thrombocythemia, autoimmune thyroiditis, hyperthyroidism, Hashimoto's disease, autoimmune atrophie hypothyroidism, primary myxedema, phacogenic uveitis, primary vasculitis, vitiligo, acute liver diseases, chronic liver diseases, allergies, asthma, mental disorders (including dépressive syndrome and schizophrenia) , Th2-type and Thl-type mediated diseases, conjuctivitis, allergie contact dermatitis, allergie rhinitis, alpha-I antitrypsin deficiency, amyotrophie latéral sclerosis, anaemia, cystic fibrosis, diseases associated with cytokine therapy, demielinising diseases, dermatitis, iridocyclitis/uveitis/ophthalmic neuritis, ischemia reperfusion injury, ishemic stroke, juvénile rheumatoid arthritis, autoimmune enteropathy, autoimmune hearing loss, autoimmune lymphoproliférative syndrome, autoimmune myocarditis, autoimmune prématuré ovarian failure and blepharitis. The antibody can also treat any combination of the above-listed disorders.
As used herein, the term disorder in which the TNFa activity is detrimental includes diseases and other disorders in which the presence of TNFa in a subject suffering from the disorder has been shown to be or is suspected of being either responsible for the pathophysiology of the disorder or a factor that contributes to a worsening of the disorder. Accordingly, a disorder in which the TNFa activity is detrimental is a disorder in which inhibition of TNFa activity is expected to alleviate the symptoms and/or progression of the disorder. Such disorders may be detected, for example, by an increase in the concentration of TNFa in the biological fluid of a subject suffering from the disorder [e.g., an increase in the concentration of TNFa in sérum, plasma, synovial fluid and the like of the subject) that can be detected, for instance, using an anti-TNFa antibody as described above. There are many examples of disorders in which the TNFa activity is detrimental. The use of antibodies and antibody fragments appropriate to the disorder in the treatment of spécifie disorders is further discussed below:
A. Sepsis
Tumor necrosis factor plays an established rôle in the pathophysiology of sepsis, with biological effects that include hypotension, myocardial suppression, vascular leakage syndrome, organ necrosis, stimulation of the release of toxic secondary mediators and activation of the clotting cascade (see e.g., Moeller A., et al. (1990), Cytokine 2:162-169; U.S. Patent No. 5231024 Moeller et at. ; European Patent No. 260610 B1 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). Correspondingly, the human antibodies and antibody fragments according to the invention can be used to treat sepsis in any of its clinical manifestations, including septic shock, endotoxic shock, sepsis caused by Gram-negative bacteria, and toxic shock syndrome.
Furthermore, for sepsis treatment, the anti-TNFa antibodies and antibody fragments according to the invention can be coadministered with one or more therapeutic agents that may further alleviate 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 methods of combination therapy for sepsis treatment are discussed below in subsection III.
Additionally, in a preferred embodiment, anti-TNFa antibody and antibody fragments according to the invention is administered to a human from a subgroup of sepsis patients having a sérum or plasma concentration of IL-6 above 500 pg/mL and more preferably 1000 pg/mL during treatment (see PCT Publication No. WO 95/20978 Daum, L., et al.).
B. Autoimmune Diseases
Tumor necrosis factor has been shown to play a rôle in the pathophysiology of a variety of autoimmune diseases. For example, TNFa has also been involved in activating tissue inflammation and caused joint destruction in rheumatoid arthritis (see e.g., Moeller A. et al. (1990), Cytokine 2:162169; US Patent No. 5231024 Moeller et al.; European Patent No. 260610 B1 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) . TNFa has also been involved in promoting the death of islet cells and in the formation of insulin résistance in diabètes (see e.g., Tracey and Cerami, supra; PCT publication No. WO 94/08609) . TNFa has also been involved in the formation of cytotoxicity to oligodendrocytes and in the induction of inflammatory plaques in multiple sclerosis (see e.g., Tracey and Cerami, supra). Chimeric and humanized murine anti-hTNFa antibodies hâve 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. Rheumatol. 34:334-342).
Human antibodies and antibody fragments according to the invention can be used to treat autoimmune diseases, in particular, those associated with inflammation, including rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis and gouty arthritis, allergy, multiple sclerosis, autoimmune diabètes, autoimmune uveitis and rénal syndrome. Typically, the antibody or antibody fragment is administered systemically, although, for certain disorders, local administration of the antibody or antibody fragment at a site of inflammation may be bénéficiai (e.g., local administration into the joints in rheumatoid arthritis or topical application to diabetic ulcers, alone or in combination with a cyclohexane-ylidene dérivative as described in PCT Publication No. WO 93/19751). The antibody and antibody fragment according to the invention can also be used with one or more additional therapeutic agents used in the treatment of autoimmune diseases, as further described in subsection III.
C. Infectious Diseases
Tumor necrosis factor has been involved in the production of biological effects observed in a variety of infectious diseases. For example, TNFa has been involved in the development of brain inflammation and capillary thrombosis and infarction in malaria. TNFa has also been involved in the development of brain inflammation, the induction blood-brain barrier breakdown, the development of septic shock syndrome, and the activation of venons infarction in meningitis. TNFa has also been involved in the induction of cachexia, stimulation of viral prolifération and formation of central nervous System injuries in acquired immune deficiency syndrome (AIDS). Correspondingly, the antibodies and antibody fragments according to the invention can be used in the treatment of infectious diseases, including bacterial meningitis (see e.g., Européen Patent Application No. EP 585 705), cérébral malaria, AIDS and AIDS-related complex (aC) (see e.g., European Patent Application No. EP 230 574), as well as cytomégalovirus infection secondary to transplantation (see e.g., Fietze, E., et al. (1994) Transplantation 58:675-680). The antibodies and antibody fragments according to the invention can also be used to alleviate symptoms associated with infectious diseases, including fever and myalgias due to infection (such as influenza) and cachexia secondary to the infection {e.g., secondary to AIDS or aC).
D. Transplantation
Tumor necrosis factor has been considered as a key mediator in allograft rejection and graft versus host disease (GVHD) and in the formation of an adverse reaction that was observed when the rat antibody OKT3, directed against the T cell receptor CD3 complex, is used to inhibit rejection of rénal transplants (see e.g., Eason J.D. et al. (1995) Transplantation 59:300-305; Suthanthiran M. and Strom T. B. (1994) New Engl. J. Med. 331:365-375). Correspondingly, the antibodies and antibody fragments according to the invention can be used to inhibit transplant rejection, including allograft and xenograft rejections, and to inhibit GVHD. Although the antibody or antibody fragment can be used alone, more preferably, they are used in combination with one or more other agents that inhibit the immune response against the allograft or inhibit GVHD. For example, in one embodiment, the antibody or antibody fragment of the invention is used in combination with one or more antibodies directed at other targets involved in the régulation of immune responses, such as the cell surface molécules CD25 (interleukin-2 receptor-β), CDlla (LFA-1), CD54 (ICAM-1), CD4, CD45, CD28/CTLA4, CD80 (B71) and/or CD86 (B7-2). In yet another embodiment, the antibody or antibody fragment according to the invention is used in combination with one or more general immunosuppressive agents, such as cyclosporin A or FK506.
E. Malignancies
Tumor necrosis factor has been involved in the induction of cachexia, the stimulation of tumor growth, the enhancement of metastatic potential and the development of cytotoxicity in malignancies. Correspondingly, the antibodies and antibody fragments according to the invention are used in the treatment of malignancies, to inhibit tumor growth or metastasis and/or to alleviate cachexia secondary to malignancies. The antibody or antibody fragment may be administered systemically or topically to the tumor site.
F. Pulmonary Disorders
Tumor necrosis factor has been involved in the pathophysiology of adult respiratory distress syndrome (aDS), including the stimulation of endothélial leukocyte activation, the direction of cytotoxicity to pneumocytes, and the induction of vascular leakage syndrome. Correspondingly, the antibodies and antibody fragments according to the invention can be used to treat varions 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 antibody and antibody fragment according to the invention can be administered with one or more additional therapeutic agents used in the treatment of pulmonary disorders, as described in subsection III below.
G. Intestinal Disorders
Tumor necrosis factor has been involved in the pathophysiology of inflammatory bowel disorders (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-TNFa antibodies hâve been clinically tested for the treatment of Crohn's disease (van Dullemen H.M. et al. (1995) Gastroenterology 109:129-135). The human antibody or antibody fragment according to the invention can also be used to treat intestinal disorders, such as idiopathic inflammatory bowel disease, which includes two syndromes, Crohn's disease, and ulcerative colitis. The antibody and antibody fragment according to the invention can also be administered with one or more additional therapeutic agents used in the treatment of intestinal disorders, as described in subsection III below.
H. Cardiac Disorders
The antibodies or antibody fragments according to the invention can also be used to treat various cardiac disorders, including cardiac ischemia (see e.g., European Patent Application No. EP 453 898) and heart failure (amyocardia) (see e.g., PCT Publication No. WO 94/20139).
I. Others
The antibodies or antibody fragments according to the invention can also be used to treat various disorders in which TNFa activity is detrimental. Examples of other diseases and disorders in which TNFa activity is involved in the pathophysiology, and which can be thus treated using the antibody or antibody fragment according to the invention, include inflammatory bone disorders and bone résorption disease (see e.g., Bertolini D.R., et al. (1986) Nature
319:516-518; Konig A., et al. (1988) J. Bone Miner. Res. 3:621-627; Lerner U.H. and Ohiin A. (1993) J. Bone Miner. Res. 8:147-155; and Shanka G. and Stern P.H. (1993) Bone 14:871876), hepatitis, including alcoholic hepatitis (see e.g., McClain C.J. and Cohen D.A. (1989) Hepatology 9:349-351; Felver M.E. et al. (1990) Alcohol Clin. Exp. Res. 14:255-259; and Hansen J. et al. (1994) Hepatology 20:461-474), viral hepatitis (Shéron N. et al. (1991) J. Hepatol. 12:241-245; and Hussain M.J. et al. (1994) J. Clin. Pathol. 47:1112-1115), and fulminant hepatitis; coagulation disorders (see e.g., van der Poil T. et al. (1990) N. Engl. J. Med. 322:1622-1627; van der Poil 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:H118124; 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 C. et al. (1994) Transplantation 58:1158-1162; Yao Y.M. et al. (1995) Resuscitation 29:157168), keloid formation (see e.g., McCauley R. L et al. (1992) J. Clin. Immunol. 12:300-308), scar tissue formation;
hyperthermia; periodontal disease; obesity and radiation toxicity.
Therapeutically effective amount is considered as the amount of therapeutic agent administered during the treatment that will relieve to some extent one or more symptoms of the disease being treated. The term chronic administration refers to the continuons (long-term) administration of the agent(s) as opposed to the acute (short-term) administration mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time.
Intermittent administration refers to the treatment that is not performed consecutively without interruption, but rather is cyclic by nature.
In the présent spécification and in the following claims, unless the context requires otherwise, the words hâve, include and comprise, or variations thereof, such as has, having, includes, including, comprises or comprising should be understood as the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
Detailed Description of the Invention
Aqueous Composition
The présent invention relates to novel, improved aqueous compositions, that can optionally be lyophilized, comprising an appropriate amount of a recombinant monoclonal anti-TNFa antibody in an appropriate buffer(s), one or more appropriate stabilizing agents and other excipients selected from appropriate surface-active substances and isotonicity agents. Said composition prevents the formation of protein (antibody) aggregates and maintains the efficacy and stability of the therapeutic compound for a désirable period of time.
Adalimumab and infliximab are examples of a recombinant monoclonal anti-TNFa antibody. Infliximab is an example of a chimeric antibody. Adalimumab is an example of a human antibody. In a preferred embodiment, the antibodies used in the composition are human antibodies. In a more preferred embodiment, the antibodies used in the composition are human anti-human TNFa antibodies. In yet another embodiment, the composition comprises D2E7 and a combination of D2E7 with other antibodies. The D2E7 antibody, known generically as adalimumab, with its high affinity for binding to TNFa, low dissociation kinetics, and high neutralizing ability. Adalimumab is available in the pharmaceutical market under the trademark Humira®. The names adalimumab and D2E7 used throughout the présent spécification 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-TNFa antibodies or antigen-binding portions thereof are generally présent in a therapeutic amount up to 200 mg/mL. In a preferred embodiment, the therapeutic amount is from about 1 mg/mL to about 150 mg/mL. In a more preferred embodiment, the therapeutic amount is from about 1 mg/mL to about 100 mg/mL. In a more preferred embodiment, the therapeutic amount is from about 50 mg/mL to about 100 mg/mL. In an even more preferred embodiment, the therapeutic amount is about 100 mg/mL.
The aqueous composition in question comprises an appropriate buffer solution in combination with other pharmaceutically acceptable excipients that stabilize the pharmaceutical préparation. Appropriate buffer solutions that can be used are selected from the group that is known in the art and can be found in the literature. In one embodiment, appropriate buffer solutions comprise, but are not limited to, an acetate ion based buffer agent (or buffer System). In a preferred embodiment, the appropriate buffer comprises an acetate buffer. In an even more preferred embodiment, the acetate buffer is selected from sodium acetate trihydrate combined with acetic acid.
Buffer solutions are generally used in concentrations from about 1 mM to about 100 mM. In a preferred embodiment, the buffer concentration is from about 1 mM to about 50 mM. In a more preferred embodiment, the buffer concentration is from about 1 mM to about 20 mM. In an even more preferred embodiment, the buffer concentration is about 5 mM.
In some embodiments, sodium acetate trihydrate is présent in an amount of up to 1.2 mg/mL. In a preferred embodiment, the amount of sodium acetate trihydrate is from about 0.4 mg/mL to about 1.2 mg/mL. In a more preferred embodiment, the amount of sodium acetate trihydrate is from about 0.4 mg/mL to about 0.5 mg/mL. In a more preferred embodiment, the amount of sodium acetate trihydrate is 0.436 mg/mL.
In one embodiment, the liquid composition maintains pH in the range of from 4.0 to about 7.0, depending on the monoclonal antibody used. In a preferred embodiment, the buffer used maintains pH of the composition in the range of about 4.5 to 6.0. In a more preferred embodiment, pH is maintained at about from 5.0 to 6.0. In a more preferred embodiment, pH is maintained at about 5.5.
Said aqueous composition comprises appropriate surfaceactive substances that are pharmaceutically acceptable excipients used to protect protein compositions from various stress effects such as stirring, shearing, high température, freezing, etc. Appropriate surface-active substances include, but are not limited to, Polysorbates or Poloxamers, or a combination of Polysorbate and Poloxamer. In a preferred embodiment, the appropriate surface-active substance is Polysorbate. In a preferred embodiment, the appropriate surface-active substance is Poloxamer. In a preferred embodiment, the appropriate surface-active substance is a combination of Polysorbate and Poloxamer. In a more preferred embodiment, Polysorbate is Polysorbate 20 or Polysorbate 80 (also distributed under the trademark Tween® 20 or Tween® 80). In a more preferred embodiment, Poloxamer is Poloxamer 188 (also distributed under the trademark Kolliphor P188®) . In another preferred embodiment, the appropriate surface-active substance is a combination of polysorbate 20 / polysorbate 80 and poloxamer 188.
In some embodiments, Polysorbate 20 is présent in an amount of up to 10 mg/mL. In a preferred embodiment, the amount of polysorbate 20 is from about 0.05 mg/mL to about 10 mg/mL. In a more preferred embodiment, the amount of Polysorbate 20 is from about 0.1 mg/mL to about 1 mg/mL. In a more preferred embodiment, the amount of polysorbate 20 is about 0.5 mg/mL.
In some embodiments, Polysorbate 80 is présent in an amount of up to 10 mg/mL. In a preferred embodiment, the amount of polysorbate 80 is from about 0.05 mg/mL to about 10 mg/mL. In a more preferred embodiment, the amount of Polysorbate 80 is from about 0.1 mg/mL to about 1 mg/mL. In a more preferred embodiment, the amount of polysorbate 80 is about 0.5 mg/mL.
In some embodiments, poloxamer 188 is présent in an amount of up to 10 mg/mL. In a preferred embodiment, the amount of poloxamer 188 is from about 0.05 mg/mL to about 10 mg/mL. In a more preferred embodiment, the amount of poloxamer 188 is from about 0.1 mg/mL to about 1 mg/mL. In a more preferred embodiment, the amount of poloxamer 188 is about 0.5 mg/mL.
In some embodiments, the combination of polysorbate 20 / polysorbate 80 and poloxamer 188 is Polysorbate 80 in an amount of up to 10 mg/mL and poloxamer 188 in an amount of up to 10 mg/mL. In a preferred embodiment, the amount of poloxamer 188 is from about 0.05 mg/mL to about 10 mg/mL and the amount of Polysorbate 20 / polysorbate 80 is from about 0.05 mg/mL to about 10 mg/mL. In a more preferred embodiment, the amount of poloxamer 188 is from about 0.1 mg/mL to about 1 mg/mL and the amount of Polysorbate 20 / polysorbate 80 is from about 0.1 mg/mL to about 1 mg/mL. In a more preferred embodiment, the amount of Polysorbate 20 / polysorbate 80 is about 0.5 mg/mL and the amount of Poloxamer 188 is about 1.0 mg/mL.
The aqueous composition comprises one or more appropriate stabilizing agents that are pharmaceutically acceptable excipients and protect the pharmaceutically active ingrédient from Chemical and/or physical dégradation during manufacture, storage, and use. In one embodiment, the stabilizing agents include, but are not limited to, amino acids, oligosaccharides, or appropriate dérivatives or mixtures thereof. In another preferred embodiment, the appropriate stabilizing agent is trehalose. In another preferred embodiment, the appropriate stabilizing agent is proline. In another preferred embodiment, the appropriate stabilizing agent is a combination of trehalose and proline. In another preferred embodiment, the appropriate stabilizing agent is a combination of trehalose and arginine.
In another embodiment, oligosaccharides that can also be used as stabilizing agents include trehalose.
In some embodiments, trehalose is présent in an amount of up to 150 mg/mL. In a preferred embodiment, the amount of trehalose is from about 25 mg/mL to about 150 mg/mL. In a more preferred embodiment, the amount of trehalose is from about 50 mg/mL to about 100 mg/mL. In a more preferred embodiment, the amount of trehalose dihydrate is about 80 mg/mL.
In another embodiment, the combination of trehalose and arginine is selected as the stabilizing agent. In some embodiments, trehalose is présent in an amount of up to 150 mg/mL, and arginine is in an amount of up to 1.0 mg/mL. In a preferred embodiment, the amount of trehalose is from about 25 mg/mL to about 150 mg/mL, and the amount of arginine is from 0.05 to 1.0 mg/mL. In a more preferred embodiment, the amount of trehalose is from about 50 mg/mL to about 100 mg/mL, and the amount of arginine is from 0.05 to 0.2 mg/mL. In a more preferred embodiment, the amount of trehalose dihydrate is about 80 mg/mL, and the amount of arginine hydrochloride is about 0.1 mg/mL.
In another such embodiment, an amino acid that can be used as stabilizing agents is proline.
In some embodiments, proline is présent in an amount of up to 40 mg/mL. In a preferred embodiment, the amount of proline is from about 5 mg/mL to about 40 mg/mL. In a more preferred embodiment, the amount of proline is from about 20 mg/mL to about 35 mg/mL. In a more preferred embodiment, the amount of proline is about 27 mg/mL.
In some embodiments, the combination of trehalose and proline is trehalose in an amount of up to 150 mg/mL and proline in an amount of up to 40 mg/mL. In a preferred embodiment, the amount of trehalose is from about 25 mg/mL to about 150 mg/mL, and the amount of proline is from about 5 mg/mL to about 40 mg/mL. In a more preferred embodiment, the amount of trehalose is from about 20 mg/mL to about 50 mg/mL, and the amount of proline is from about 10 mg/mL to about 20 mg/mL. In a more preferred embodiment, the amount of trehalose dihydrate is about 40 mg/mL, and the amount of proline is about 13.5 mg/mL.
In some embodiments, the aqueous composition comprises from 50 to 200 mg/mL of the recombinant monoclonal anti-TNFa antibody, from 0.4 to 1.2 mg/mL of sodium acetate trihydrate, from 25 to 150 mg/mL of trehalose and/or from 5 to 40 mg/mL of proline or from 25 to 150 mg/mL of trehalose, and from 0.05 to 0.5 mg/mL of arginine, glacial acetic acid until pH 5.0 to 6.0, from 0.1 mg/mL to 1 mg/mL of Polysorbate 20. In a preferred embodiment of the aqueous composition, the recombinant monoclonal anti-TNFa antibody is adalimumab. In a more preferred embodiment, the composition comprises from 50 to 150 mg/mL of adalimumab, 0.436 mg/mL of sodium acetate trihydrate, 80 mg/mL of trehalose dihydrate, glacial acetic acid until pH 5.5, 0.5 mg/mL of polysorbate 20. In a more preferred embodiment, the composition comprises from 50 to 150 mg/mL of adalimumab, 0.436 mg/mL of sodium acetate trihydrate, 80 mg/mL of trehalose dihydrate, 0.1 mg/mL of arginine hydrochloride, glacial acetic acid until pH 5.5, 0.5 mg/mL of polysorbate 20. In a more preferred embodiment, the composition comprises from 50 to 150 mg/mL of adalimumab, 0.436 mg/mL of sodium acetate trihydrate, 27 mg/mL of proline, glacial acetic acid until pH 5.5, 0.5 mg/mL of Polysorbate 20. In a more preferred embodiment, the composition comprises from 50 to 150 mg/mL of adalimumab, 0.436 mg/mL of sodium acetate trihydrate, 40 mg/mL of trehalose dihydrate, 13.5 mg/mL of proline, glacial acetic acid until pH 5.5, 0.5 mg/mL of polysorbate 20.
In some embodiments, the aqueous composition comprises from 50 to 200 mg/mL of the recombinant monoclonal anti-TNFa antibody, from 0.4 to 1.2 mg/mL of sodium acetate trihydrate, from 25 to 150 mg/mL of trehalose and/or from 5 to 40 mg/mL of proline or from 25 to 150 mg/mL of trehalose, and from 0.05 to 0.5 mg/mL of arginine, glacial acetic acid until pH 5.0 to 6.0, from 0.1 mg/mL to 1 mg/mL of polysorbate 80. In a preferred embodiment of the aqueous composition, the recombinant monoclonal anti-TNFa antibody is adalimumab. In a more preferred embodiment, the composition comprises from 50 to 150 mg/mL of adalimumab, 0.436 mg/mL of sodium acetate trihydrate, 80 mg/mL of trehalose dihydrate, glacial acetic acid until pH 5.5, 0.5 mg/mL of Polysorbate 80. In a more preferred embodiment, the composition comprises from 50 to 150 mg/mL of adalimumab, 0.436 mg/mL of sodium acetate trihydrate, 80 mg/mL of trehalose dihydrate, 0.1 mg/mL of arginine hydrochloride, glacial acetic acid until pH 5.5, 0.5 mg/mL of Polysorbate 80. In a more preferred embodiment, the composition comprises from 50 to 150 mg/mL of adalimumab, 0.436 mg/mL of sodium acetate trihydrate, 27 mg/mL of proline, glacial acetic acid until pH 5.5, 0.5 mg/mL of Polysorbate 80. In a more preferred embodiment, the composition comprises from 50 to 150 mg/mL of adalimumab, 0.436 mg/mL of sodium acetate trihydrate, 40 mg/mL of trehalose dihydrate, 13.5 mg/mL of proline, glacial acetic acid until pH 5.5, 0.5 mg/mL of polysorbate 80.
In some embodiments, the aqueous composition comprises from 50 to 200 mg/mL of the recombinant monoclonal anti-TNFa antibody, from 0.4 to 1.2 mg/mL of sodium acetate trihydrate, from 25 to 150 mg/mL of trehalose and/or 5 to 40 mg/mL of proline or from 25 to 150 mg/mL of trehalose, and from 0.05 to 0.5 mg/mL of arginine, glacial acetic acid until pH 5.0 to 6.0, from 0.1 mg/mL to 1 mg/mL of Poloxamer 188. In a preferred embodiment, the recombinant monoclonal anti-TNFa antibody is adalimumab. In a more preferred embodiment, the composition comprises from 50 to 150 mg/mL of adalimumab, 0.436 mg/mL of sodium acetate trihydrate, 80 mg/mL of trehalose dihydrate, glacial acetic acid until pH 5.5, 1 mg/mL of Poloxamer 188. In a more preferred embodiment, the composition comprises from 50 to 150 mg/mL of adalimumab, 0.436 mg/mL of sodium acetate trihydrate, 80 mg/mL of trehalose dihydrate, 0.1 mg/mL of arginine hydrochloride, glacial acetic acid until pH 5.5, 1 mg/mL of Poloxamer 188. In a more preferred embodiment, the composition comprises from 50 to 150 mg/mL of adalimumab, 0.436 mg/mL of sodium acetate trihydrate, 27 mg/mL of proline, glacial acetic acid until pH 5.5, 1.0 mg/mL of Poloxamer 188. In a more preferred embodiment, the composition comprises from 50 to 150 mg/mL of adalimumab, 0.436 mg/mL of sodium acetate trihydrate, 40 mg/mL of trehalose dihydrate, 13.5 mg/mL of proline, glacial acetic acid until pH 5.5, 1.0 mg/mL of poloxamer 188.
Further, said composition may additionally comprise one or more other appropriate excipients that are well-known to those skilled in the art.
The above compositions are suitable for intravenous, subcutaneous or intramuscular administration.
In some embodiments, the liquid composition maintains storage stability to the effect that there are no further protein aggregation processes or modifications compared to the measure of stability at the time zéro.
Methods for treatment and use of an aqueous composition
In another embodiment, the invention relates to a method for treating a mammal comprising administering to a mammal a therapeutically effective amount of the pharmaceutical composition of the invention wherein the mammal may hâve a disease or disorder that can be effectively treated with adalimumab.
In a preferred embodiment, the mammal is a human.
Diseases or disorders that can be treated with the compositions provided 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, axial spondyloarthritis, active hidradenitis suppurativa, juvénile idiopathic arthritis, (moderate or severe) Crohn's disease, uveitis, active enthesitis-associated arthritis. Additional diseases or disorders . that can be treated with the compositions of the présent invention include those described in US Patent Nos.
6090382 and 8216583, the relevant portions of which are incorporated herein by reference.
The provided pharmaceutical compositions may be administered to a subject in need of treatment by systemic injection, for example by intravenous or subcutaneous, or by intramuscular injection; or by injection or application to a relevant site, for example by direct injection or direct application to the site when the site is exposed during surgery; or by topical application.
In one embodiment, the invention relates to a method for treating and/or preventing rheumatoid arthritis comprising administering to a mammal in need thereof a therapeutically effective amount of one of the provided adalimumab compositions.
The therapeutically effective amount of adalimumab in the provided compositions dépends on the condition to be treated, the severity of the condition, prior therapy, and the patient's medical history, and the response to the therapeutic agent. An appropriate dose can be adjusted according to the judgment of the attending physician so that it can be administered to the patient at once or over multiple administrations .
In one embodiment, the effective amount of adalimumab per adult dose is from about 1 to 500 mg/m2, or about 1 to 200 mg/m2, or about 1 to 40 mg/m2, or about 5 to 25 mg/m2.
Alternatively, a fiat dose can be administered, the amount of which can be in the range of 2 to 500 mg/dose, 2 to 100 mg/dose or about 10 to 80 mg/dose.
If the dose is to be administered more than once a week, the exemplary dose range is the same as the af orement ioned dose ranges or lower, and it is preferably administered two or more times per week at a dose range of 25 to 100 mg/dose.
In another embodiment, an acceptable dose for administration by injection comprises 80 to 100 mg/dose or alternatively comprises 80 mg per dose.
The dose can be administered weekly, biweekly or separated by several weeks (for example, from 2 to 8) .
In one embodiment, adalimumab is administered at the dose of 40 mg by a single subcutaneous (SC) injection.
In some cases, improvements in a patient's condition can be achieved by administering a dose of up to about 100 mg of the pharmaceutical composition one to three times a week for a period of at least three weeks. To achieve the desired level of improvement, treatment for longer periods may be necessary. For incurable chronic conditions, the treatment regimen can be continued indefinitely. For pédiatrie patients (âge 4 to 17 years), a suitable treatment regimen may include administering a dose of 0.4 mg/kg to 4 mg/kg of adalimumab one or more times per week.
In another embodiment, the pharmaceutical formulations of the invention may be prepared in a bulk formulation, and as such, the components of the pharmaceutical composition are présent in the amounts higher than it would be required for administration and are diluted appropriately prior to the administration.
The pharmaceutical compositions can be administered as a single therapeutic agent or in combination with additional therapeutic agents as needed. Thus, in one embodiment, the provided methods for treating and/or preventing are used in combination with administering a therapeutically effective amount of another active agent. The other active agent may be administered before, during or after administering the pharmaceutical compositions of the invention. The other active agent may be administered as part of the provided composition or, alternatively, as a separate formulation.
The administration of the provided pharmaceutical compositions can be carried out in various ways, including parentéral, 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 présent invention are particularly useful for parentéral administration, i.e., subcutaneously, intramuscularly, intravenously, intraperitoneally, intramedullary, intraarticulary, intrasynovialy and/or intrathecally. The parentéral administration can be carried out by bolus injection or continuous infusion. The pharmaceutical compositions for injection may be in a unit dosage form, for example, but is not limited to in ampoules, vials, prefilled syringes or in multi-dose containers with an added preservative. In addition, a number of recent drug delivery approaches hâve been developed, and the pharmaceutical compositions of the présent invention are suitable for administration by these new methods, e.g., Inject-ease®, Genject®, injectons such as GenPen®, and needleless devices such as MediJector® and BioJector®. The pharmaceutical composition of the présent invention can also be adapted for yet to be discovered modes of administration. See also Langer, 1990, Science, 249:15271533 .
The provided pharmaceutical compositions can also be formulated as a depot préparation. Such long-acting formulations can be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the formulations can be modified using suitable polymeric or hydrophobie materials (e.g., as an émulsion in an acceptable oil), or ion exchange resins, or as sparingly soluble dérivatives, for example, as a sparingly soluble sait.
The pharmaceutical compositions may, if desired, be provided in a vial, package or dispenser device, which may contain one or more unit dosage forms comprising the active ingrédient. In one embodiment, the dispenser device may comprise a syringe comprising a single dose of a ready for injection liquid formulation. The syringe can be accompanied by the instruction for administration.
In another embodiment, the présent invention provides a kit or container containing an aqueous pharmaceutical composition of the invention. The polypeptide concentration in the aqueous pharmaceutical composition can vary over a wide range, but generally within the range of about 1 to about 200 mg/mL of the aqueous formulation. The kit can also be accompanied by the instruction for use.
Method for Producing
A method for producing the above compositions comprises the addition of acetate buffering agents to an aqueous phase followed by the addition, in any order, of the following components: an osmolyte and/or a stabilizing agent selected from the group of trehalose, proline or a combination thereof; a recombinant monoclonal anti-TNFa antibody; a surface-active substance selected from the group of polysorbate 20, polysorbate 80, poloxamer 188, or a combination thereof.
Techniques
1. Protein Concentration Détermination in Test Samples.
The protein concentration was determined using UV spectrophotometry at a wavelength of 280 nm in UVspectrophotometry plates.
Each sample was diluted with appropriate placebo solution to a concentration of ~ 0.5 mg/mL. 150 pL of the diluted sample was placed into a well of the UV spectrophotometry plate. The optical density of the plate solutions was measured using a plate-reader at the wavelength of 280 nm. The appropriate placebo solution was used as the reference solution.
The protein concentration (C) at mg/mL was calculated by the following formula:
_ A(280) * b ε * l
A280 is optical density value at the wavelength of 280 nm;
ε is extinction value for the study protein;
b is total dilution ratio of the sample;
is layer thickness per plate well; for 150 pL, 1 = 0.42 cm.
2. Buffer Replacement and Sample Concentration.
Diafiltration and concentration of the samples were performed in Stirred Cell (Millipore) concentration cells under pressure.
3. PEG aggregation.
Préparation of PEG 6000 Solutions.
A PEG 6000 solution having a mass concentration of 20-25% in the study additive formulation was prepared. The resulting solutions were filtered through a 0.45 pm Durapore filter.
The calculated amount of the sample, the additive solution, and 20-25% PEG 6000 solution were transferred into 96-well UV-spectrophotometry plates such that the PEG6000 concentration per well was from 0 to 18%, and the protein concentration per well was 1 mg/mL. Ail the resulted solutions in wells were well mixed by pipetting.
Next, the solution degree of turbidity was assessed visually, and the optical density of the solutions was measured at a wavelength of 400 nm. The less stable the sample, the lower PEG 6000 concentration will form visible aggregates (opalescence). The solution degree of turbidity was also evaluated following one or more days after the solution préparation.
4. Protein Homogeneity and Aggregation Point Détermination using Dynamic Light Scattering (DLS) Technique.
Study sample homogeneity détermination was performed on a Zetasizer Nano ZSP in the Size measurement mode. For this purpose, 0.05 mL of the solution was placed into a dust-free disposable plastic cuvette.
• Analytical model: Protein analysis.
• Holding at the température for 30 seconds before measuring.
• At each point, an average of 13 measurements in 3 réplications.
Détermination of the study protein aggregation point was performed on the Zetasizer Nano ZSP. For this purpose, the solution was placed into a quartz dust-free cuvette, which was gradually heated in the instrument while continuously measuring the intensity of the scattered light in the Température trend measurement mode.
• Analytical model: Protein analysis.
• Température trend, mod: Protein aggregation point. From 50 to 85°C at the heating step of 1.5°C.
• Holding at the température for 30 seconds before measuring.
• At each point, an average of 15 measurements in 1 réplication.
A température trend was plotted using the instrument software, which automatically calculâtes the protein aggregation point (Aggregation point).
5. Thermal Stability Détermination by Thermostress 50°C Technique.
The test samples were divided into 2 parts and placed in separate tubes: 1 tube for each formulation was stored in a refrigerator at + 4°C, the rest were placed into an incubator and incubated at 50°C for the specified time. On completing the warm-up period, the tubes were removed from the incubator, allowed to stand at room température for about 15 minutes, and submitted for analysis according to the spécification.
6. Colloïdal Stability Détermination by Shake-Test Technique.
The test samples were divided into 2 parts of 200 pL each and placed in glass vials, 1 vial of each formulation was stored in the refrigerator at +4°C, the rest were placed into a thermo-shaker and shook at 800 rpm at 2 to 8°C for the specified time. On completing the stress, the tubes were removed from the thermo-shaker and submitted for analysis according to the spécification.
7. Colloïdal Stability Détermination by Cryoconcentration Technique.
The test samples were divided into 2 parts and placed in polymeric tubes: 1 tube for each formulation was stored in a refrigerator at +4°C, the rest were placed into a freezing chamber and stored at minus 16 to 20°C for the specified time. On completing the stress, the tubes were removed from the freezing chamber, allowed to stand at room température until the contents completely thawed, the solutions were mixed using Vortex and submitted for analysis according to the spécification.
8. Sample Purity Détermination by Size Exclusion HighPerformance Liquid Chromatography (SE-HPLC) Technique.
Column: Tosoh TSK-GelG3000SWXL 7.8 mm ID χ 30 cm, cat. # 08541.
Column température: 25 °C.
Mobile phase flow rate: 0.7 mL/min.
Injection volume: 20 pL.
Sample concentration: 5 mg/mL.
Detector wavelength: 220 nm.
Elution time: 23 min.
Mobile phase: Anhydrous disodium hydrophosphate: 7.1 mg/mL.
Sodium chloride: 17.54 mg/mL.
pH of the mobile phase was adjusted to 7.0 with orthophosphoric acid.
9. Sample Acid-Base Profile Détermination using Caliper LabChip GX II Instrument.
9.1. Sample préparation.
Samples were diluted to a concentration of 1 mg/mL. 2 pL of 5 mg/mL carboxypeptidase B (CpB) solution was added to 200 pL of the resulting solution. It was stirred and incubated for 2 hours at 37°C. The test samples were dialyzed against three water changes in Amicon Ultra centrifuge tubes and concentrated to 2 mg/mL.
9.2. Work solution préparation.
Work solutions and a plate with the assay samples were prepared according to the manufacturer' s procedure using the HT Protein Charge Variant Labeling Kit.
9.3. Chip préparation.
Préparation of the chip and tube with buffer was carried out according to the manufacturer' s procedure using buffer solutions from the Protein Charge Variant Buffer Kit.
The assay initiation is a standard operation. Protein Charge Variant 68s assay.
10. Sample Purify Détermination using Caliper Labchip GX II Instrument under Reducing and Non-Reducing Conditions.
10.1. Assay sample préparation.
700 pL of HT Protein Express Sample Buffer supplemented with 24.5 pL of 1M DTT for buffer under reducing conditions and 24.5 pL of IM IAM for buffer under non-reducing conditions was used to préparé denaturing and reducing solutions. Samples were diluted to a concentration of 2 mg/mL. Two microtubes were prepared for each sample - 35 pL of denaturing buffer was added to one, 35 pL of reducing buffer was added to another. The samples were denatured at 100°C for 5 minutes. Tubes were vortexed, then 70 pL of water was added to each tube and mixed. 44 pL of each sample was transferred to a 96-well plate for analysis.
10.2. Work solution préparation and chip loading.
Work solution and chip préparation were carried out according to a standard procedure using the HT Protein Express Reagent Kit. The assay initiation is a standard operation. HT Protein Express 200 assay.
11. Sample Acid-Base Profile Détermination using lonExchange (IE) HPLC.
Column: DIONEX ProPack WCX-10 4 mm x 250 mm (Tosoh bioscience, Japan);
Pre-column: Dionex ProPack WCX-10G
Mobile phase: Eluent A: 0.01 M 2-(Nmorpholino)ethanesulfonic acid (MESA), pH 6.0 Eluent B: 0.01 M MESA, 0.4 M sodium chloride, pH 5.5; Injection volume: 40 pL; Flow rate: 0.5 mL/min;
Column 25°C;
température : Autosampler 5°C;
température : Détection 280 nm.
wavelength: Gradient: Phase A 95 - 0 - 95 %.
. Purity Détermination by Polyacrylamide Gel Electrophoresis (PAGE) under Reducing and Non-Reducing Conditions.
PAG was prepared in the presence of sodium dodecyl sulfate in glass plates consisting of a concentrating layer of 4% PAGE and a separating layer: 12.5% PAG for reducing conditions, 8% PAG for non-reducing conditions.
The electrophoresis chamber was assembled and installed according to the operational manual for the vertical electrophoresis device. Specimens were prepared by diluting the samples with purified water to a final concentration of 1 mg/mL. A volume équivalent to 40 pg was taken and the prepared specimens of the test sample were mixed in a ratio of 3:1 (v/v) with a sample application buffer solution containing 2-mercaptoethanol (reducing conditions) and containing no 2mercaptoethanol (non-reducing conditions), mixed. The resulting solutions were incubated at a température of (99 ± 1)°C for 3 minutes (samples containing 2-mercaptoethanol) and at a température of (99 + 1)°C for 1 minute (samples containing no 2-mercaptoethanol). The solutions were cooled to room température, mixed and transferred to the wells of PAG under the layer of running buffer solution.
Electrophoresis was run in a direct current mode using a water cooling System. The power source parameters were set as following: when the dye front was passing through the stacking gel, the voltage was 110 V. Once the dye front entered the lower running gel for 5 to 7 mm, the voltage was increased to 180 V. The power source was turned off when the dye front reached the lower limit of the gel.
Once the electrophoresis finished, the gels were separated from the glasses and proteins were fixed with the fixing solution for 16 to 18 hours at room température. Further, the gels were stained (in acid blue 83 solution) and washed until a clear view of the bands. The gels were scanned. The purity and impurities in the test samples were evaluated using the GelPro software.
13. Spécifie Activity Détermination.
Sample-specific activity was assessed by the ability to bind specifically and neutralize TNFa, which in turn has a cytotoxic effect and causes the death of WEHI-13 var culture (fibrosarcoma, Mus musculus), a variant of the culture that has increased sensitivity to TNFa in the presence of actinomycin D. Sample préparation was performed using a TecanEvo 200 robotic station, RPMI1640, 2 mM Gin, 10% FBS, 2.5 pg/mL of actinomycin D, 5 pg/mL of gentamicin were used as the QM (quantification medium).
The test antibody sample was diluted to 50 pg/mL with a dilution step of no more than 20 and placed into the robotic station. Using the TecanEvo200, three independent dilutions of RS and IS in the range of 10,000 to 0.5 ng/mL were prepared in culture plates, 500 pg/mL of TNFa solution was added to prepared dilutions. The resulting mixture was stirred and incubated at room température for 1 hour.
After the incubation, (0.5 ± 0.1) χ 106 cells/mL of WEHI13 var cell suspension was added. The plates were placed in a
CO2 incubator and incubated at the température of (37 ± 1)°C, 5% C02 in humidified air for 20-24 h.
At the end of the incubation period, the vital dye Alamar Blue was added to the culture plates and the plates were incubated under the same conditions until the color development. The fluorescence intensity was evaluated at an excitâtion/emission wavelength of 544/590 nm using an Infinité M200Pro reading device. Fluorescence intensity versus protein concentration curves were plotted using Magellan 7.2 software; the parallel alignment of the resulted curves was evaluated. The relative spécifie activity of the test samples was determined as the reference sample ED50 to test sample ED50 ratio, expressed as a percentage.
The following examples are provided for the best understanding of the invention. These examples are provided for illustrative purposes only and are not to be construed as limiting in any way the scope of the invention.
Ail publications, patents and patent applications cited in this spécification are included herein by reference. While the above invention has been described in some detail by way of illustration and example for purpose of clarity, as will be appreciated by those skilled in the art based on the teaching disclosed herein, certain changes and modifications can be made without departing from the spirit and scope of the enclosed embodments of the invention.
Examples
Example 1. Buffer Solution Nature Sélection.
Study Formulations.
Four buffer solutions were selected for this study, and 5 the original Humira préparation formulations (for formulations having protein content of 50 mg/mL and 100 mg/mL) were used as a control.
Humira 1 Disodium hydrophosphate dihydrate 1.530 mg/mL Sodium dihydrophosphate dihydrate 0.860 mg/mL Mannitol 12.0 mg/mL Citric acid monohydrate 1.305 mg/mL Polysorbate 80 1.0 mg/mL Sodium citrate 0.305 mg/mL Sodium chloride 6.165 mg/mL Sodium hydroxide up to pH 5.2
Humira 2 Mannitol 42.0 mg/mL Polysorbate 80 1.0 mg/mL
Acet, pH 5.0 Sodium acetate trihydrate 0.436 mg/mL Glacial acetic acid up to pH 5.0
Cit, pH 5.0 Sodium citrate dihydrate 0.932 mg/mL Citric acid anhydrous 0.352 mg/mL
His, pH 6.0 L-histidine 0.40 mg/mL Histidine hydrochloride monohydrate 0.40 mg/mL
PB, pH 6.0 Sodium dihydrophosphate monohydrate 1.21 mg/mL Sodium phosphate anhydrous 0.17 mg/mL
1.1. Colloïdal Stability Détermination by PEG Aggregation.
The PEG aggregation test was performed in triplicate for 10 each sample. The analysis was performed according to the procedure 3. The data on the average optical density of the solutions are presented in Table 1. The results are also shown in Fig. 1.
Table 1. The average optical density of the solutions after préparation.
PEG 6000, % 0 % 2 % 4 % 6 % 8 % 10 % 12 % 14 % 16 % 18 %
Acet, pH 5.0 0.0597 0.0519 0.0531 0.0593 0.0590 0.0598 0.0502 0.0513 0.0591 0.0608
Cit, pH 5.0 0.0582 0.0620 0.0620 0.0639 0.6261 1.0340 1.4279 1.4765 1.3668 1.4133
His, pH 6.0 0.0583 0.0615 0.0621 0.0634 0.0637 0.0664 0.0781 0.9847 1.3867 1.5626
PB, pH 6.0 0.0598 0.0619 0.0654 0.0656 0.0712 0.9003 1.3453 1.5324 1.4662 1.4297
Humira 1 0.0563 0.0583 0.0594 0.0614 0.3005 0.9315 1.1182 1.3925 1.3980 1.4120
Humira 2 0.0605 0.0579 0.0608 0.0834 0.0845 0.0685 0.0673 0.0658 0.0803 0.1320
There is visible aggregation
1.2. Thermal Stability Détermination by Protein Aggregation Point using Dynamic Light Scattering (DLS) Technique.
The analysis was performed according to the procedure 4.
The results are shown in Table 2 and Fig. 4-8.
1.3. Thermal Stability Détermination under Long-Term Exposure using Thermostress 50°C Method.
The analysis was performed according to the procedure 5. The results are shown in Table 2.
1.4. Results.
Table 2. Summary of the buffer solution nature sélection.
Formulation Thermal stress 50°C, 24 h Visible aggregation at PEG 6000 O O (SP type) Aggregation point, °C (DLS)
Increase in impurities, o. O (SE-HPLC) Increase in fragments, O, O (SE-HPLC)
Protein concentration 5 mg/mL 1 mg/mL
Acet, pH 5.0 0.02 A o.oi j > 18 74.0. ;
Cit, pH 5.0 1.50 0.08 8 69.5
His, pH 6.0 0.75 0.20 14 69.5
PB, pH 6.0 0.55 0.11 10 71.0
Humira 1 0.38 0.15 8 69.5
Humira 2 0.02 ; 0.01 ; > 18 72.5
positive results négative results average results
1.5. Conclusions from Example 1.
Based on the results of this study, the recommended storage formulation (excluding additional additives) is:
Acet, pH 5.0 Sodium acetate trihydrate 0.436 mg/mL Glacial acetic acid up to pH 5.0
The présent formulation showed the best stabilizing properties among ail the test sample: the lowest increase in impurities during thermal stress (0.02% on a par with Humira 2 20 formulation), the absence of aggregation at 18% PEG 6000, and the highest aggregation température (74°C).
Adalimumab with Humira 1 formulation has a lower thermal stability than the recommended formulation. Minimal colloïdal and thermal stability was noted for the formulation based on 5 mM citrate buffer solution with pH of 5.0.
Example 2. Composition pH and Buffer Capacity Optimization.
Based on the results of the first part of the study, adalimumab showed the best stability in acetate buffer solution with pH 5.0. According to the prior art, the most patents and patent applications for pharmaceutical 10 compositions comprising adalimumab protect solutions with pH of 4.0 to 8.0. Thus, the purpose of the présent section was to study the possibility for obtaining stable compositions comprising acetate ions with pH of less than 4.
Study Formulations.
Abbreviation Concentration, mM PH
Acet 5 mM pH 6 5 6.0
Acet 5 mM pH 5.5 5 5.5
Acet 5 mM pH 5 5 5.0
Acet 5 mM pH 4 5 4.0
Acet 5 mM pH 3.75 5 3.75
Acet 5 mM pH 3.5 5 3.5
Acet 10 mM pH 6 10 6.0
Acet 10 mM pH 5.5 10 5.5
Acet 10 mM pH 5 10 5.0
Acet 10 mM pH 4 10 4.0
Acet 10 mM pH 3.75 10 3.75
Acet 10 mM pH 3.5 10 3.5
Acet 20 mM pH 6 20 6.0
Acet 20 mM pH 5.5 20 5.5
Acet 20 mM pH 5 20 5.0
Acet 20 mM pH 4 20 4.0
Acet 20 mM pH 3.75 20 3.75
Acet 20 mM pH 3.5 20 3.5
2.1. Colloïdal Stability Détermination by PEG Aggregation.
The PEG aggregation test was performed in triplicate for each sample. The analysis was performed according to the procedure 3. The data on the average optical density of the 20 solutions are presented in Table 3. The results are also shown in Fig. 2.
Table 3. The average optical density (400 nm) of the solutions after préparation.
PEG 6000, Acet 5 mM pH 6 Acet 5 mM pH 5.5 Acet 5 mM pH 5 Acet 5 mM pH 4 Acet 5 mM pH 3.75 Acet 5 mM pH 3.5 Acet 10 mM pH 6 Acet 10 mM pH 5.5 Acet 10 mM pH 5 Acet 10 mM pH 4
0 % 0.0341 0.0344 0.0299 0.0303 0.0312 0.0285 0.0365 0.0314 0.0289 0.0290
6 % 0.0346 0.0341 0.0293 0.0294 0.0290 0.0288 0.0315 0.0316 0.0316 0.0296
8 % 0.0344 0.0342 0.0290 0.0291 0.0295 0.0289 0.0355 0.0322 0.0312 0.0289
10 % 0.0346 0.0331 0.0298 0.0298 0.0297 0.0296 0.0344 0.0331 0.0299 0.0296
12 % 0.0348 0.0338 0.0302 0.0313 0.0319 0.0358 0.0371 0.0318 0.0303 0.0295
14 % 0.0361 0.0346 0.0313 0.0327 0.0331 0.0327 0.0367 0.0316 0.0313 0.0326
16 % 0.0359 0.0341 0.0341 0.0331 0.0333 0.0329 0.0346 0.0316 0.0339 0.0334
18 % 0.0348 0.0350 0.0308 0.0308 0.0310 0.0310 0.0344 0.0355 0.0307 0.0314
PEG 6000, % Acet 10 mM pH 3.75 Acet 10 mM pH 3.5 Acet 10 mM pH 3.5 Acet 20 mM pH 6 Acet 20 mM pH 5.5 Acet 20 mM pH 5 Acet 20 mM pH 4 Acet 20 mM pH 3.75 Acet 20 mM pH 3.5
0 % 0.0281 0.0292 0.0292 0.0344 0.0310 0.0295 0.0289 0.0289 0.0289
6 % 0.0292 0.0288 0.0288 0.0346 0.0322 0.0289 0.0304 0.0294 0.0285
8 % 0.0299 0.0337 0.0337 0.0381 0.0328 0.0292 0.0307 0.0296 0.0298
10 % 0.0295 0.0306 0.0306 0.0346 0.0316 0.0295 0.0291 0.0302 0.0312
12 % 0.0319 0.0321 0.0321 0.0346 0.0318 0.0300 0.0308 0.0301 0.0308
14 % 0.0309 0.0331 0.0331 0.0398 0.0320 0.0322 0.0312 0.0315 0.0306
16 % 0.0306 0.0320 0.0320 0.0361 0.0310 0.0325 0.0321 0.0315 0.0320
18 % 0.0313 0.0313 0.0313 0.0356 0.0311 0.0322 0.0321 0.0324 0.0362
2.2. Thermal Stability Détermination under Long-Term Exposure using Thermostress 50°C Method.
The analysis was performed according to the procedure 5 .
2.3. Colloïdal Stability Détermination by Shake-Test Technique.
The analysis was performed according to the procedure 6.
2.4. Colloïdal Stability Détermination by Cryoconcentration Technique.
The analysis was performed according to the procedure 7 .
Summary is provided in Table 4, where the sample quality measures before and after the thermal stress, shake test and cryoconcentration are demonstrated.
2.5. Results.
Table 4. Summary of the sample quality measures before and after stress.
Input control
pH placebo pH after dialysis Δ pH after dialysis Protein c, mg/mL Turbidity, OD 400 nm SP-type
5 mM acet pH 6.0 6.00 6.10 0.10’ 10 0.0506
10 mM acet pH 6.0 6.00 6.06 0.0 6 10 0.0531
20 mM acet pH 6.0 6.00 6.04 0.04 10 0.0511
5 mM acet pH 5.5 5.50 5.60 ο.Ίό 10 0.0513
10 mM acet pH 5.5 5.50 5.53 0.03 10 0.0531
20 mM acet pH 5.5 5.50 5.51 ô. ôi 10 0.0514
5 mM acet pH 5.0 5.00 5.15 Ô.15 10 0.0504
10 mM acet pH 5.0 5.00 5.12 0.12 10 0.0501
20 mM acet pH 5.0 5.00 5.09 Cm 09 10 0.0488
5 mM acet pH 4.0 4.00 4.35 0.35 10 0.0446
10 mM acet pH 4.0 4.00 4.22 0.22 10 0.0471
20 mM acet pH 4.0 4.00 4.20 0.20 10 0.0489
5 mM acet pH 3.75 3.75 4.04 0.29 10 0.0504
10 mM acet pH 3.75 3.75 4.01 0.26 10 0.0501
20 mM acet pH 3.75 3.75 3.95 0.20 10 0.0473
5 mM acet pH 3.5 3.50 3.90 0.40 10 0.0511
10 mM acet pH 3.5 3.50 3.84 0.34 10 0.0476
2 0 mM acet pH 3.5 3.50 3.79 0.29 ____12____ 0.0492
positive results négative results average results/baseline data
Table 5.
Thermal stress 50°C, 96 h Shake test 800 rpm, 96 h
Δ pH* Change in purity*, % Turbidity, 400 nm Acid-base profile Δ abs. Δ pH* Change in purity*, % Turbidity, OD 400 nm Acid-base profile Δ abs. % * *
SE-HPLC Caliper EP, red. Caliper EP, nonred. SP type Caliper SE-HPLC Caliper EP, red. Caliper EP, nonred. SP type Caliper
5 mM acet pH 6.0 0.03 -0.58 -1.88; -0.81 0 30446 28.11 0.03 -0.26 -0.49 +0.03 0.0490 2.66
10 mM acet pH 6.0 0.04 -0.66 -1.99 -0.94 0.0479 32.34 0.05 -0.31 -0.87 ; +0.19 0.0471 3.41
20 mM acet pH 6.0 0.01 -0.74 ' -2.09 7 -1.59 0.0487 33.09 0.02 -0.38 -0.96 +0.21 0.0468 3.98
5 mM acet pH 5.5 0.05' -0.47 -1.741 ' -1.01 0.0455 25.41 : 0.04 -0.22 -0.99 0.55 0.0413 1.38
10 mM acet pH 5.5 0,06 - -0.76.! / -1,61/ï -0.76 0.0489 26.19 0.06 -0.28 -0.87 +0.51 0.0482 1.55
20 mM acet pH 5.5 0.03 ' ' -0.88? -2.33 ' -0.81 0.0494 28.88 • 0.09 -0.31 ‘ -0.79 +0.63 O.O460 1.49
5 mM acet pH 5.0 0.00 -0.55 -1.99 -1.13 0.0506 26.57 0.07 -0.33 -1.17 +0.49 0.0480 2.45
10 mM acet pH 5.0 0.05 2. -0.73 -2.06 -0.07, 0.0517 22.04 ’ 0.06 -0.50 -1.07 0.55 0.0499 3.22
20 mM acet pH 5.0 0.03 r -0.91 -a. 65' -1.90 0.0527 32.92 0.03 -0.51 +0.29C -0.21 0.0520 7.02
5 mM acet pH 4.0 -0.06 -2.00 -2.67 -0.59 0.0494 33.07 0.05 ' -0.24 -1.49 +2.05 0.0466 / 1.51
10 mM acet pH 4.0 - 0.03 -2.57 -2.55 -1.63 0.0507 32.88 0.07 -0.29 -0.91 +0.45 0.0505 3.44
20 mM acet pH 4.0 -0.03'. -2.85 -1·24 -0.99 0.:0511 30.75 0.01 -0.40 +1.34 +0.85 0.0497 2.80
5 mM acet pH 3.75 -0.01 ' -3.32 -5.00 -2.63 0.0492 29.11 0.07 -/ ////-0.. 4 2 +1/33/0 // -1.41 0.0484 0.99
10 mM acet pH 3.75 0.02' -3.09 -2.62 -2.50 0.0488 30.85 0.10 -0.31 -0.19: / +0.67 / 0.0452 1.29
20 mM acet pH 3.75 -0.01 · -4.09 -2.36 -3.23 0.0535 30.95 0.06 ' -0.51 +12.11 +0.37 0/0504 0.37
5 mM acet pH 3.5 /0.00 -4.28 -5.50 -2.72 : 0.0489 /Αβ/ΐΐΟ/ - 0.08 -0.38 -2.19 ' + 1.03 0.0468 r 0.76
10 mM acet pH 3.5 0.02 ' -4.61 -6.22 -2.88 0.0494 32.07 0.10 -0.38 -0.01 +0.50 0.0471 3.78
20 mM acet pH 3.5 -0.05 -6.87 -7.32 -3.11 / :0//0500 / 38.64 -/0:.02 . -0.67 +0.63 +1.01 0.0498 2.67 : 7/
Table 5. Continuation.
Freezing -20 ° C, defrosting + 25 ° C
Δ pH* * Change in purity*, % Turbidity, OD 400 nm Acid-base profile Δ abs. % * *
SE-HPLC Caliper EP, red. Caliper EP, nonred. SP type Caliper
5 mM acet pH 6.0 -0.06 -0.24 -0.44 -0.21 0.0487 3.11
10 mM acet pH 6.0 -0J08 -0.31 „ ' -0.74 -0.38 493 3.54 .ijy,
20 mM acet pH 6.0 -0.04 -1.23 -0.59 -0.36 ΌΛ511 4.11
5 mM acet pH 5.5 -0.09 -0.39 -0.56 -0.31 0.0486 2.66
10 mM acet pH 5.5 -0.07 -0.60 -0.41 -0.28 0.0473 2.19
20 mM acet pH 5.5 -0.06 -0.45 -0.09 . -0.41 0.,lÿ80 4.01
5 mM acet pH 5.0 -0.05 -0.20 -0.46 -0.39 0.0494 2.67
10 mM acet pH 5.0 -0.01 -0.49 -0.87 + 1.09 0.0466 4.46
20 mM acet pH 5.0 0.01 -0.59 -0.08 -0.09 0.0536 3.69
5 mM acet pH 4.0 -0.09 -1.29 -0.75 +0.99 0.0475 2.06 F
10 mM acet pH 4.0 0.00 -1.77 -2.11 -0.79 0.0440 3.18
20 mM acet pH 4.0 -0.04 -1.62 +0.73 +0.72 0.0513 4.54
5 mM acet pH 3.75 -0.05 -1.49 -1.74 -0.01 0.0463 ,:3.07
10 mM acet pH 3.75 -0.02 -1.76 -1.68 +0.70 0.0473 3.02
20 mM acet pH 3.75 0.00 -2.16 +2.42 +1.03 0.0544 4.70
5 mM acet pH 3.5 -0.04 -1.22 -2.09 +0.72 0.0479 2.60
10 mM acet pH 3.5 0107 -1.97 -1.59 . +0.07 0.0492 6.83-
20 mM acet pH 3.5 -0.06 -2.98 -1.13 +1.02 0.0427 4.88
* Change was calculated by the formula Δ = (Value after stress - Value before stress) ** Absolute change was calculated by the formula
Δ = (Content of acid fraction before stress - Content of acid fraction after stress| +
négative results average results / baseline data (Content of basic fraction before stress Content of basic fraction after stress | + (Content of dominant fraction before stn
Content of dominant fraction after stress|
2.6. Conclusions from Example 2.
• The study showed that the presence of adalimumab in acid solutions of placebo leads to a significant increase in the pH level. In addition, compositions with pH of less than 5.0 5 demonstrated poor stability during dialysis (low purity on Labchip Caliper under reducing conditions) and thermal stress (low purity after stress on ail assay methods used).
• The most stable sample among ail studied is adalimumab in 5 mM acetate buffer solution with pH of 5.0 to 6.0. They 10 demonstrated a minimal change in purity and in acid-base profile during the stresses. To create an adalimumab composition, however, it is possible to use solutions with a larger buffer capacity.
Example 3. Osmotic and Stabilizer Agent Sélection.
Based on the results of the first part of the study, adalimumab showed the best stability in acetate buffer solution with pH 5.0-6.0. The formulation with pH 5.5 was used as a basis for the osmolyte and stabilizing agent sélection.
Study Formulations.
Humira 1 Disodium hydrophosphate dihydrate 1.530 mg/mL Sodium dihydrophosphate dihydrate 0.860 mg/mL Mannitol 12.0 mg/mL Citric acid monohydrate 1.305 mg/mL Polysorbate 80 1.0 mg/mL Sodium citrate dihydrate 0.305 mg/mL Sodium chloride 6.165 mg/mL Sodium hydroxide up to pH 5.2
Humira 2 Mannitol 42.0 mg/mL Polysorbate 80 1.0 mg/mL
Acet, pH 5.5 Sodium acetate trihydrate 0.436 mg/mL Glacial acetic acid up to pH 5.5
Acet + NaCl, pH 5.5 Sodium acetate trihydrate 0.436 mg/mL Glacial acetic acid up to pH 5.5 Sodium chloride 9 mg/mL
Acet + Tre, pH 5.5 Sodium acetate trihydrate 0.436 mg/mL Glacial acetic acid up to pH 5.5 Trehalose dihydrate 100 mg/mL
Acet + Suc, pH 5.5 Sodium acetate trihydrate 0.436 mg/mL Glacial acetic acid up to pH 5.5 Sucrose 100 mg/mL
Acet + Mannitol, pH 5.5 Sodium acetate trihydrate 0.436 mg/mL Glacial acetic acid up to pH 5.5
Mannitol 50 mg/mL
Acet + Sorb, pH 5.5 Sodium acetate trihydrate 0.436 mg/mL Glacial acetic acid up to pH 5.5 Sorbitol 50 mg/mL
Acet + lOOmM Gly , pH 5.5 Sodium acetate trihydrate 0.436 mg/mL Glacial acetic acid up to pH 5.5 Glycine 7.5 mg/mL
Acet + 50 mM Arg, pH 5.5 Sodium acetate trihydrate 0.436 mg/mL Glacial acetic acid up to pH 5.5 Arginine hydrochloride 10.5 mg/mL
Acet + 10 mM Meth, pH 5.5 Sodium acetate trihydrate 0.436 mg/mL Glacial acetic acid up to pH 5.5 Méthionine 1.5 mg/mL
Acet + 250 mM Prol, pH 5.5 Sodium acetate trihydrate 0.436 mg/mL Glacial acetic acid up to pH 5.5 L-Proline 27.0 mg/mL
Acet + 10 mM EDTA, pH 5.5 Sodium acetate trihydrate 0.436 mg/mL Glacial acetic acid up to pH 5.5 EDTA disodium sait dihydrate 3.72 mg/mL
Acet + lOOmM Lys, pH 5.5 Sodium acetate trihydrate 0.436 mg/mL Glacial acetic acid up to pH 5.5 Lysine 14.6 mg/mL
Acet + lOOmM Guan, pH 5.5 Sodium acetate trihydrate 0.436 mg/mL Glacial acetic acid up to pH 5.5 Guanidine hydrochloride 9.55 mg/mL
Acet + lOOmM Glu, pH 5.5 Sodium acetate trihydrate 0.436 mg/mL Glacial acetic acid up to pH 5.5 Glucose monohydrate 19.82 mg/mL
Acet + lOOmM Lact, pH 5.5 Sodium acetate trihydrate 0.436 mg/mL Glacial acetic acid up to pH 5.5 Lactose monohydrate 36.03 mg/mL
Acet + lOOmM Mannose, pH 5.5 Sodium acetate trihydrate 0.436 mg/mL Glacial acetic acid up to pH 5.5 Mannose 18.02 mg/mL
Acet + Tre + 0. Cyst, pH 5.5 Sodium acetate trihydrate 0.436 mg Glacial acetic acid up to pH 5.5 Trehalose dihydrate 100 mg Cysteine hydrochloride monohydrate 0.088 mg
Acet + Tre + 2. Cyst, pH 5.5 Sodium acetate trihydrate 0.436 mg Glacial acetic acid up to pH 5.5 Trehalose dihydrate 100 mg Cysteine hydrochloride monohydrate 0.44 mg
Acet + lOmM Cyst, pH 5.5 Sodium acetate trihydrate 0.436 mg/mL Glacial acetic acid up to pH 5.5 Cysteine hydrochloride monohydrate 1.76 mg/mL
Acet + Tween 20 0.5, pH 5.5 Sodium acetate trihydrate 0.436 mg/mL Glacial acetic acid up to pH 5.5 Polysorbate 20 0.5 mg/mL
Acet + Tween20 1.0, pH 5.5 Sodium acetate trihydrate 0.436 mg/mL Glacial acetic acid up to pH 5.5 Polysorbate 20 1.0 mg/mL |
Acet + TweenS0 0.5, pH 5.5 Sodium acetate Glacial acetic Polysorbate 80 trihydrate acid up to 0.436 mg/mL pH 5.5 0.5 mg/mL
Acet + Tween80 1.0, pH 5.5 Sodium acetate Glacial acetic Polysorbate 80 trihydrate acid up to 0.436 mg/mL pH 5.5 1.0 mg/mL
Acet + Pol.188 0.5, pH 5.5 Sodium acetate Glacial acetic Poloxamer 188 trihydrate acid up 0.5 to m 0.436 mg/mL pH 5.5 g/mL
Acet + Pol.188 1.0, pH 5.5 Sodium acetate Glacial acetic Poloxamer 188 trihydrate acid up 1.0 to m 0.436 mg/mL pH 5.5 g/mL
3.1. Colloïdal Stability Détermination by PEG Aggregation.
The PEG aggregation test was performed in triplicate for each sample. The analysis was performed according to the procedure 3. The data on the average optical density of the 5 solutions are presented in Table 6. The results are also shown in Fig. 3.
Table 6. The average optical density of the adalimumab solutions after préparation.
PEG% 0.5 Tween 20 1 Tween 20 0.5 Tween 80 1 Tween 80 0.5 Pol.18 8 1 Pol.188 NaCl Tre Mann Sort Suc
0 0.064 8 0.063 8 0.071 4 0.073 3 0.0588 0.0599 0.073 7 0.059 9 0.069 9 0.059 9 0.062 1
6 0.063 2 0.065 4 0.068 7 0.075 5 0.0552 0.0566 0.057 0 0.063 8 0.066 4 0.061 4 0.064 9
8 0.062 0 0.065 8 0.065 4 0.076 9 0.0590 0.0565 0.062 2 0.066 1 0.065 2 0.058 2 0.064 6
10 0.067 4 0.067 0 0.070 0 0.077 6 0.0644 0.0669 0.233 9 0.063 0 0.060 0 0.061 5 0.069 6
12 0.064 9 0.067 1 0.070 4 0.075 4 0.0650 0.0673 0.706 6 0.066 3 0.063 7 0.061 1 0.070 0
14 0.066 0 0.070 9 0.070 4 0.081 9 0.0668 0.0710 1.202 7 0.073 3 0.063 3 0.061 8 0.072 7
16 0.066 3 0.070 3 0.104 9 0.147 9 0.0687 0.0660 1.233 9 0.066 0 0.062 0 0.062 3 0.073 3
18 0.067 4 0.070 7 0.123 0 0.168 1 0.0689 0.0773 1.275 4 0.062 1 0.065 8 0.062 0 0.076 0
PEG % Gly Arg Meth Prol EDTA Lys Guan Glu Lact Mannose lOCys t
0 0.061 1 0.086 3 0.058 1 0.065 0 0.056 1 0.069 8 0.063 6 0.052 9 0.069 3 0.0582 0.061 6
6 0.061 3 0.061 3 0.056 7 0.069 6 0.057 3 0.069 7 0.052 5 0.056 5 0.061 0 0.0601 0.065 0
8 0.064 6 0.061 4 0.062 0 0.072 2 0.058 4 0.072 3 0.060 6 0.056 3 0.062 3 0.0613 0.067 0
10 0.068 2 0.070 0 0.067 6 0.073 4 0.695 8 0.076 5 0.056 5 0.057 3 0.065 0 0.0633 0.073 3
12 0.071 0 0.068 7 0.064 5 0.079 8 1.035 5 0.085 5 0.224 2 0.059 3 0.066 3 0.0612 0.070 8
14 0.072 6 0.076 9 0.065 7 0.083 7 1.096 4 0.886 7 0.906 0 0.061 1 0.066 3 0.0638 0.083 6
16 0.073 9 0.948 3 0.067 6 0.086 3 1.293 5 1.294 2 1.311 2 0.061 8 0.066 8 0.0630 0.075 8
18 0.077 7 1.238 3 0.077 5 0.086 9 1.319 9 1.433 8 1.413 8 0.058 5 0.067 3 0.0657
Ο . 072
There is visible aggregation
3.2. Thermal Stability Détermination by Protein Aggregation Point using Dynamic Light Scattering (DLS) Technique.
The analysis was performed according to the procedure 4.
3.3. Thermal Stability Détermination under Long-Term Exposure using Thermostress 50°C Method.
The analysis was performed according to the procedure 5.
3.4. Results.
Table 7. Summary on the sample quality measures before and after stress.
Protein C, mg/mL Thermal stress 50°C, 72 h Aggregation température, C Aggreg. at % PEG 6000 Conclusion
Change in purity, %* Increase in fragments, %* Turbidity, OD 400 nm Acid-base profile Δ abs. %**
SE-HPLC SP type Labchip Caliper
Humira 1 5 -1.10 0.94 0,0544- 21.44 69.5*** 8
Humira 2 5 -o:i8 0.10 ' L 0.0487 - 18.38 72.5 >18 j
Acet 5 -0.46 0.43 0.0464 17.01 74.0 16
Acet + NaCl 5 -2.03 1.86 0.0457 ” 22.05 66.5*** » 1Q
Acet + Tre 5 1 -0.28 ' ‘ 0.15 0.0581 î 14.55 75.5 Ί > 18 .+ ' J
Acet t Suc 5 -0.46 0.34 0.0505 19.78 74.0 >18
Acet + Mannitol 5 -0.48 0.30 0.0521 19.03 75.5 1 ' > 18
Acet + Sort 5 -0.53 0.34 0.0574 20.78 75.5 18
Acet + lOOmM Gly 5 -0139 T/l l·20 - : 0.0457 20.70 74.0 ; >18 +
Acet + 50 mM Arg 5 -1.31 1.15 0.0497 ; 14.90 69.5*** 16 , ... + .
Acet + 10 mM Meth 5 -0-42 ’ 0.3\ ; , ' 0.0432^ 16.27 - . >18 +
Acet + 250 mM Prol 5 -0.38 0.27 ' 0.0448 15.43 74.0 > 18 +
Acet + 10 mM EDTA 5 -1.24 0.72 0.0488 ; 12.08 : 68.0*** 10
Acet + lOOmM Lys 5 -3.2 6 2.19 0.0424 17.67 ,1 68.0*** 14
Acet + lOOmM Guan 5 -3.98 2.36 ‘ '0.0480 17.51 66.5*** 12
Acet + lOOmM Glu 5 -1.60 1.54 0.0496c 55.64 74.0 > 18
Acet + lOOmM Lact 5 -1.86 1.60 0.0457 ' 29.43 74.0 > 18
Acet + lOOmM Mannose 5 -1.73 1.57 '0.0450 44.16 74.0 > 18
Protein C, mg/mL Thermal stress 50°C, 72 h Aggregation température, °C Aggreg. at % PEG 6000 Conclusion
Change in purity, %* Increase in fragments, %* Turbidity, OD 400 nm Acid-base profile Δ abs. %**
SE -HPLC ΞΡ type Labchip Caliper
Acet + 0. Cyst 5 -5.08 3.55 0.0425 - - -
Acet + 2. Cyst 5 -8.99 5.78 0.0440 - - -
Acet + lOmM Cyst 5 -100 93.20 0.0467 - - > 18
Acet + Tween20 0.5 5 - - 0.0425 19.25 74.0*** > 18
Acet + Tween20 1.0 5 -0.41 0.33 . 0.0452 20.72 74.0*** . > 18 :
Acet + Tween80 0.5 5 -0.46 0.39 0.0475 19.52 72.5*** > 18
Acet + TweenSO 1.0 5 -0.48 0.31 0.0436 17.36 72.5*** >18 - + ,Ί
Acet + Koll 0.5 5 , -0.31 0.30 0.0452 J 17.65 74.0 >18 1
Acet + Koll 1.0 5 -0.40 . Ό.44 -c 0.0487 1 19.46 74.0 > 18 + '
* Change was calculated by the formula Δ = (Value after stress — Value before stress) ** Absolute change was calculated by the formula
Δ = | Content of acid fraction before stress — Content of acid fraction after stress | + |Content of basic fraction before stress — Content of basic fraction after stress] + |Content of dominant fraction before stress — Content of dominant fraction after stress] *** There is a marked aggregation upon heating (dramatic improvement in particle size and scattered light intensity > 10,000 kcps).
négative results average data results/baseline
3.5. Conclusions from Example 3.
Based on the results of the présent study, promising additives for adalimumab compositions are trehalose dihydrate, glycine, proline, méthionine, and arginine hydrochloride. Polysorbate 80, Polysorbate 20, Poloxamer 188 were taken as surface-active substances for the final pharmaceutical composition screening.
Sodium chloride, arginine, EDTA, lysine, guanidine hâve an adverse impact on the colloïdal stability of adalimumab.
Sodium chloride, lysine, guanidine hâve an adverse impact on the thermal stability of adalimumab. Cysteine-containing in the formulation leads to complété fragmentation of the antibody. A shift in the protein acid-base profile was noted when EDTA additive was added to the formulation.
Example 4. Final Pharmaceutical Composition Sélection.
Based on the results of the third part of the study, trehalose dihydrate, glycine, proline, méthionine, and arginine hydrochloride were chosen as promising additives. Polysorbate 80, Polysorbate 20, Poloxamer 188 were taken as surface-active substances for the final pharmaceutical composition screening.
Study formulation (mg/mL).
* Buffer solution Protein conc. Trehalose dihydrate L-Proline Glycine Méthionine Polysorbate 80 Polysorbate 20 Poloxamer 188 Arginine hydrochloride
1 15 Humira formulation 1 (see item 1)
2 15 Humira formulation 2 (see item 1)
3 Acetate buffer, pH 5.5 15 80 -
4 15 80 0.1
5 15 80 0.5
6 15 80 1.0
Ί 15 80 0.1
8 15 80 1.0
9 15 80 0.1
10 15 80 0.5
11 15 80 1.0
12 15 80 0.5
13 15 80 0.1 0.5
14 Acetate 15 80 0.5 0.5
* Buffer solution Protein conc. Trehalose dihydrate L-Proline Glycine Méthionine Polysorbate 80 Polysorbate 20 Poloxamer 188 Arginine hydrochloride
15 buffer, pH 5.5 15 80 - 1.0 0.5
16 15 80 0.1 0.5
17 15 80 1.0 0.5
18 15 80 0.1 0.5
19 15 80 0.5 0.5
20 15 80 t 1.0 0.5
21 15 27
22 15 27 0.1 :
23 15 27 0.5
24 15 27 . - 1.0 -
25 15 27 0.1
26 15 27 * 1.0
27 15 27 - 0.1
28 15 27 S 0.5
29 15 27 1.0 -
30 15 8.0 7.5
31 15 - 8.0 7.5 0.1
32 15 8.0 7.5 0.5
33 15 8.0 7.5 1.0
34 15 8.0 7.5 0.1
35 15 8.0 7.5 1.0
36 15 8.0 7.5 0.1
37 15 8.0 7.5 0.5
38 15 8.0 7.5 r 1.0
39 15 27 1.5
40 15 27 1.5 0.1
41 15 27 1.5 0.5
42 15 27 1.5 1.0
43 15 27 1.5 0.1
44 15 27 1.5 1.0
45 15 27 1.5 0.1
46 15 27 1.5 0.5
47 15 27 1.5 1.0
48 15 40 13.5 -
49 15 53.3 9.0 - -
50 15 26.7 18 - r
4.1. Thermal Stability Détermination under Long-Term Exposure using Thermostress 50°C Method.
The analysis was performed according to the procedure 5 .
4.2. Colloïdal Stability Détermination by Shake-Test Technique.
The analysis was performed according to the procedure 6.
4.3. Colloïdal Stability Détermination by Cryoconcentration 5 Technique.
The analysis was performed according to the procedure 7.
Summary is provided in Tables 8 and 9, where the sample quality measures before and after the thermal stress, shake test and cryoconcentration are demonstrated.
4.4. Results.
Table 8. Summary on SE-HPLC and UV-spectrophotometry of the samples before and after stress.
# Additive formulation, mg/mL Change in purity SE-HPLC, %* Turbidity, OD 400 nm
anti TNFa Trehalose dihydrate L-Proline Glycine Methionin e Polysorba te 80 Polysorba te 20 Poloxamer 188 Arginine hydrochlori de Thermal stress, 50°C,120 h Freezing -18°C, thawing t25°C Shake test 120 h Shake test 120 h Freezing -18°C, thawing t25°C
1 15 Humira formulation 1 (see item 1) -1.12 0.02 0.03 0.0410 0.0501
2 15 Humira formulation 2 (see item 1) -0.38 -7.49 0.02 0.0408 0.0503
3 15 80 -0.33: 0.04 0.03 0.0409 0.0521
4 15 80 0.1 -0.34 -0.09 -0.09 0.0446 0.0479
5 15 80 0.5 -0.35 -0.04 -0.02 0.0406 0.0531
6 15 80 1.0 -0.38 -0.04 -0.21 0.0407 0.0489
7 15 80 0.1 -0.39 0.05 0.07 0.0413 0.0474
8 15 80 1.0 -0.44 0.07 -0.01 0.0454 0.0465
9 15 80 0.1 -0.54 0.02 -0.07 0.0465 0.0494
10 15 80 0.5 -0.45 -0.09 -0.18 0.0465 0.0516
11 15 80 1.0 -0.56 -0.04 0.05 0.0412 0.0494
12 15 80 0.1 -0.40 -0.03 0.08 0.0448 0.0446
13 15 80 0.1 0.1 -0.44 0.03 -0.07 0.0479 0.0468
14 15 80 0.5 0.1 -0.46 -0.09 -0.09 0.0445 0.0510
15 15 80 1.0 0.1 -0.49 -0.01 0.07 0.0434 0.0512
16 15 80 0.1 0.1 -0.47 0.05 0.08 0.0445 0.0455
17 15 80 1.0 0.1 -0.33 0.04 0.07 0.0478 0.0444
18 15 80 0.1 0.1 -0.46 0.09 0.05 0.0497 0.0447
19 15 80 0.5 0.1 -0.48 -0.01 0.01 0.0441 0.0518
20 15 80 1.0 0.1 -0.40 -0.01 0.03 0.0434 0.0470
# Additive formulation, mg/mL Change in purity SE-HPLC, %* Turbidity, OD 400 nm
anti TNFa Trehalose dihydrate L-Proline Glycine Methionin e Polysorba te 80 Polysorba te 20 Poloxamer 188 ; Arginine ’hydrochlori de Thermal stress, 50°C,120 h Freezing -18°C, thawing +25°C Shake test 120 h Shake test 120 h Freezing -18°C, thawing +25°C
21 15 27 -0.36 0.00 0.02 0.0400 0.0409
22 15 27 0.1 -0.51 -0.03 -0.06 0.0440 0.0475
23 15 27 0.5 -0.78 -0.03 -0.04 ' 0.0464 0.0745
24 15 27 1.0 -0.39 0.03 0.01 0.0445 0.0443
25 15 27 0.1 -0.46 -0.01 0.08 ' 0.0412 0.0457 j
26 15 27 1.0 -0.48 -0.07 -0.07 0.0434 0.0476
27 15 27 0·. 1 -0.66 -0.09 0.00 0.0445 ” '0.0449
28 15 27 0.5 -0.36 -0.04 0.06 0.0410 / 0.0469
29 15 27 1.0 -0.39 0.02 0.06 0.0409 0.0501
30 15 8.0 7.5 -0.46 -0.02 0.00 0.0402 0.0467
31 15 8.0 7.5 0.1 -0.48 -0.05 -0.01 0.0403 0.0439
32 15 8.0 7.5 0.5 -0.40 -0.04 -0.01 0.0412 0.0496
33 15 8.0 7.5 1.0 -0.20 0.02 0.08 0.0555 0.0474
34 15 8.0 7.5 0.1 -0.46 -0.01 -0.03 0.0585 0.0479
35 15 8.0 7.5 1.0 -0.37 0.01 -0.02 0.0565 0.0443
36 15 8.0 7.5 0.1 -0.49 -0.02 0.04 0.0408 0.0457
37 15 8.0 7.5 0.5 -0.36 -0.01 0.06 0.0478 0.0464
38 15 8.0 7.5 1.0 -0.31 0.00 0.03 0.0432 0.0498
39 15 27 1.5 -0.36 0.01 -0.01 0.0512 0.0463
40 15 27 1.5 0.1 -0.38 0.06 0.05 0.0509 » 0.0503
41 15 27 1.5 0.5 -0.36 -0.01 H 0.08 0.0549 0.0417
42 15 27 1.5 1.0 -0.30 0.01 0.04 0.0520 > - 0.0473 ;
# Additive formulation, mg/mL Change in purity SE-HPLC, %* Turbidity, OD 400 nm
anti TNFa Trehalose dihydrate L-Proline Glycine Methionin e Polysorba te 80 Polysorba te 20 Poloxamer 188 Arginine hydrochlori de Thermal stress, 50°C,120 h Freezing -18°C, thawing +25°C Shake test 120 h Shake test 120 h Freezing -18°C, thawing +25°C
43 15 27 1.5 0.1 -0.37 0.00 -0.08 0.0512 0.0444
44 15 27 1.5 , ; 1.0 , -0.32 -0.03 0.03 0.0513 0.0465
45 15 27 1.5 0.1 -0.33 -0.01 -0.03 0.0510 0.0411
46 15 27 1.5 0.5 -0.36 -0.02 -0.02 0.0550 0.0477
47 15 27 1.5 - 1.0 -0.32 0.01 -0.03 0.0552 0.0498
48 15 40 13.5 -0.50 -0.09 -0.08 0.0494 0.0469
49 15 53.3 9.0 ! ' I -0.55 -0.01 0.03 0.0414 0.0464
50 15 26.7 18 -0.59 -0.02 -0.03 0.0410 0.0466
* Change was calculated by the formula Δ = (Value after stress — Value before stress) positive results négative results average data results/baseline
Table 9. Summary on the acid-base profile of the samples on the LabChip instrument and the homogeneity using the dynamic üght scattering technique before and after stress.
# Additive formulation, mg/mL Homogenicit y DLS, % (by intensity) Homogenicity DLS after stresses, % Total change in acid-base profile, in modulus**, %
anti TNFa Trehalose dihydrate L-Proline Glycine Méthionine Polysorbate 80 Polysorbate. 20 Poloxamer 188 Arginine hydrochlori de Thermal stress, 50°C, 120 h Shake test 120 h Freezing -18°C, thawing +25°C Thermal stress, +50°C 120 h Shake test 120 h Freezing -18°C, thawing +25°C
1 15 Humira formulation 1 (see item 1) 100 99.6 100 100 33.16 1.13 1.84
2 15 Humira formulation 2 (see item 1) 98.8 96.0 100 80.1 29.55 1.03 2.88
3 15 80 100 100 93.3 100 24.56 2.88 2.46
4 15 80 · 0.1 100 100 93.6 100 27.26 1.09 2.82
5 15 80 0.5 100 100 94.8 100 28.27
6 15 80 1.0 100 100 96.9 100 27.64
Ί 15 80 0.1 100 100 97.0 100 24.10
8 15 80 1.0- 100 98.5 98.8 100 26.17
9 15 80 0.1 100 100 98.6 100 26.26 1.44 3.55
10 15 80 0.5 100 100 95.1 100 26.98
11 15 80 1.0 100 97.6 98 . 9 100 26.13
12 15 80 0.1 100 100 99.0 100 24.88 0.88 3.10
13 15 80 0.1 0.1 100 100 100 100 25.58 1.43 1.99
14 15 80 0.5 0.1 100 100 100 100 26.16
15 15 80 1.0 0.1 99.4 100 100 100 24.96
16 15 80 0.1 0.1 100 100 100 100 28.10
17 15 80 1.0 0.1 100 100 100 100 24.74
# Additive formulation, mg/mL Homogenicit y DLS, % (by intensity) Homogenicity DLS after stresses, % Total change in acid-base profile, in modulus**, %
anti TNFa Trehalose dihydrate L-Proline Glycine Méthionine 08 e^pqjosÀTOj Polysorbate 20 Poloxamer 188 Arginine hydrochlori de Thermal stress, 50°C, 120 h Shake test 120 h Freezing -18°C, thawing +25°C Thermal stress, +50°C 120 h Shake test 120 h Freezing -18°C, thawing +25°C
18 15 80 0.1 0.1 100 100 100 100 24.50 2.30 3.68
19 15 80 0.5 0.1 100 100 100 10O . 24.55 .
20 15 80 1.0 0.1 100 100 90.0 100 24.64
21 15 26.5 100 100 100 100 26.79 1.03 1.20
22 15 26.5 0.1 100 ...... 100 100 • 100 24.60 1.85 2.49
23 15 26.5 0.5 100 100 100v 100 21.96
24 15 26.5 1.0 100 100 95.8 100 22.20
25 15 26.5 0.1 100 100 100 100 24.22
26 15 26.5 1.0 100 98.5 100 100 26.07
27 15 26.5 0.1 100 100 100 100 22.38 2.74 4.67
28 15 26.5 0.5 100 100 100 100 21.70
29 15 26.5 1.0 100 100 100 100 21.16
30 15 8.0 7.5 100 100 100 100 24.87 2.90 2.91
31 15 8.0 7.5 0.1 100 100 100 1007- 29.76 2.78 0.18 ·
32 15 8.0 7.5 0.5 100 100 100 100 31.09
33 15 8.0 7.5 1.0 100 100 100 100 29.96
34 15 8.0 7.5 0.1 100 100 100 100 27.30
35 15 8.0 7.5 1.0 100 100 100 100 32.39
36 15 8.0 7.5 0.1 100 100 100 100 31.69 2.49 0.49
37 15 8.0 7.5 0.5 100 100 100 100 29.55
38 15 8.0 7.5 1.0 100 100 100 100 30.00
39 15 26.5 1.5 ïoo 100 100 100 28.52 1.44 1.62
40 15 26.5 1.5 0.1 100 100 100 100 28.41 1.09 0.79
41 15 26.5 1.5 0.5 100 100 100 100 28.16
42 15 26.5 1.5 1.0 100; 100 94.6 :· 100 28.56
# Additive formulation, mg/mL Homogenicit y DLS, % (by intensity) Homogenicity DLS after stresses, % Total change in acid-base profile, in modulus**, %
anti TNFa Trehalose dihydrate L-Proline Glycine Méthionine : Polysorbate 80 Polysorbate 20 Poloxamer 188 ! Arginine hydrochlori de Thermal stress, 50°C, 120 h Shake test 120 h Freezing -18°C, thawing +25°C Thermal stress, +50°C 120 h Shake test 120 h Freezing -18°C, thawing +25°C
43 15 26.5 1.5 0 .1 100 100 ( 92.0 100 33.88
44 15 26.5 1.5 1 < 1.0 100 100 88.4 100 ' 30.46
45 15 26.5 1.5 0.1 , 100 100 95.4 100 26.82 0.33 0.81
46 15 26.5 1.5 0.5 100 100 92.7 100 24.57
47 15 26.5 1.5 1.0 100 100 100 100 28.22
48 15 40 13.5 100 100 90.7 100 26.85 1.74 0.77
49 15 53.3 9.0 100 100 100 100 26.58 1.33 4.37
50 15 26.7 18 100 100 99.9 100 26.73 0.42 5.87
* Change was calculated by the formula Δ = (Value after stress — Value before stress) ** Absolute change was calculated by the formula
Δ = (Content of acid fraction before stress — Content of acid fraction after stress] + (Content of basic fraction before stress — Content ofbasic fraction after stress| + (Content of dominant fraction before stress — Content of dominant fraction after stress| positive results négative results average results / baseline data
4.4. Conclusions from Example 4.
The study showed that adalimumab in the Humira formulation 1 is the most thermolabile among ail the test sample. Thermostress at 50°C for 120 hours leads to the maximum increase in impurities (1.2%) on SE-HPLC, and to the maximum change in the acid-base profile (the total absolute change of ail fractions is more than 33% on the LabChip Caliper instrument) . The colloïdal stability of the sample in the Humira formulation 1 is comparable to the alternative formulations.
The Humira formulation 2 has a thermal stability comparable with one of the alternative formulations (loss of purity during thermal stress 0.38%). However, upon freeze-thawing of adalimumab in the présent formulation, there is a marked aggregation of the protein: the increase in aggregates in the Size Exclusion HPLC is more than 7%, which makes the préparation unusable in case of its accidentai freezing. A marked increase in impurities after freezing was also noted using DLS.
Alternative formulations based on the acetate buffer solution with the addition of a number of additives showed good thermal and colloïdal stability during stresses. Size Exclusion HPLC showed no différence in impurity increase in the présent formulations. Also, there was no significant effect of surfaceactive substances on the stability of adalimumab at the concentration of 15 mg/mL.
Analysis of the stressed samples using DLS showed that the presence of arginine hydrochloride in the formulation reduces the impurity increase during the shake-test, and the presence of méthionine increases the impurity accumulation during shaking.
Minimal change in the acid-base profile was noted for formulations 12-29 (formulations containing trehalose and arginine hydrochloride, and also containing L-proline). Based on the results of the présent study, promising formulations for adalimumab are:
Sodium acetate trihydrate 0.436 mg/mL Glacial acetic acid up to pH Sodium acetate trihydrate 0.436 mg/mL Glacial acetic acid up to pH 5.0-6.0 Trehalose dihydrate 80 mg/mL
5.0-6.0 Trehalose dihydrate 80 mg/mL Polysorbate 20 / Polysorbate 80 /
Poloxamer 188 0.5 - 1.0 mg/mL
Sodium acetate trihydrate mg/mL Glacial acetic acid up 5.0-6.0 Trehalose dihydrate 80 Arginine hydrochloride 0. 0.4 to mg/mL 1 mg/mL 36 pH Sodium acetate trihydrate 0.436 mg/mL Glacial acetic acid up to pH 5.0-6.0 Trehalose dihydrate 80 mg/mL Arginine hydrochloride 0.1 mg/mL Polysorbate 20 / Polysorbate 80 / Poloxamer 188 0.5 - 1.0 mg/mL
Sodium acetate trihydrate mg/mL Glacial acetic acid up 5.0-6.0 L-Proline 27 0.4 to mg/mL 36 pH Sodium acetate Glacial acetic L-Proline Polysorbate 2 Poloxamer 188 trihydrate 0.436 mg/mL acid up to pH 5.0-6.0 27 mg/mL 0 / Polysorbate 80 / 0.5 - 1.0 mg/mL
Example 5. Final High Concentrated Adalimumab Pharmaceutical Composition Confirmation.
To confirm the stability of the recommended pharmaceutical 5 compositions, adalimumab at the concentration of 50 mg/mL, 100 mg/mL, and 150 mg/mL was exposed to a thermal stress at 50°C for 6 days.
Study Formulations.
# Buffer solution Protein conc. Trehalose dihydrate L-Proline Polysorbate 80 Polysorbate 20 Poloxamer 188 Arginine hydrochlorid e
1 Acetate buffer, pH 5.0-6.0 50 Humira formulation 1 (see item 1)
2 Humira formulation 2 (see item 1)
3 80 -- 0.5 0.1
4 80 1.0 0.1
5 80 0.5 0.1
6 80 1.0 0.1
7 80 - 0.5 0.1
8 80 1.0 0.1
9 80 0.5 0.5 0.1
10 27 0.5
11 27 1.0
12 - 27 0.5
13 27 1.0
14 27 0.5
15 27 1.0
16 Λ 27 0.5 0.5
17 100 Humira formulation 1 (see item 1)
18 Humira formulation 2 (see item 1)
19 80 - 0.5 0.1
20 80 1.0 0.1
21 80 0.5 0.1
22 80 1.0 0.1
# Buffer solution Protein conc. Trehalose dihydrate L-Proline 08 Polysorbate 20 Poloxamer 188 Arginine hydrochlorid e
23 80 - 0.5 0.1
24 80 1.0 0.1
25 80 0.5 0.5 0.1
26 27 0.5
27 27 1.0 -
28 27 0.5
29 27 1.0
30 27 0.5
31 27 1.0
32 27 0.5 0.5
Buffer solution Protein conc. Trehalose dihydrate L-Proline Polysorbate 80 Polysorbate 20 Poloxamer 188 Arginine hydrochlorid e
33 Acetate buffer, pH 5.0-6.0 150 Humira formulation 1 (see item 1)
34 Humira formulation 2 (see item 1)
35 80 0.5 0.1
37 80 1.0 0.1
38 80 0.5 0.1
39 80 1.0 • 0.1
40 80 0.5 0.1
41 80 1.0 0.1
42 80 0.5 0.5 0.1
43 27 0.5
44 27 1.0
45 27 0.5
46 27 1.0
47 27 0.5
48 27 1.0
49 27 0.5 0.5
5.1. Thermal Stability Détermination under Long-Term Exposure using Thermostress 50°C Method.
The analysis was performed according to the procedure 5.
5.2. Results.
Table 10. Summary on the concentrated samples with pH 5.0 before and after stresses.
Formulation SE-HPLC IE-HPLC Polyacrylamide gel electrophoresis Spec. activity
Monomer content, Change in purity after thermal stress, 50°C 144 h* Fraction content, % Total change in acid-base profile, modulus** Monomer content, % Change in purity after thermal stress, 50°C, 144 h* Activit y before stress Value after thermal stress, 50°C, 144 h
# Protein conc. Trehalose dihydrate L-Proline 08 s^eqjosÂTOd Polysorbate 20 Poloxamer 188 Arginine hydrochlorid e acid base alka li Red. Nonred. Red. Nonred.
1 50 Humira formulation 1 (see item 1) 99.85 2.67 11.30 86.90 1.80 61.87 98.46 94.64 -1.68 -6.46 96 92
2 Humira formulation 2 (see item 1) 99.85 1.65 11.19 87.16 1.66 61.66 98.73 94.89 -1.46 -3.48 97 95
3 80 0.5 0.1 99.30 1.55 11.48 86.88 1.65 64.49 97.99 94.16 -1.46 -2.69 94 92
4 80 1.0 0.1 99.04 1.62 9.59 88.92 1.49 68.05 -0.99 -2.46 93
5 80 0.5 0.1 99.38 1.79 11.46 86.85 1.69 62.11 -1.41 -3.19 91
6 80 1.0 0.1 99.34 1.72 11.89 86.80 1.31 63.01 -1.37 -3.49 90
7 80 0.5 0.1 99.48 1.54 11.02 87.38 1.60 63.66 -1.31 -3.10 91
8 80 1.0 0.1 99.45 1.62 11.02 87.58 1.40 64.68 -1.67 -3.79 91
9 80 0.5 0.5 0.1 99.35 1.66 11.09 86.97 1.94 63.79 -1.78 -3.91 90
10 27 0.5 99.29 1.76 11.27 87.19 1.55 62.47 98.10 94.78 -1.46 -3.49 96 97
11 27 1.0 99.93 1.81 11.43 87.03 1.54 62.11 -1.78 -2.41 96
12 27 0.5 99.44 1.69 11.41 87.18 1.41 62.46 -1.41 -2.46 94
13 27 1.0 99.65 1.79 11.98 86.53 1.49 62.41 -1.29 -3.01 93
14 27 0.5 99.41 1.72 11.95 86.42 1.63 60.36 -1.29 -2.64 94
15 27 1.0 99.22 1.58 11.40 87.15 1.44 62.77 -1.38 -2.04 95
16 27 0.5 0.5 99.03 1.79 11.46 87.00 1.54 64.66 -1.43 -3.14 93
Formulation SE-HPLC IE-HPLC Polyacrylamide gel electrophoresis Spec. activity
Monomer content, % Change in purity after thermal stress, 50“C 144 h* Fraction content, % Total change in acid-base profile, in modulus** Monomer content, % Change in purity after thermal stress, 50°C, 144 h* Activit y before stress Value after thermal stress, 50°C, 144 h
# Protein conc. Trehalose dihydrate L-Proline Polysorbate ; 80 Polysorbate 20 Poloxamer 188 Arginine hydrochlorid e acid base alka li Red. Nonred. Red. Nonred.
17 100 Humira formulation 1 (see item 1) 99.75 2.99 11.63 86.41 1.96 60.84 97.95 94.10 -1.46 -7.23 98 99
18 Humira formulation 2 (see item 1) 99.84 2.26 11.20 87.10 1.70 61.30 98.26 94.74 -1.68 -3.15 99 75
19 80 0.5 0.1 99.26 2.01 10.63 87.77 1.60 64.94 98.38 94.11 -1.54 -2.86 98 91
20 80 1.0 0.1 99.90 2.48 9.55 89.06 1.40 69.09 -1.82 -4.52 84
21 80 0.5 0.1 99.30 2.06 11.46 87.04 1.50 65.03 -2.06 -4.61 89
22 80 1.0 0.1 99.10 2.09 11.98 86.39 1.63 65.87 -2.21 -4.13 84
23 80 0.5 0.1 99.38 2.01 11.15 87.25 1.61 63.18 -2.27 -4.06 81
24 80 1.0 0.1 99.29 2.36 10.85 87.57 1.58 65.63 -2.57 -4.28 86
25 80 0.5 0.5 0.1 99.41 2.46 11.86 86.98 1.16 63.64 -2.66 -4.49 88
26 27 0.5 99.08 2.21 11.21 87.31 1.48 62.43 98.35 94.09 -3.09 -4.25 97 91
27 27 1.0 99.95 1.87 10.62 87.82 1.57 60.90 -2.68 -3.44 87
28 27 0.5 99.43 2.62 11 .41 86.93 1.66 64.13 -2.64 -3.46 89
29 27 1.0 99.46 2.49 11.32 86.90 1.78 63.89 -1.89 -3.49 88
30 27 0.5 99.28 2.48 10.99 87.31 1.71 62.47 -1.44 0.89 89
31 27 1.0 99.33 1.92 11.52 87.08 1.40 60.28 -0.84 (-2.09 89
32 27 0.5 0.5 99.38 2.31 10.46 87.79 1.75 62.48 -1.98 -3.01 86
Formulation SE-HPLC IE-HPLC Polyacrylamide gel electrophoresis Spec. activity
Monomer content, Change in purity after thermal stress, 50°C 144 h* Fraction content, % Total change in acid-base profile, in modulus * * Monomer content, % Change in purity after thermal stress, 50°C, 144 h* Activit y before stress Value after thermal stress, 50°C, 144 h
# Protein conc. Trehalose dihydrate L-Proline Polysorbate 80 Polysorbate 20 Poloxamer ' 188 Arginine hydrochlorid e acid base alka li Red. Nonred. Red. Nonred.
33 150 Humira formulation 1 (see item 1) 99.88 3.22 11.45 86.73 1.82 59.91 98.29 94.64 -1.92 -6.61 99 98
34 Humira formulation 2 (see item 1) 99.72 2.81 11.19 87.16 1.65 60.64 98.43 94.06 -1.75 -7.52 99 101
35 80 0.5 0.1 99.09 2.40 10.75 87.64 1.61 63.48 98.32 94.85 -1.82 -4 .13 98 113
37 80 1.0 0.1 99.92 2.67 11.28 87.01 1.71 62.90 -2.00 -6.09 98
38 80 0.5 0.1 99.24 2.45 11.28 87.25 1.47 62.30 -2.28 -4.34 99
39 80 1.0 0.1 99.16 2.80 10.73 87.75 1.52 64.32 -2.70 -4.24 97
40 80 0.5 0.1 99.74 2.89 11.39 86.92 1.69 64.11 -2.20 -4 .11 94
41 80 1.0 0.1 99.12 2.80 11.74 86.80 1.46 63.79 -2.67 -4.06 93
42 80 0.5 0.5 0.1 99.20 2.79 11.79 86.52 1.69 64.39 -2.78 -4.08 96
43 27 0.5 99.88 2.48 11.28 87.10 1.63 62.63 98.45 94.14 -3.13 -2.37 97 86
44 ,1 27 1.0 1 99.61 2.64 11.35 87.07 1.58 61.45 -2.92 -2.50 88
45 27 0.5 !l 99.46 2.71 11.49 86.78 1.73 63.44 -2.01 ,-2.03 89
46 27 1.0 99.42 2.83 11.13 86.99 1.88 63.79 -2.03 -1.95 87
47 27 0.5 99.12 2.36 10.91 87.27 1.82 62.17 -1.45 0.52 86
48 27 1.0 99.12 2.52 11.30 87.14 1.57 61.01 -2.08 -1.73 92
49 27 0.5 0.5 99.42 2.80 11.98 86.56 1.46 61.09 -2.35 -1.79 91
* Change was calculated by the formula Δ = (Value after stress — Value before stress) ** Absolute change was calculated by the formula | Content of acid fraction before stress — Content of acid fraction after stress | + |Content of basic fraction before stress - Content of basic fraction after stress| + | Content of dominant fraction before stress - Content of dominant fraction after stress | positive results négative results average data results baseline
Table 11. Summary on the concentrated samples with pH 6.0 before and after stresses.
Formulation SE-HPLC IE-HPLC Polyacrylamide gel electrophoresis Spec. activity
Monomer content, Change in purity after thermal stress, 50°C 144 h* Fraction content, % Total change in acid-base profile, in modulus**, Monomer content, % Change in purity after thermal stress, 50°C, 144 h* Activit y before stress Value after thermal stress, 50°C, 144 h
# Protein 1 conc . Trehalose dihydrate L-Proline Polysorbate 80 Polysorbate 20 Poloxamer 188 Arginine hydrochlorid e acid base alka li Red. Nonred. Red. Nonred.
1 50 Humira formulation 1 (see item 1) 99.85 2.67 11.30 86.90 1.80 61.87 98.46 94.64 -1.68 -6.46 96 92
2 Humira formulation 2 (see item 1) 99.85 1.65 11.19 87.16 1.66 61.66 98.73 94.89 -1.46 -3.48 97 95
50 80 0.5 0.1 99.31 1.66 11.38 86.47 2.15 61.44 97.66 94.06 -1.66 -2.41 94 92
51 80 1.0 0.1 99.41 1.74 11.46 86.99 1.55 62.01 -1.45 -3.08 88
52 80 0.5 0.1 99.64 1.61 11.84 87.01 1.15 64.21 -1.11 -2.49 94
53 80 1.0 0.1 99.41 1.45 11.35 86.86 1.79 64.26 -1.65 -2.77 96
54 80 0.5 0.1 99.29 1.56 11. 65 86.88 1.47 61.87 -1.82 -2.09 89
55 80 1.0 0.1 99.56 1.59 11.98 86.79 1.23 61.94 -1.97 -2.67 91
56 80 0.5 0.5 0.1 99.16 1.67 11.71 87.16 1.13 61.81 -1.19 -2.48 96
57 27 0.5 99.64 1.47 11.22 87.19 1.59 60.20 98.02 94.41 -0.88 -2.37 95 95
58 27 1.0 99.67 1.88 11.64 86.68 1.68 62.11 -0.91 -2.48 94
59 27 0.5 99.73 1.62 11.58 86.63 1.79 63.53 -1.19 -2.61 89
60 27 1.0 99.48 1.40 11.27 87.15 1.58 60.84 -1.41 -2.00 96
61 27 0.5 99.35 1.89 11.34 86.81 1.85 60.84 -1.06 -3.09 91
62 27 1.0 99.47 1.85 11.65 87.09 1.26 61.01 -0.94 -2.14 90
63 27 0.5 0.5 99.58 1.74 11.82 87.14 1.04 60.06 -1.23 -2.90 93
Formulation SE-HPLC IE-HPLC Polyacrylamide gel electrophoresis Spec. activity
Monomer content, Change in purity after thermal stress, 50°C 144 h* Fraction content, % Total change in acid-base profile, in modulus * *, Monomer content, % Change in purity after thermal stress, 50°C, 144 h* Activit y before stress Value after thermal stress, 50°C, 144 h
# Protein conc. Trehalose dihydrate L-Proline Polysorbate 80 Polysorbate 20 Poloxamer 188 Arginine hydrochlorid e acid base alka li Red. Nonred. Red. Nonred.
17 100 Humira formulation 1 (see item 1) 99.75 2.99 11.63 86.41 1.96 60.84 97.95 94.10 -1.46 -7.23 98 99
18 Humira formulation 2 (see item 1) 99.84 2.26 11.20 87.10 1.70 61.30 98.26 94.74 -1.68 -3.15 99 75
64 80 0.5 0.1 99.31 2.34 11.55 86.65 1.80 69.84 98.12 94.66 -1.35 -2.06 98 94
65 80 1.0 0.1 99.26 2.41 11.64 86.84 1.52 66.44 -1.82 -3.49 96
66 80 0.5 0.1 99.64 2.43 11.20 86.69 2.11 62.59 -2.31 -4.11 89
67 80 1.0 0.1 99.53 .....'1.89 11.68 86.72 1.60 61.49 -2.48 -3.69 90
68 80 0.5 0.1 99.16 1.84 11.54 86.69 1.77 61.48 -2.64 -3.17 91
69 80 1.0 0.1 99.60 1.91 11.20 86.51 2.29 62.03 -2.41 -3.76 92
70 80 0.5 0.5 0.1 99.22 2.06 11.28 87.12 1.60 62.07 -2.37 -3.19 93
71 27 0.5 99.18 2.24 11.62 87.30 1.08 61.34 98.32 94.21 -3.11 -2.22 102 94
72 27 1.0 99.67 2.16 11.59 86.88 1.53 61.55 -2.46 -3.49 96
73 27 0.5 99.48 2.19 11.41 86.84 1.75 60.48 -2.80 -3.85 88
74 27 1.0 99.49 1.99 11.60 86.70 1.70 60.68 -1.42 -3.41 89
75 27 0.5 99.56 2.09 11.26 86.90 1.84 61.89 -0.84 -2.18 90
76 27 1.0 99.62 2.22 11.41 86.66 1.93 60.76 -0.98 -2.14 93
77 27 0.5 0.5 99.44 2.37 11.73 86.58 1.69 63.18 -1.49 -3.11 98
Formulation SE-HPLC IE-HPLC Polyacrylamide gel electrophoresis Spec. activity
Monomer content, Change in purity after thermal stress, 50°C 144 h* Fraction content, % Total change in acid-base profile, modulus**, Monomer content, % Change in purity after thermal stress, 50°C, 144 h* Activât y before stress Value after thermal stress, 50°C, 144 h
# Protein conc. Trehalose dihydrate L-Proline Polysorbate 80 Polysorbate 20 Poloxamer 188 ! Arginine hydrochloric! e acid base alka li Red. Nonred. Red. Nonred.
33 150 Humira formulation 1 (see item 1) 99.88 3.22 11.45 86.73 1.82 59.91 98.29 94.64 -1.92 -6.61 99 98
34 Humira formulation 2 (see item 1) 99.72 2.81 11.19 87.16 1.65 60.64 98.43 94.06 -1.75 -7.52 99 101
78 80 0.5 0.1 99.18 2.87 11.44 86.88 1.68 59.22 98.13 94.54 -0.82 -4.41 99 94
79 80 1.0 ' l 0.1 99.25 2.66 11.52 86.57 1.91 ' 60.16 -2.08 -4.09 92
80 80 0.5 0.1 99.35 2.28 11.68 86.69 1.63 63.11 -2.14 -4 .44 92
81 80 1.0 0.1 99.41 1.87 11.97 86.81 1.22 62.59 -2.62 -4 . 14 93
82 80 0.5 0.1 99.64 2.44 11.44 86.76 1.80 63.00 -2.33 -4.16 93
83 80 1.0 0.1 99.12 2.57 11.50 86.58 1.92 62.41 -2.57 -4.90 94
84 80 0.5 0.5 0.1 99.16 2.14 11.64 86.59 1.77 63.49 -2.60 -4.16 91
85 27 0.5 99.26 5.49 11.63 86.74 1.63 61.18 98.04 94.10 -2.13 -3.37 97 94
86 27 1.0 99.61 1.88 11.80 86.59 1.61 60.66 -1.87 -2.11 96
87 27 0.5 99.34 2.91 11.68 86.44 1.88 60.49 -2.06 -2.18 92
88 27 1.0 99.55 2.60 11.91 86.58 1.51 61.27 -2.74 -2.95 88
89 27 0.5 99.16 2.58 11.11 86.93 1.96 61.08 -1.73 -1.99 89
90 27 1.0 99.20 2.78 11.54 86.97 1.49 62.54 -2.13 -2.60 90
91 27 0.5 0.5 99.44 2.43 11.67 86.77 1.56 60.20 -2.01 -2.06 93
* Change was calculated by the formula Δ = (Value after stress — Value before stress) ** Absolute change was calculated by the formula
Δ = | Content of acid fraction before stress — Content of acid fraction after stress| + | Content of basic fraction before stress — Content of basic fraction after stress| + | Content of dominant fraction before stress — Content of dominant fraction after stress | positive results négative results average results / baseline data
General conclusions
1. Screening for the stable adalimumab pharmaceutical composition was carried out in several stages: sélection of the buffer solution nature, sélection of pH and buffer capacity of the solution, sélection of the osmolyte and the stabilizing agent, sélection of the final pharmaceutical composition and confirmation of the final high-concentrated adalimumab pharmaceutical composition.
2. During the investigation, the adalimumab thermal and colloïdal stability in more than 90 formulations were studied using the following methods: PEG aggregation, shake-test, freeze-thawing, thermal stress with subséquent analysis of the degree of turbidity (UV-spectrophotometry), purity (exclusion HPLC, dynamic light scattering and gel electrophoresis, including using the LabChip, Caliper System), and acid-base profile (LabChip, Caliper and ion-exchange HPLC).
3. The study showed a low thermal stability of the original Humira préparation formulation (Humira 1), used in the commercial préparation with adalimumab concentration of 50 mg/mL, compared to the formulations of the présent invention.
4. The experiment showed the significant impact of the freezing and thawing processes on adalimumab aggregation in the original Humira préparation formulation (Humira 2), used in the commercial préparation of adalimumab 100 mg/mL, compared to the formulations of the présent invention.
5. Adalimumab pharmaceutical compositions based on acetate buffer solution having pH 5.0 to 6.0 supplemented with trehalose dihydrate, arginine hydrochloride, proline, and surface-active substances from the group of Polysorbate 20, Polysorbate 80 and Poloxamer 188 exhibit a greater thermal and colloid stability at concentrations of 50-150 mg/mL compared to the original formulations and are the subject matters of the présent invention.
The optimal additive formulation of adalimumab was selected for solutions having pH 5.5. The stability of adalimumab in the formulations with a pH range of 5.0 to 6.0 was confirmée! on the samples at protein concentrations of 50, 100 and 150 mg/mL.
6. The préparation process of the pharmaceutical compositions of the présent invention with the adalimumab 5 concentration of 150 mg/mL enables concentrâting up to 200 mg/mL without loss of protein quality for subséquent dilution upon adding SAS and washing the ultrafiltration device after concentrating.

Claims (27)

1 An aqueous pharmaceutical composition for intravenous or
5 subcutaneous administration comprising:
a) adalimumab in concentration from 50 to 200 mg/ml;
b) an acetate ion-based buffer agent (or buffer system);
c) trehalose and/or proline, or trehalose and arginine;
d) polysorbate 20, Polysorbate 80 or Poloxamer 188, or a 10 combination thereof.
2 The composition of claim 1 wherein the acetate buffer solution concentration is from 1 to 100 mM.
15
3 The composition of claim 1 wherein pH of the composition is from 4 to 7 .
4 The composition of claim 1 wherein the trehalose concentration is from 25 to 150 mg/ml.
5 The composition of claim 1 wherein the proline concentration is from 5 to 40 mg/mL.
6 The composition of claim 1 wherein the trehalose
25 concentration is from 25 to 150 mg/mL, and the arginine concentration is from 0.05 to 0.5 mg/mL.
7 The composition of claim concentration is from 0.05 mg/mL
8 The composition of claim concentration is from 0.05 mg/mL
1 wherein the polysorbate 20 to 10 mg/mL.
1 wherein the Polysorbate 80 to 10 mg/mL.
9 The composition of claim 1 wherein the Poloxamer 188
35 concentration is from 0.05 mg/mL to 10 mg/mL.
10 The composition of claim 1 comprising:
a) from 50 to 200 mg/ml adalimumab;
b) from 0.4 to 1.2 mg/mL of sodium acetate trihydrate;
c) from 25 to 150 mg/ml of trehalose and/or from 5 to 40 mg/mL of proline, or from 25 to 150 mg/mL of trehalose and from 0.05 to 0.5 mg/mL of arginine;
d) glacial acetic acid until pH 5.0 to 6.0;
e) from 0.1 mg/mL to 1 mg/mL of polysorbate 80.
11 The composition of claim 10 comprising:
a) from 50 to 150 mg/ml adalimumab;
b) 0.436 mg/ml of sodium acetate trihydrate;
c) 80 mg/mL of trehalose dihydrate;
d) glacial acetic acid until pH 5.5;
e) 0.5 mg/mL of Polysorbate 80.
12 The composition of claim 10 comprising:
a) from 50 to 150 mg/mL of adalimumab;
b) 0.436 mg/mL of sodium acetate trihydrate;
c) 80 mg/mL of trehalose dihydrate and 0.1 mg/mL of arginine hydrochloride;
d) glacial acetic acid until pH 5.5;
e) 0.5 mg/mL of polysorbate 80.
13 The composition of claim 10 comprising:
a) from 50 to 150 mg/mL of adalimumab;
b) 0.436 mg/mL of sodium acetate trihydrate;
c) 27 mg/mL of proline;
d) glacial acetic acid until pH 5.5;
e) 0.5 mg/mL of Polysorbate 80.
14 The composition of claim 10 comprising:
a) from 50 to 150 mg/mL of adalimumab;
b) 0.436 mg/mL of sodium acetate trihydrate;
c) 40 mg/mL of trehalose dihydrate;
d) 13.5 mg/mL of proline;
e) glacial acetic acid until pH 5.5;
f) 0.5 mg/mL of Polysorbate 80.
15 The composition of claim 1 comprising:
a) from 50 to 200 mg/mL adalimumab;
b) from 0.4 to 1.2 mg/mL of sodium acetate trihydrate;
c) from 25 to 150 mg/mL of trehalose and/or from 5 to 40 mg/mL of proline, or from 25 to 150 mg/mL of trehalose and from 0.05 to 0.5 mg/mL of arginine;
d) glacial acetic acid until pH 5.0 to 6.0;
e) from 0.1 mg/mL to 1 mg/mL of polysorbate 20.
16 The composition of claim 15 comprising:
a) from 50 to 150 mg/mL of adalimumab;
b) 0.436 mg/mL of sodium acetate trihydrate;
c) 80 mg/mL of trehalose dihydrate;
d) glacial acetic acid until pH 5.5;
e) 0.5 mg/mL of polysorbate 20.
17 The composition of claim 15 comprising:
a) from 50 to 150 mg/mL of adalimumab;
b) 0.436 mg/mL of sodium acetate trihydrate;
c) 80 mg/mL of trehalose dihydrate and 0.1 mg/mL of arginine hydrochloride;
d) glacial acetic acid until pH 5.5;
e) 0.5 mg/mL of polysorbate 20.
18 The composition of claim 15 comprising:
a) from 50 to 150 mg/mL of adalimumab;
b) 0.436 mg/mL of sodium acetate trihydrate;
c) 27 mg/mL of proline;
d) glacial acetic acid until pH 5.5;
e) 0.5 mg/mL of polysorbate 20.
19 The composition of claim 15 comprising:
a) from 50 to 150 mg/mL of adalimumab;
b) 0.436 mg/mL of sodium acetate trihydrate;
c) 40 mg/mL of trehalose dihydrate;
d) 13.5 mg/mL of proline;
e) glacial acetic acid until pH 5.5;
f) 0.5 mg/mL of polysorbate 20.
20 The composition of claim 1 comprising:
a) from 50 to 200 mg/mL of adalimumab;
b) from 0.4 to 1.2 mg/mL of sodium acetate trihydrate;
c) from 25 to 150 mg/mL of trehalose and/or 5 to 40 mg/mL of proline, or from 25 to 150 mg/mL of trehalose and from 0.05 to 0.5 mg/mL of arginine;
d) glacial acetic acid until pH 5.0 to 6.0;
e) from 0.1 mg/mL to 1 mg/mL of Poloxamer 188.
21 The composition of claim 20 comprising:
a) from 50 to 150 mg/mL of adalimumab;
b) 0.436 mg/mL of sodium acetate trihydrate;
c) 80 mg/mL of trehalose dihydrate;
d) glacial acetic acid until pH 5.5;
e) 1.0 mg/mL of Poloxamer 188.
22 The composition of claim 20 comprising:
a) from 50 to 150 mg/mL of adalimumab;
b) 0.436 mg/mL of sodium acetate trihydrate;
c) 80 mg/mL of trehalose dihydrate and 0.1 mg/mL of arginine hydrochloride;
d) glacial acetic acid until pH 5.5;
e) 1.0 mg/mL of Poloxamer 188.
23 The composition of claim 20 comprising:
a) from 50 to 150 mg/mL of adalimumab;
b) 0.436 mg/mL of sodium acetate trihydrate;
c) 27 mg/mL of proline;
d) glacial acetic acid until pH 5.5;
e) 1.0 mg/mL of Poloxamer 188.
24 The composition of claim 20 comprising:
a) from 50 to 150 mg/mL of adalimumab;
b) 0.436 mg/mL of sodium acetate trihydrate;
c) 40 mg/mL of trehalose dihydrate
d) 13,5 mg/mL of proline;
e) glacial acetic acid until pH 5.5;
f) 1.0 mg/mL of Poloxamer 188.
25 A method for treating TNFa mediated diseases comprising administering an effective amount of the composition of daims 1-24, wherein the TNFa mediated disease is selected from the group of:
a) active (moderate to severe) rheumatoid arthritis,
b) active psoriatic arthritis,
c) active ankylosing spondylitis,
d) chronic (moderate to severe) plaque psoriasis,
e) (moderate to severe) ulcerative colitis,
f) axial spondylarthritis,
g) active hidradenitis suppurativa,
h) juvénile idiopathic arthritis,
i) (moderate or severe) Crohn's disease,
j) uveitis,
k) active enthesitis-associated arthritis.
26 Use of the composition of claims 1-24 for treating TNFa mediated diseases, wherein the disease is selected from the group of:
a) active (moderate to severe) rheumatoid arthritis,
b) active psoriatic arthritis,
c) active ankylosing spondylitis,
d) chronic (moderate to severe) plaque psoriasis,
e) (moderate to severe) ulcerative colitis,
f) axial spondylarthritis,
g) active hidradenitis suppurativa,
h) juvénile idiopathic arthritis,
i) (moderate or severe) Crohn's disease,
j) uveitis,
k) active enthesitis-associated arthritis.
27 A method for producing the composition of any one of claims 1-24 comprising the addition of acetate buffering agents to an aqueous phase followed by the addition, in any order, of the following components:
a) an osmolyte and/or a stabilizing agent selected from the group of trehalose, proline or a combination thereof;
b) a recombinant monoclonal anti-TNFa antibody;
c) a surface-active substance selected from the group of Polysorbate 20, Polysorbate 80, Poloxamer 188, or a combination thereof.
OA1201900260 2016-12-30 2017-12-29 Aqueous pharmaceutical composition of a recombinant monoclonal antibody to FNOα. OA19427A (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
RU2016152691 2016-12-30
RU2017146821 2017-12-29

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Publication Number Publication Date
OA19427A true OA19427A (en) 2020-09-18

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