OA17126A - Pharmaceutical formulations of TNF-alpha antibodies - Google Patents

Abstract

The present invention provides certain improved formulations of proteins. Specifically, the present invention provides use of certain excipients that are useful for stabilization of antibody preparations. Additionally, the novel formulation of the present invention prevents the formation of aggregates or fragments or modification of protein in solution.

Description

PHARMACEUTICAL FORMULATIONS OF TNF-ALPIIA ANTIBODIES

Field of the Invention

The présent invention relates to certain improved formulations of proteins. Specifically, the présent invention provides use of certain excipients that arc useful for stabilization of antibody préparations. Additionally, the novel formulation of the présent invention prevents the formation of aggregates or fragments or modification of protein in solution during storage.

Background of the Invention

Proteins are large and complex molécules. They are required to be in their native confirmation in order to remain biologically active and not be immunogenic. Proteins having a high pi value or distinct polarity may not show stabilïty in solution at or around physiologîcal pH conditions. Further, at high concentration, protein molécules in solution are susceptible to undergo aggregation or dégradation or certain modifications with time during storage. Most common protein dégradation pathways known from literature are protein aggregation, deamidation and oxidation [Cleland et al. Critical Reviews in Therapeutic Drug Carrier Systems 10(4): 307-377 (1993)]. Dégradation of protein during storage may take place due to chemical instability (i.e. any process which involves modification of the protein by bond formation or cleavage resulting in a new çhemical entîty) or physical instability (i.e. changes in the higher order structure of the protein). Chemical instability majorly can be resuit of deamidation, racemization, hydrolysis, oxidation, beta élimination or disulfide exchange. Physical instability can resuit from dénaturation, aggregation, précipitation or adsorption. ln an aspect, the présent invention discloses suitable formulation of antibody proteins. Antibodies are highly complex molécules and are the fastest growing class of biologïcs in the pharmaceutical industry due to. their therapeutic eftectiveness in humans. However, antibodies are subject to undergo aggregation or dégradation or dénaturation or chemical modifications resulting in the loss of biological activity during the manufacturing process and / or during storage with time. Such protein modifications can also make them immunogenic resulting in· the génération of anti-drug antibodies by the patient which can reduce the drug availability during subséquent injections or worse induce an autoimmune reaction. Therefore, there is a need to hâve stable new formulations for antibody préparations which protect the molécules from aggregation or dégradation or chemical modification during the manufacturing process and / or storage in liquid solution even at high protein concentration while preserving the active biological conformation of the antibody molécules.

Liquid pharmaceutical formulation is a primary choice for manufacturera to préparé a 10 stable, safe and effective pharmaceutical préparation of antibody for therapy. Liquid pharmaceutical préparation is also considered to be easy-to-handle for the patients and by the patients. However, a long appreciated problem with liquid formulations of protein therapeutics is that of aggregation, where protein molécules physically stick together, for exampte, resulting in the formation of either soluble high molecular weight protein aggregates or 15 insoluble protein aggregates, which may cause undesired immunological reactions in patients upon administration. Additionally, a major problem caused by the aggregate formation is that during the administration the formulation may block syringes or pumps rendering it unsafe to patients. Aggregation of protein can also significantly impact its potency, immunogénieity and stability. Another reason of dégradation is that unfolding mediated adsorption at interfaces can 20 often be an initiating step for irréversible aggregation in solution. In this respect, proteins tend to adsorb at liquid-solid, liquid-air, and liquid-liquid interfaces. Sufficient exposure of a protein's core at a hydrophobie surface can resuit in adsorption as a conséquence of agitation, température or pH induced stresses. Further, proteins also are sensitive to, for example, pH, ionic strength, thermal stress, shear and interfacial stresses, ail of which can lead to 25 aggregation and resuit in instabîlity. Another conséquence of aggregation is particle formation, an important considération in liquid and tyophîlized protein pharmaceuticals.

WO 2004/016286 discloses a liquid formulation for stabilizing antibodies which treat TNF a mediated diseases, comprising the antibody, a buffer system, mannitol, polysorbates and tonicity agents. The formulation uses a citrate-phosphate huiler system. This formulation 30 could be thawed / frozen at least 3 times without any detrimental effect on either the chemical and physicochemicat properties or biological activity.

In order to realize the clinical potency of an antibody protein, there is a need for new and improved formulations comprising the antibody molecule(s) in its native conformation, which can be stored under a desired / suitable condition for long-term storage without formation of significant amount of aggregates or fragments or modified variants of the antibody protein, even at high protein concentration. The present invention addresses the 10 above-identified need by providing novel stable formulations comprising an antibody molécule, preferably a monoclonal antibody, along with suitable excipients which makes the formulation stable and having sufficiently low viscosity at around physiological osmolality and which is therefore suitable for administration to mammals, particulariy human subjects.

We herein disclose some such formulations which prevent the formation of aggregates 15 during and after formulation while providing a suitable condition for long term storage.

Embodiments of the Invention

The invention provides improved liquid formulation comprising therapeutic amount of proteins preferably antibodies and suitable excipients.

In certain embodiments the present invention provides a liquid formulation suitable for 20 human use which comprises a therapeutic amount of monoclonal antibody or antigen binding portion thereof, which binds to tumor necrosis factor (TNF) and suitable excipients optionally selected from suitable buffers, stabilizer(s), surfactant(s) and suitable additives for maintaining osmolality, optionally with other excipients.

In an embodiment the present invention provides a liquid formulation which comprises 25 of monoclonal antibodies or antigen binding portion thereof along with suitable buffer(s) and other excipients optionally selected from one or more stabilizers, surfactants and tonicity agents such as sodium chloride or potassium chloride. In an embodiment, such formulations can also optionally be lyophilized. Lyophilization can be performed by a skilled person using the techniques available in the art which includes various steps like freezing, annealing, 30 primary drying and secondary drying.

In yet another embodiment the présent invention provides a liquid formulation also suitable for lyophilization which comprises from about I mg / mL to about 160 mg / mL of monoclonal antibody or antigen binding portion thereof and suitable buiTers at a concentration of about 5 mM to 100 mM, optionally suitable stabliizers with a concentration of about 1% to 10%, optionally suitable surfactants at a concentration of about 0.001% to 1% and optionally 10 suitable tonicity agents at a concentration of about 10 mM to about 150 mM.

In an embodiment, the présent invention provides a liquid formulation buflered between pH 4 to 8.

In another embodiment, the présent invention provides a liquid formulation which can be used for parentéral administration. Parentéral administration includes intravenous, 15 subcutaneous, intra peritoneal, intramuscular administration or any other route of delivery generally considered to be falling under the scope of parentéral administration and as is well known to a skilled person.

In another embodiment, the présent invention provides a liquid formulation which improves stability and prevents formation of aggregates of protein in the said formulation.

Generally, a stable formulation is the one which retains its physical stability and/or chemical stability and/or biological activity over a period of time upon storage.

In a further embodiment, the présent invention provides a liquid formulation which can be used for administering to a patient suffering from a disorder in which TNFa activity is detrimental. Such disorders include sepsîs, infections, autoimmune diseases, transplant 25 rejection, malignancy, pulmonary disorders, cardiac disorders, intestinal disorders, graftversus-host disease and the like. Such disorders occur due to TNFa activity and are well described in literature such as in US patent No. 6,09,382.

Description of figures

Fig 1 shows the level of aggregates of Adalïmumab protein formulated in citrate-phosphate buffer with different stabilïzerfs), bulking agent and an isotonicîty agent during storage under stressed condition. Formulation compositions are described with Examples 1 to 6.

Fig 2 shows the level of principal charged variant of Adalimumab protein formulated in citrate-phosphate bufîer with different stabilizer(s), bulking agent and an isotonicity agent during storage under storage condition. Formulation compositions are described with Examples l to 6.

Fig 3 shows the level of aggregates of Adalimumab protein formulated in sodium succinate bufîer with different stabilizer(s), bulking agent and an isotonicity agent during storage under stressed condition. Formulation compositions are described with Examples 7 to 13.

Fig 4 shows the level of principal charged variant of Adalimumab protein formulated in sodium succinate bufîer with different stabilizer(s), bulking agent and an isotonicity agent during storage under stressed condition. Formulation compositions are described with Examples 7 to 13.

Fig 5 shows the level of aggregates of Adalimumab protein formulated in sodium succinate bufîer with different stabilizer(s), bulking agent and an isotonicity agent during storage under stressed condition. Formulation compositions are described with Examples 14 to 16.

Fig 6 shows the level of principal charged variant of Adalimumab protein formulated in sodium succinate bufîer with different stabilizer(s), bulking agent and an isotonicity agent during storage under stressed condition. Formulation compositions are described with Examples I4to 16.

Detailed description of the Invention

The présent invention provides novel and improved liquid formulations which can optionally be lyophilized, comprising of suitable amount of therapeutic protein(s), preferably monoclonal antibodies, in suitable bufîer(s), one or more suitable stabilizers, and other excipients which are optionally selected from suitable surfactants and tonicity agents. The said formulation prevents formation of aggregates of protein (antibody) and maintains the potency and stability of the therapeutic compound for up to the desired period of time.

In such embodiment the protein is an antibody or antigen binding portion thereof. In a preferred embodiment the antibody is selected from suitable polyclonal, monoclonal, recombinant antibodies, single chain antibodies, hybrid antibodies, chimeric antibodies, humanized antibodies, or fragments thereof, isolated human antibodies or antibody portions thereof or antibody-like molécules containing one or two binding sites for an antigen and an Fc-part of an immunoglobulin. Adaîimumab and Infliximab are exampîes of antibodies or more suitably, monoclonal antibodies. Infliximab is example of chimeric antibody. Adaîimumab is example of human antibody. Etanercept is an exampie of antibody like molécules. In a preferred embodiment, antibodies used in the formulation are human antibodies. In a more preferred embodiment, the antibodies used in the formulation are human antibodies directed to TNFa including human TNFa. In a still further embodiment, the formulation includes D2E7 and combination of D2E7 with other antibodies. D2E7 antibody which is known by the generic name Adaîimumab, with its high affînity binding to hTNFa with low dissociation kinetics and high neutralïzing capacity is described in US patent Nos. 6,090,382 and US 6,258,562, as well as several publications such as Ann Rhettm Dis l999;58:(Suppl I) I70-I72, Ann Rhettm Dis 2000; 59(suppl. I):i44—i45 etc. ail of which are incorporated herein in their entirety. Adaîimumab is available in the market with the brand name HUMIRA®. The names Adaîimumab and D2E7 whenever used in the spécification represent the same human monoclonal antibody as described in the above référencés.

In a preferred embodiment, the monoclonal antibody is Adaîimumab or antigen binding portion, thereof.

In some embodiments the monoclonal antibodies or antigen binding portion thereof is generally présent in a therapeutic amount of up to I60 mg / mL. In a preferred embodiment the therapeutic amount is about l mg / mL to about I00 mg / mL. In a more preferred embodiment the therapeutic amount is about I mg / mL to about 50 mg / mL.

The liquid formulation comprises a suitable buffer along with other pharmaceutically acceptable excipients, which stabilizes the pharmaceutical préparation. Suitable buiîers which can be used are selected from those which are known in the art and can be found in the literature. In an embodiment the suitable buffers comprise but are not limited to histidinebuffers, citrate-buffers, succinate-buffers, acetate-buffers, phosphate-buffers, phosphate buffered saline, citrate and phosphate buffer, tromethamine buffers and the like or their suitable mixtures.

In a preferred embodiment the suitable buffer comprises of a succinate-buffer or acetate-buffer or histidine buffer. In a still preferred embodiment the suitable buffer comprises a succinate-buffer or an acetate-buffer. In a further preferred embodiment the suitable buffer comprises a succinate-buffer. Succinate buffer can be prepared by dissolving sodium succinate in stérile water or Water for Injection (WFI) or by titrating succinic acid with sodium hydroxi de.

The buffers are generally used in concentrations of about ImM to about 100 mM. In a preferred embodiment the buffer concentration is about 5 mM to about 50 mM. In a more preferred embodiment the buffer concentration is about 10 mM to about 20 mM. In a still more preferred embodiment the buffer concentration is about 10 mM.

In an embodiment the liquid formulation maintains a pH value ranging from 4.0 to about 8.0 dependîng on the monoclonal antibody being used. In a preferred embodiment the buffer used maintains the pH of the formulation in the range of about 5.0 to 5.5. In a more preferred embodiment the pH is maintained to about 5.2.

The liquid formulation further comprises suitable surfactants which are pharmaceutically acceptable excipients used to protect the protein formulations against various stress conditions, like agitation, shearing, exposure to high température etc. The suitable surfactants inciude but are not limited to polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (e.g. Brij), alkylphenylpolyoxycthylene ethers (e.g. Triton-X), polyoxyethylene-polyoxypropylene copolymer (e.g. Poloxamer, Pluronîc), sodium dodecyl sulphate (SDS) and the like. In a preferred embodiment the suitable surfactant is polyoxyethylenesorbitan-fatty acid esters (Tweens). In a more preferred embodiment the polyoxyethylenesorbitan-fatty acid esters are polysorbate 20, (sold under the trademark Tween 20™) and polysorbate 80 (sold under the trademark Tween 80™), In another preferred embodiment the suitable surfactant is polyethylene-polypropylene copolymers which are sold under the names Pluronîc (R) F68 or Poloxamer 188™. In another preferred embodiment the suitable surfactant is alkylphenolpolyoxyethylene esters which are sold under the trade name Triton-X.

In a preferred embodiment the surfactant is Polysorbate 80 or Polysorbate 20. In a more preferred embodiment the surfactant is Polysorbate 80.

The surfactants are generally used in concentrations of about 0.001 % to about 1 %. In a preferred embodiment surfactant concentration is about 0.01 % to about 1%.

The liquid formulation further comprises one or more suitable stabilizer(s) which are pharmaceutically acceptable excipients, which protect the active pharmaceutical ingrédient from chemical and/or physical dégradation during manufacturing, storage and application. In an embodiment the stabilizers include but are not limited to suitable sugars, amino acids, polyols, cyclodextrines and the like or suitable dérivative or mixtures thereof.

In one such embodiment the sugar is a monosaccharide or an oligosaccharide. Monosaccharide sugars include but are not limited to glucose, fructose, galactose, mannose, sorbose, ribose, deoxyribose and the like or amino sugars, like neuraminic acid. An oligosaccharide includes but is not limited to sucrose, trehalose, lactose, maltose and raffinose and the like or suitable mixtures, thereof.

In another embodiment the polyols which can be used as stabilizers include but are not limited to mannitol, sorbitol, dextran, glycerol, arabîtol, propylenc glycol, polyethylene glycol and the like or suitable combinations thereof. In a preferred embodiment the suitable polyol is sorbitol.

In another preferred embodiment the stabilîzer is a polyol preferably sorbitol. In an embodiment the sorbitol is présent in amount about 1% to about 10%.

In another such embodiment the amino acids which can be used as stabilizers include but are not limited to arginine, glycine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanïne, tyrosine, tryptophane, méthionine, serine, proline, cysteine / cystine and the like or suitable combination of any the above. In a preferred embodiment the suitable amino acid is arginine or lysine.

In an embodiment the amino acid is présent in amount about 0.5% to about 10%.

In another embodiment cyclodextrines or dérivative thereof, which can be used as stabilizers, includes but are not limited to a-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, or their hydroxypropylated, hydroxyethylated, ethylated or methylated dérivatives thereof or Sulfobutyl ether beta-cyclodextrin (SBE-beta-CD) or a branched cyclodextrins or cyclodextrin polymers or suitable mixture thereof. In a preferred embodiment the suitable cyclodextrin variant is hydroxypropylated cyclo beta-dextrin (ΗΡ-β-CD).

In a preferred embodiment the cyclodextrin or dérivative is présent in amount about 0.2% to about 10%.

In another embodiment the liquid formulation optionally comprises tonicity agents such as sodium chloride or potassium chloride. In a preferred embodiment, the tonicity agent is sodium chloride which is présent in amount about 10 mM to about 150 mM.

The formulation may additionally further comprise one or suitable other excipients which are well known to a person skilled in the art.

In some embodiments, the liquid formulation maintains the storage stability in terms of not allowing any further protein aggregation or modifications as compared to time point zéro of stability.

In some embodiments, the liquid formulation maintains the stability during the process of formulation.

To estimate the level of aggregates and the principal charged variant of Adalimumab protein, analytical HP-size exclusion chromatography and HP-ion exchange chromatography were performed, respectively.

The said analytical methods used in the présent invention are well known to a skilled person and a brief description of the same is provided below merely for the sake of reference only.

ΗΡ-Size exclusion chromatography (HP-SEC):

Samples were analyzed to estimate the aggregates by HP-size exclusion chromatography (HP-SEC) using TSK. gel G3000 SWXL column (7.8 mm I.D ^χ 30 cm L). Samples were loaded and eluted isocratically using sodium phosphate buffer at a flow rate of 0.5 mL ! min. Elution was monitored at UV 214 nm.

HP-Ion Exchange Chromatography (HP-1EC):

Samples were analyzed to estimate the principal charged variant by HP-1EC using analytical cation exchange column. Samples were loaded and eluted using sait gradient at a flow rate of 1.0 mL ! min. Elution was monitored at UV 280 nm.

The présent invention is illustrated further in the following examples which are provided for illustration purpose and should not be construed as being a limitation to the scope of the invention.

Examples

The following non-limiting examples describe the different formulations which can be prepared as per the présent invention. It will be appreciated that other excipients may be added as are necessary to these formulations and such addition of excipients are within the scope of a person skilled in the art and are to be included within the scope of the présent invention.

Screening of excipients is carried out by formulating the Adalimumab protein in different compositions with different excipients and exposing them to higher température over the period of time. Formulations of Adalimumab protein were prepared in the presence of different buffering agent(s), stabilizer(s), bulking agent(s) and isotonicity agent(s) as exemplified below.

Example 1

Formulation composition
Active Ingrédient
Adalimumab	50 mg / mL
Inactive Ingrédients
Dibasic sodium phosphate dihydrate	1.53 mg / mL
Monobasic sodium phosphate dihydrate	0.86 mg/mL
Sodium citrate	0.3 mg / mL
Citric acid	1.3 mg/mL
Mannitol	12 mg/mL
Sodium Chloride	6.16 mg/mL
Polysorbate 80	1 mg / mL

Adalimumab protein was purified as per the technique known in the art. In this exampie, the purified Adalimumab protein was formulated in the presence of citrate-phosphate butter along with a stabilizer, a bulking agent and an isotonicity agent at a desired concentration as described above. pH of the formulation medium was adjusted to around pH 10 5.2. Excipients were added to the protein solution from respective stock solutions to adjust the final concentration and the volume was made up to the desired level with stérile water or Water for Injection. The formulated bulk was distributed in suitable container-closure Systems (like vials, syringes etc.) for storage. Samples at different time points were taken out and analyzed to estimate the aggregates and the principal charged variant of Adalimumab protein 15 by ΗΡ-Size exclusion chromatography and ΗΡ-IEC, respectively. Results are shown in Figures 1 and 2.

Example 2

Formulation composition

Active Ingrédient

Adalimumab 50 mg / mL

Inactive Ingrédients

Dibasic sodium phosphate dihydrate	1.53 mg/mL
Monobasic sodium phosphate dihydrate	0.86 mg / mL
Sodium citrate	0.3 mg ! mL
Citric acid	1.3 mg ! mL
Mannitol	12 mg/mL
Arginine	5 mg / mL
Sodium Chloride	6.16 mg/mL
Polysorbate 80	1 mg / mL

Adalimumab protein was prepared as per the technique known in the art and formulated in the same formulation medium as described with Example 1 in the presence of Arginine. pH of the formulation medium was adjusted to around pH 5.2. Excipients were added to the protein solution from respective stock solutions to adjust the final concentration and volume was made up to the desired level with Water for Injection. The formulated bulk 10 was distributed in suitable container-closure Systems (like vials, syringes etc.) for storage.

Samples were exposed to the higher température for checking the dégradation over a period of time. Samples at different time points were taken out and analyzed to estimate the aggregates and the principal charged variant of Adalimumab protein by ΗΡ-Size exclusion chromatography and HP-1EC, respectiveiy. Results are shown in Figures 1 and 2.

IS Example 3

Formulation composition
Active Ingrédient
Adalimumab	50 mg / mL
Inactive Ingrédients
Dibasic sodium phosphate dihydrate	1.53 mg/mL
Monobasic sodium phosphate dihydrate	0.86 mg/mL
Sodium citrate	0.3 mg/mL
Citric acid	1.3 mg/mL
Mannitol	12 mg/mL

Arginine

Sorbitol

Sodium Chloride

Polysorbate 80 mg / mL lOmg/mL 6.16 mg/mL mg / mL

Adalimumab protein was prepared as per the technique known in the art and formulated in the same formulation medium as described in Example 1 with the addition of arginine and sorbitol. pH of the formulation medium is adjusted to around pH 5.2. Excipients were added to the protein solution from respective stock solutions to adjust the final concentration and volume was made up to the desired level with stérile water or Water for 10 Injection. The formulated bulk was distributed in suitable container-closure Systems (like vials, syringes etc.) for storage. Samples were exposed to the higher température for checking the dégradation over a period of time. Samples at different time points were taken out and analyzed to estimate the aggregates and the principal charged variant of Adalimumab protein by ΗΡ-Size exclusion chromatography and ΗΡ-IEC, respectively. Results are shown in 15 Figures 1 and 2.

Example 4

Formulation composition
Active Ingrédient
Adalimumab	50 mg / mL
Inactive Ingrédients
Dibasîc sodium phosphate dihydrate	1.53 mg/mL
Monobasic sodium phosphate dihydrate	0.86 mg / mL
Sodium citrate	0.3 mg / mL
Citric acid	1.3 mg/mL
Mannitol	12 mg/mL
Hydroxypropyl beta cyclodextrin	20 mg / mL
Sodium Chloride	6.16 mg/mL
Polysorbate 80	1 mg / mL

Adalimumab protein was prepared as per the technique known in the art and formulated in the same formulation medium as described in Example l with the addition of hydroxypropyl beta cyclodextrin. pH of the formulation medium was adjusted to around pH 5.2. Excipients were added to the protein solution from respective stock solutions to adjust the 10 final concentration and volume was made up to the desired level with stérile water or Water for Injection. The formulated bulk was distributed in suitable container-closure Systems (like vials, syringes etc.) for storage. Samples were exposed to the higher température for checking the dégradation over a period of time. Samples at different time points were taken out and analyzed mainiy for presence of aggregates and HC-LysO content by ΗΡ-Size exclusion 15 chromatography and HP-1EC respectively. Obtained results are shown in Figures 1 and 2.

Example 5

Formulation composition
Active Ingrédient
Adalimumab	50 mg / mL
Inactive Ingrédients
Dibasic sodium phosphate dihydrate	1.53 mg/mL
Monobasic sodium phosphate dihydrate	0.86 mg / mL
Sodium citrate	0.3 mg / mL
Citric acid	1.3 mg / mL
Mannitol	12 mg/ mL
Hydroxypropyl beta cyclodextrin	20 mg / mL
Sorbitol	10 mg / mL
Sodium Chloride	6.16 mg/ mL
Polysorbate 80	1 mg/mL

Adalimumab protein was prepared as per the technique known in the art and formulated in the same formulation medium as described with Example 1 in the presence of hydroxypropyl beta cyclodextrin and sorbitol. pH of the formulation medium was adjusted to around pH 5.2. Excipients were added to the protein solution from respective stock solutions to adjust the final concentration and volume was made up to the desired level with stérile water or Water for Injection. The formulated bulk was distributed in suitable container-closure Systems (like vials, syringes etc.) for storage. Samples were exposed to the higher température for checking the dégradation over a period of time. Samples at different time points were taken out and analyzed to estimate the aggregates and the principal charged variant of Adalimumab protein by ΗΡ-Size exclusion chromatography and ΗΡ-IEC, respectively. Results are shown in Figures 1 and 2.

Example 6

Formulation composition
Active Ingrédient
Adalimumab	50 mg / mL
Inactive Ingrédients
Dibasic sodium phosphate dihydrate	1.53 mg/mL
Monobasic sodium phosphate dihydrate	0.86 mg/mL
Sodium citrate	0.3 mg / mL
Citric acid	1.3 mg/mL
Mannitol	12 mg / mL
Lysine	5 mg / mL
Sodium Chioride	6.16 mg / mL
Polysorbate 80	1 mg/mL

Adalimumab protein was prepared as per the technique known in the art and formulated in the same formulation medium as described in Example ! with the addition of lysine. pH of the formulation medium was adjusted to around pH 5.2. Excipients were added to the protein solution from respective stock solutions to adjust the final concentration and volume was made up to the desired level with stérile water or Water for Injection. The formulated bulk was distributed in suitable container-closure Systems (like vials, syringes etc.) for storage. Samples were exposed to the higher température for checking the dégradation over a period of time. Samples at different time points were taken out and analyzed to estimate the aggregates and the principal charged variant of Adalimumab protein by ΗΡ-Size exclusion chromatography and ΗΡ-IEC, respectively. Results are shown in Figures l and 2.

Example 7

Formulation composition

Active Ingrédient
Adalimumab	50 mg / mL
Inactive Ingrédients
Sodium succinate	20 mM
Sodium chloride	110 mM
Sorbitol	50 mg / mL
Arginine	lOmg/mL
Polysorbate 80	0.1 mg / mL

Adalimumab protein was prepared as per the technique known in the art and formulated in the presence of succinate buffer containing arginine and sorbitol as stabilizers, a bulking agent and an isotonicity agent at a desired concentration as described above in formulation composition. pH of the formulation medium was adjusted to around pH 5.2. Excipients were added to the protein solution from respective stock solutions to adjust the final concentration and volume was made up to the desired level with stérile water or Water for Injection. The formulated bulk was distributed in suitable container-closure Systems (like vials, syringes etc.) for storage. Samples were exposed to the higher température for checking the dégradation over a period of time. Samples at different time points were taken out and analyzed to estimate the aggregates and the principal charged variant of Adalimumab protein by ΗΡ-Size exclusion chromatography and ΗΡ-IEC, respectively. Results are shown in Figures 3 and 4.

Example 8

Formulation composition

Active Ingrédient
Adalimumab	50 mg / mL
Inactive Ingrédients
Sodium succinate	20 mM
Sodium chloride	110 mM
Polysorbate 80	0.1 mg / mL

Adalimumab protein was prepared as per the technique known in the art and formulated in the absence of stabilizers. The formulation medium comprises a succinate butter, a bulking agent and an isotonicity agent at a desired concentration as described above. pH of the formulation medium was adjusted to around pH 5.2. Excipients were added to the protein solution from respective stock solutions to adjust the final concentration and volume 10 was made up to the desired level with stérile water or Water for Injection. The formulated bulk was distributed in suitable container-closure système (like vials. syringes etc.) for storage. Samples were exposed to the higher température for checking the dégradation over a period of time. Samples at different time points were taken out and analyzed to estimate the aggregates and the principal charged variant of Adalimumab protein by HP-Size exclusion 15 chromatography and ΗΡ-IEC, respectively. Results are shown in Figures 3 and 4.

Example 9

Formulation composition
Active Ingrédient
Adalimumab	50 mg ! mL
Inactive Ingrédients
Sodium succinate	20 mM
Sodium chloride	liOmM
Arginine	i 0 mg / mL
Polysorbate 80	0.1 mg/mL

Adalimumab protein was prepared as per the technique known in the art and formulated in the presence of succinate buffer further comprising arginine as a stabilizer, a bulking agent and an isotonicity agent at a desired concentration as described above. pH of the formulation medium was adjusted to around pH 5.2. Excipients were added to the protein solution from respective stock solutions to adjust the final concentration and volume was made up to the desired level with stérile water or Water for Injection. The formulated bulk was distributed in suitable container-closure Systems (like vîals, syringes etc.) for storage. Samples at different time points were taken out and analyzed to estimate the aggregates and the principal charged variant of Adalimumab protein by ΗΡ-Size exclusion chromatography and HP-lEC, respectively. Results are shown in Figures 3 and 4.

Example 10

Formulation composition

Active Ingrédient
Adalimumab	50 mg / mL
Inactive Ingrédients
Sodium succinate	20 mM
Sodium chloride	IlOmM
Trehalose	50 mg ! mL
Arginine	lOmg/mL
Polysorbate 80	0.1 mg / mL

Adalimumab protein was prepared as per the technique known in the art and formulated in the presence of succinate buffer along with arginine and trehalose as stabilizers, a bulking agent and an isotonicity agent at a desired concentration as described above in the formulation composition. pH of the formulation medium was adjusted to around pH 5.2. Excipients were added to the protein solution from respective stock solutions to adjust the final concentration and volume was made up to the desired level with stérile water or Water for Injection. The formulated bulk was distributed in suitable container-closure Systems (like vials, syringes etc.) for storage. Samples were exposed to the higher température for checking the dégradation over a period of time. Samples at different time points were taken out and analyzed to estimate the aggregates and the principal charged variant of Adalimumab protein by ΗΡ-Size exclusion chromatography and ΗΡ-IEC, respectively. Results are shown in Figures 3 and 4.

Example 11

Formulation composition

Active Ingrédient
Adalimumab	50 mg / mL
Inactive Ingrédients
Sodium succinate	20 mM
Sodium chloride	IIOmM
Raffinose	50 mg / mL
Arginine	10mg/ mL
Polysorbate 80	0.1 mg / mL

Adalimumab protein was prepared as per the technique known in the art and formulated in the presence of succinate buffer with arginine and raffinose as stabilizers, a suitable bulking agent and an isotonicity agent at a desired concentration as described above in the formulation composition. pH of the formulation medium was adjusted to around pH 5.2. Excipients were added to the protein solution from respective stock solutions to adjust the final concentration and volume was made up to the desired level with stérile water or Water for Injection. The formulated bulk was distributed in suitable container-closure Systems (like vials, syringes etc.) for storage. Samples were exposed to the higher température for checking the dégradation over a period of time. Samples at different time points were taken out and analyzed to estimate the aggregates and the principal charged variant of Adalimumab protein by ΗΡ-Size exclusion chromatography and ΗΡ-IEC, respectively. Results are shown in Figures 3 and 4.

Exemple 12

Formulation composition

Active Ingrédient
Adalimumab	50 mg / mL
Inactive Ingrédients
Sodium succinate	20 mM
Sodium chloride	HOmM
Sorbitol	10 mg/mL
Arginine	5 mg/mL
Polysorbate 80	0.1 mg / mL

Adalimumab protein was prepared as per the technique known in the art and formulated in the presence of succinate buffer containing arginine and sorbitol as stabilizers at a reduced concentration compared to the earlïer Example 7 to reduce the osmolality. A bulking agent and an isotonicity agent were added at a desired concentration as described above. pH of the formulation medium was adjusted to around pH 5.2. Excipients were added to the protein solution from respective stock solutions to adjust the final concentration and volume was made up to the desired level with stérile water or Water for Injection. The formulated bulk was distributed in suitable container-closure Systems (like vials, syringes etc.) for storage. Samples were exposed to the higher température for checking the dégradation over a period of time. Samples at different time points were taken out and analyzed to estimate the aggregates and the principal charged variant of Adalimumab protein by ΗΡ-Size exclusion chromatography and HP-IEC, respectiveiy. Results are shown in Figures 3 and 4.

Example 13

Formulation composition

Active Ingrédient
Adalimumab	50 mg / mL
Inactive Ingrédients
Sodium succinate	10 mM
Sodium chloride	100 mM
Sorbitol	10 mg/mL
Arginine	5 mg / mL
Polysorbate 80	0. i mg / mL

Adalimumab protein was prepared as per the technique known in the art and formulated in the presence of succinate buffer with arginine and sorbitol as stabilizers, a suitable bulking agent and an isotonicity agent as described above in the formulation composition. The concentration of an isotonicity agent was reduced in comparison to the earlier Example 12 to further reduce the osmolality of the formulation medium. pH of the formulation medium is adjusted to around pH 5.2. Excipients were added to the protein solution from respective stock solutions to adjust the final concentration and volume was made up to the desired level with stérile water or Water for Injection. The formulated bulk was distributed in suitable container-closure Systems (like viats, syringes etc.) for storage. Samples were exposed to the higher température for checking the dégradation over a period of time. Samples at different time points were taken out and analyzed to estimate the aggregates and the principal charged variant of Adalimumab protein by ΗΡ-Size exclusion chromatography and ΗΡ-IEC, respectively. Results are shown in Figures 3 and 4.

Exemple 14

Formulation composition
Active Ingrédient
Adalimumab	50 mg / mL
Inactive Ingrédients
Sodium succinate	20 mM
Sodium chioride	IIOmM
Hydroxypropyl beta cyclodextrin	20 mg / mL

Adalimumab protein was prepared as per the technique known in the art and formulated in the presence of succinate buffer further comprising hydroxypropyl beta cyclodextrin as a stabilizer along with a suitable isotonicity agent at a desired concentration as described above in the formulation composition. pH of the formulation medium was adjusted to around pH 5.2. Excipients were added to the protein solution from respective stock solutions to adjust the final concentration and volume was made up to the desired level with stérile water or Water for Injection. The formulated bulk was distributed in suitable container-clo sure Systems (like vials, syringes etc.) for storage. Samples were exposed to the higher température for checking the dégradation over a period of time. Samples at different time points were taken out and analyzed to estimate the aggregates and the principal charged variant of Adalimumab protein by ΗΡ-Size exclusion chromatography and ΗΡ-IEC, respectively. Results are shown in Figures 5 and 6.

Example 15

Formulation composition

Active Ingrédient

Adalimumab 50 mg / mL

Inactive Ingrédients

Sodium succinate Sodium chioride mM

IIOmM

Hydroxypropyl beta cyclodextrin 20 mg / mL

Arginine lOmg/mL

Polysorbate 80 0.1 mg / mL

Adalimumab protein was prepared as per the technique known in the art and formuiated in the presence of succinate buffer with arginine and hydroxypropyl beta cyclodextrin as stabilizers along with a bulking agent and an isotonicity agent at a desired concentration as described above in the formulation composition. pH of the formulation medium is adjusted to around pH 5.2. Excipients were added to the protein solution from respective stock solutions to adjust the final concentration and volume was made up to the desired level with stérile water or Water for Injection. The formuiated bulk was distributed in suitable container-closure Systems (like vials, syringes etc.) for storage. Samples were exposed to the higher température for checking the dégradation over a period of time. Samples at different time points were taken out and analyzed to estimate the aggregates and the principal charged variant of Adalimumab protein by ΗΡ-Size exclusion chromatography and HP-1EC, respectively. Results are shown in Figures 5 and 6.

Example 16

Formulation composition
Active Ingrédient
Adalimumab	50 mg / mL
Inactive Ingrédients
Sodium succinate	20 mM
Sodium chloride	llOmM
Hydroxypropyl beta cyclodextrin	20 mg / mL
Polysorbate 80	0.1 mg/mL

Adalimumab protein was prepared as per the technique known in the art and formuiated in the presence of succinate buffer containing hydroxypropyl beta cyclodextrin as a stabiliser along with a bulking agent and an isotonicity agent at a desired concentration as described above in the formulation composition. pH of the formulation medium was adjusted to around pH 5.2. Excipients were added to the protein solution from respective stock solutions to adjust the final concentration and volume was made up to the desired level with stérile water or Water for Injection. The formulated bulk was distributed in suitable container-closure Systems (like vials, syringes etc.) for storage. Samples were exposed to the higher température for checking the dégradation over a period of time. Samples at different time points were taken out and analyzed to estimate the aggregates and the principal charged variant of Adalimumab protein by ΗΡ-Size exclusion chromatography and ΗΡ-IEC, respectively. Results are shown in Figures 5 and 6.

The formulations described with Examples 7 toi6 are also prepared using acetate buffers or histidine buffer. Such formulations are also to be considered as being encompassed by the présent invention.

The formulations of the présent invention are stable when kept at 2-8 °C.

The formulation of the invention can be used in similar indications as those described in U. S. Patent Nos. 6,090,382 and 6,258,562 each of which îs incorporated by reference herein.

The language effective amount of the formulation is that amount necessary or sufficient to inhibit TNFa activity, e.g. prevent the various morphological and somatic symptoms of a detrimental TNFa activity-associated state. In one embodiment, an effective amount of the formulation is the amount sufficient to inhibit detrimental TNFa activity. The effective amount can vary depending on such factors as the size and weight of the subject, or the type of illness. One of ordinary skill in the art would be able to study the aforementioned factors and make the détermination regarding the effective amount of the TNFa activity inhibiting formulation without undue expérimentation,

The formulation of the présent invention can be administered to the subject either prior to or after the onset of detrimental TNFa activity. Further, several divtded dosages, as well as staggered dosages can be administered daily or sequentially, or the dose can be continuously infused, or can be a bolus injection. The dose can be titrated based on the exigencies of the therapeutic or prophylactic situation.

Actual dosage levels of the active ingrédients (antibody) in the pharmaceutical formulation of this invention may be varied so as to obtain an amount of the active ingrédient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

The selected dosage level will dépend upon a variety of factors including the activity of the antibody found in the formulation, the route of administration, the time of administration, the rate of excrétion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the âge, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known to a medical practitioner who can easily prescribe the effective amount of the pharmaceutical composition ofthe invention.

ln general, a suitable daily dose of a formulation of the invention will be that amount of the formulation that is the lowest dose effective to produce a therapeutic efîect. Such an effective dose will generally dépend upon the factors described above and well known to a skilled practitioner.

It is to be noted that dosage values may vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, spécifie dosage regimens should be adjusted over time according to the individuai need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

The invention provides a pharmaceutical formulation with an extended shelf life, which, in one embodiment, is used to inhibit TNFa activity în a subject suffering from a disorder in which TNFa activity is detrimental, comprising administering to the subject an antibody or antibody portion of the invention such that TNFa activity in the subject is inhibited. Preferably, the TNFa is human TNFa and the subject îs a human subject.

Claims (38)

1. 5 We claim:

   1. A Iiquid pharmaceutical formulation comprising an effective amount of an antibody or antigen binding portion thereof directed to TNFa in a buffer system with a pH between 4 to 8, a surfactant, a tonicity agent and a stabilizer selected from amino acids and

   10 cyclodextrines wherein the antibody is Adalimumab or its antigen binding portion.
2. 2. The formulation as claimed in claim 1, wherein the antibody or antigen binding portion thereof is polyclonal or monoclonal or recombinant antibodies or single chain antibodies or hybrid antibodies or chimeric antibodies or humanized antibodies or

   15 fragments thereof or isolated human antibodies or antibody portions thereof.
3. 3. The formulation as claimed in claim 1, wherein the concentration of antibody is between 1 mg / mL to 160 mg / mL preferably 1 mg / mL to 100 mg / mL.
4. 4. The formulation as claimed in claim 3, wherein the concentration of antibody is 50 mg

   20 / mL.
5. 5. The formulation as claimed in claim I, wherein the pH of the formulation is between pH 5.0 and pH 5.5, preferably at pH 5.2.

   25
6. 6. The formulation as claimed in claim I, wherein the buffer system is selected from histidine-buffers, citrate-buffers, succinate-buffers, acetate-buffers, phosphate-buffers, phosphate buffered saline, citrate and phosphate buffer, tromethamine buffers and suitable mixtures thereof.

   30
7. 7. The formulation as claimed in claim 6, wherein the buffer system is a succinate- buffer or acetate-buffer or histidine buffer or suitable mixture thereof.
8. 8. The formulation as claimed in claim 7, wherein the buffer system is a succinate-buffer.
9. 9. The formulation as claimed in claim 1, wherein the buffer is in the concentration of ImM to about 100 mM, preferably 5 mM to 50 mM, more preferably 10 mM to 20 mM.
10. 10. The formulation as claimed in claim I, wherein the amino acid is selected from arginine, glycine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophane, méthionine, serine, proline, cysteine ! cystine and suitable combination thereof.
11. 11. The formulation as claimed in claim 10, wherein the stabilizer is arginine alone or in combination with other suitable amino acids.
12. 12. The formulation as claimed in claim I, wherein the cyclodextrin is selected from acyclodextrîn, β-cyclodextrin, γ-cyclodextrin, their hydroxypropylated, hydroxyethylated, ethylated and methylated dérivatives thereof, Sulfobutyl ether betacyclodextrin (SBE-beta-CD), a branched cyclodextrins, cyclodextrin polymers and suitable mixture thereof.
13. 13. The formulation as claimed in claim 11, wherein stabilizer is hydroxypropylated cyclo beta-dextrin (ΗΡ-β-CD).
14. 14. The formulation as claimed in claim I, comprising additional stabilizers selected from sugars and polyols, including their suitable combination.
15. 15. The formulation as claimed in claim 14, wherein the sugar is selected from glucose, fructose, galactose, mannose, sorbose, ribose, deoxyribose, sucrose, trehalose, lactose, maltose, rafïïnose and suitable mixtures thereof preferably trehalose or raffinose.
16. 16. The formulation as claimed in claim 15, wherein the polybl is selected from mannitol, sorbitol, dextran, glycerol, arabitol, propylene glycol, polyethylene glycol and suitable combinations thereof.
17. 17. The formulation as claimed in claim 16, wherein the stabilizer is sorbitol.
18. 18. The formulation as claimed in claim 10, wherein the amino acid is présent in the concentration of 1% to 10%.
19. 19. The formulation as claimed in 12, wherein the cyclodextrin is in the concentration of 0.2% to 10%.
20. 20. The formulation as claimed in claim 1 which further comprises suitable excipients selected from surfactants, tonicity agent and suitable combination thereof.
21. 21. The formulation as claimed in claim 20, wherein the surfactant is selected from polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers, alkylphenylpolyoxyethylene ethers, polyoxyethylene-polyoxypropylene copolymer and sodium dodecyl sulphate (SDS).
22. 22. The formulation as claimed in claim 21, wherein the surfactant is selected from polysorbate 20 or polysorbate 80.
23. 23. The formulation as claimed in claim 22, wherein the surfactant is 0.001 % to about 1 %.
24. 24. The formulation as claimed in claim 23, wherein the tonicity agent is sodium chloride or potassium chloride.
25. 25. The formulation as claimed in claim 20, wherein the tonicity agent is présent in amount about 10 mM to about 150 mM,
26. 26. A formulation as claimed in claim 1 comprising

    a) 1 - 160 mg / mL of antibody,

    b) 1-10% stabilizer

    c) A butter system with a pH of 4 to 8.
27. 27. The formulation as claimed in claim 26, wherein the stabilizer is selected from sorbitol, raffinose, trehalose, arginine, lysine, hydroxypropylated cyclo beta- dextrin (ΗΡ-β-CD) and combination thereof.
28. 28. The formulation as claimed in claim 26, wherein the butter system is a succinate or acetate or histidine butter system.
29. 29. The formulation as claimed in claim 26, wherein the antibody is directed to TNFa.
30. 30. The formulation as claimed in claim 26, wherein the antibody is Adalimumab or antigen binding portion thereof.
31. 31. The formulation as claimed in claim 26 wherein the antibody concentration is 50 mg / mL.
32. 32. The formulation as claimed in claim 26 which further comprises a surfactant and/or tonicity agent.
33. 33. The formulation as claimed in claim 27, wherein the surfactant is polysorbate 80 and the tonicity agent is sodium chloride.
34. 34. A formulation claimed in claim 1 comprising

    a) 50 mg / mL of antibody,

    b) 10 mg / mL sorbitol,

    c) 5 mg / mL arginine

    d) 0.1 mg / mL polysorbate 80

    e) 10 mM Succinate buffer of pH of 5.2.
35. 35. A lyophilized formulation comprising the composition as ciaimed in claim 1-34.
36. 36. The formulation of claim 35 wherein the lyophilized powder is reconstituted with water.
37. 37. The formulation as ciaimed in claim 1 is suitable for parentéral administration.
38. 38. The formulation of claim I which is administered to a patient sufiering from the disorder in which activity of TNFa is detrimental.