STABLE FUSION PROTEIN FORMULATION

Description

FIELD OF INVENTION

[0001] The present invention is related to stable formulation of a fusion protein molecule. In particular, the invention discloses stable cytotoxic T-lymphocyte-associated protein 4-immunoglobulin (CTLA4-Ig) fusion protein formulation, wherein the formulation comprises buffer systems and stabilizers.BACKGROUND

[0002] Over the past two decades, recombinant DNA technology has led to the commercialization of many proteins, particularly antibody therapeutics and fusion protein molecules.

[0003] Fusion proteins, in particular, Fc fusion protein molecules (in which Fc portion of human immunoglobulin (Ig) is conjugated to a particular portion of a receptor) are gaining significance, since their wide usage in treatment of various oncological and immunological disorders. Etanercept (TNFR-IgFc), aflibercept (VEGFR-IgFc) and abatacept and belatacept (CTLA4-IgFc) are among those Fc fusion proteins approved by Food and Drug Administration (FDA) to treat various disorders. The effectiveness of fusion protein molecule is majorly dependent on the stability, route of administration and their dosage forms and concentrations. This in turn, necessitates these protein molecules to be formulated appropriately to retain stability and activity.

[0004] Proteins in general, and Fc fusion proteins in particular, are typically unstable in solution and sensitive to pH, temperature and oxidation and hence can undergo a variety of covalent and non-covalent reactions, modifications or degradations in solution. The more common protein degradation pathways include aggregation, deamidation and / or oxidation and these degradation pathways are known to be influenced by pH, temperature and storage conditions, including formulation conditions and excipients. These pathways thus lead to both physical and chemical instability of a protein in solution.

[0005] Aggregation in therapeutic proteins, is of particular interest, because it often results in decreased bioactivity / loss of activity over a period of time and may be immunogenic when administered to a patient. In case of fusion proteins aggregation is significant since they involve fusion of two or more proteins, are large and complex structure and tend to form aggregates at a rapid rate as compared to simple polypeptides or antibodies.

[0006] Apart from aggregation, another type of instability of a multimeric protein, specifically occurring at the regions where two or more proteins are fused, is fragmentation / clipping which can be a result of deamidation, oxidation, isomerization and / or hydrolysis. Deamidation can occur at aspargine or glutamine residues, resulting in a charge variant / s of the protein. Oxidation of fusion proteins involves mainly methionine residues, and are generally influenced by external factors such as exposure to light and transition metal ions or degradation product of an excipient (e.g., hydrogen peroxide from polysorbate degradation). Presence of these oxidized products and charge variants in a therapeutic protein molecule are known to increase instability and, thus decrease the bioactivity of the protein.

[0007] Hence, it is essential to develop a suitable mixture of formulation component / s that would stabilize a therapeutic (fusion) protein molecule against the many physical and chemical instability inducing factors. Further, the developed formulation should maintain colloidal stability during storage conditions, since it measures and ensures that the protein molecules remain suspended in an aqueous solution at equilibrium.

[0008] There are numerous class of excipients such as sugars (sugar or sugar alcohols), amino acids and surfactants which are used in stabilizing proteins and fusion protein molecules.

[0009] WO2007076354A2 (Squibb Bristol Myers Co [US]) discloses lyophilized and liquid formulations of CTLA4-lg fusion protein, more specifically recommending use of a specific ratio of sugar to fusion protein.

[0010] WO2017078385 A1 (Genexine Inc [KR]) discloses IL-7 fusion protein formulations in buffer, sugar and surfactant, and stabilization of the protein at lower concentrations (i.e., 10 mg / ml).

[0011] CN101199 484 B (National Engineering Research Center of Antibody medicine) discloses lyophilized / freeze dried formulations of lower concentrations of CTLA-4 1g fusion protein, at a concentration of 20 mg / ml.

[0012] However, the choice of excipients while formulating a protein (specifically high concentration protein) is governed by various other factors such as their compatibility with the protein and other components in the formulation, (intended) mode of administration and dosage of the therapeutic protein, etc. Therefore, the challenge behind a formulation development involves screening and selection of suitable buffer conditions and excipients, including their concentrations, to achieve a stable formulation. Further, it is also expected that the developed formulation is stable at room temperature and be suitable to be administered in either lyophilized or liquid form.SUMMARY

[0013] The present invention discloses a stable pharmaceutical formulation of a fusion protein molecule comprising phosphate buffer at a pH ranging from pH 6.0 to pH 8.0, sucrose or mannitol, histidine, and surfactant (poloxamer), wherein the fusion protein is a CTLA4-Ig molecule.

[0014] In particular, the invention discloses a stable pharmaceutical formulation of 125 mg / ml of CTLA4-Ig fusion protein comprising phosphate buffer, sucrose or mannitol, histidine, and poloxamer.

[0015] In addition, the invention discloses a method of reducing aggregation and / or fragmentation of CTLA4-Ig fusion protein by formulating in a formulation comprising histidine and sugar, wherein the sugar is sucrose or mannitol. This combination of sugar and histidine imparts colloidal stability to the fusion protein molecule present in the formulation.

[0016] Also, the inventive formulation does not require / include any other amino acids, other than histidine, that are commonly used as stabilizers in therapeutic protein formulation.

[0017] In addition, the invention also discloses a method of increasing the stability of CTLA4-Ig fusion protein formulation, comprising histidine and sugar, wherein the histidine and sugar components, are also added during the process step i.e., in particular in the tangential flow filtration process step (a step before the formulation step). Such addition during the process imparts significant stability to the formulation. The sugar used is either sucrose or mannitol.

[0018] The CTLA4-Ig fusion protein in the said formulation is stable for at least two weeks at 30°C, and contains less than 10% of the protein molecule in aggregate form.DETAILED DESCRIPTION OF THE INVENTION Definitions

[0019] The term "fusion protein" means a protein formed by fusing (i.e., joining) all or part of two polypeptides which are not the same. Typically, fusion proteins are made using recombinant DNA techniques, by end to end joining of polynucleotides encoding the two polypeptides.

[0020] The terms "CTLA4-Ig" or "CTLA4-Ig molecule" or "CTLA4Ig molecule" are used interchangeably, and refer to a protein molecule that comprises a polypeptide having a CTLA4 extracellular domain or a portion thereof, and an immunoglobulin constant region or a portion thereof. The extracellular domain and the immunoglobulin constant region can be wild-type, or mutant or modified, and mammalian, including human or mouse. The polypeptide can further comprise additional protein domains. A CTLA4-Ig molecule can also refer to multimer forms of the polypeptide, such as dimers, tetramers, and hexamers. A CTLA4-Ig molecule also is capable of binding to CD80 and / or CD86.

[0021] The term "stable" formulation refers to the formulation wherein the antibody therein retains its physical stability and / or chemical stability and / or biological activity, upon storage.

[0022] Pre-formulation steps refer to any or multiple steps performed before formulating the protein into a therapeutic product. Examples of such steps include, chromatography, filtration, (ultrafiltration, sterile filtration, nano filtration, diafiltration, depth filtration), or any other steps performed to concentrate the protein or to exchange the buffer to a different / suitable buffer. The filtration steps mentioned herein may be performed in a tangential flow filtration mode.

[0023] Stability studies provides evidence of the quality of an antibody under the influence of various environmental factors during the course of time. ICH's "Q1A: Stability Testing of New Drug Substances and Products," states that data from accelerated stability studies can be used to evaluate the effect of short-term excursions higher or lower than label storage conditions that may occur during the shipping of the antibodies.

[0024] Various analytical methods are available for measuring the physical and chemical degradation of the fusion protein in the pharmaceutical formulations. A fusion protein "retains its physical stability" in a pharmaceutical formulation if it shows substantially no signs of aggregation, precipitation and / or denaturation upon visual examination of color and / or clarity, or as measured by UV light scattering or by size exclusion chromatography. A fusion protein is said to "retain its chemical stability" in a pharmaceutical formulation when its shows no or minimal formation of product variants which may include variants as a result of chemical modification of fusion protein such as deamination, oxidation etc. Analytical methods such as ion exchange chromatography and hydrophobic ion chromatography may be used to investigate the chemical product variants.

[0025] The monomer, dimer and high molecular weight (HMW) species of CTLA4Ig molecule may be separated by size exclusion chromatography (SEC). SEC separates molecules based on the molecular size. Separation is achieved by the differential molecular exclusion or inclusion as the molecules migrate along the length of the column. Thus, resolution increases as a function of column length. In order to maintain the appropriate activity of a fusion protein, it is desirable to reduce the formation of aggregate or fragmentation (monomer / low molecular weight species) of products and hence control the dimer content to a target value. Dimer is major form present in fusion proteins and elutes as main peak in size exclusion chromatography. CTLA4Ig molecule samples may be separated using a 2695 Alliance HPLC (Waters, Milford, Mass.) equipped with TSK Gel ®< G3000SWXL (300 mm×7.8 mm) and TSK Gel ®< G3000SWXL (40 mm×6.0 mm) columns (Tosoh Bioscience, Montgomery, Pa.).

[0026] The colloidal stability of a protein gives information on interaction of proteins molecules within self, and between the surrounding molecules, in an aqueous environment. A common indicator or predictor of colloidal stability of a protein molecule in a solution is the diffusion co-efficient (k D ) value, measured by dynamic light scattering (DLS) technique. The higher the diffusion co-efficient value, the more the repulsive forces, more solubility and less aggregate formation in the protein molecule, and thus the protein exhibits colloidal stability. And colloidal stability is an indicator of protein solubility, viscosity, type of protein aggregates etc.

[0027] Pharmaceutically acceptable excipients refer to the additives or carriers, which may contribute to stability of the antibody in formulation. The excipients may encompass stabilizers and tonicity modifiers. Examples of stabilizers and tonicity modifiers include, but not limited to, sugars, salts, surfactants, and derivatives and combination thereof.

[0028] Sugar / s herein includes sugars and sugar alcohols such as polyols. Sugars can be referred to monosaccharides, disaccharides, and polysaccharides. Examples of sugars include sucrose. Examples of polyols include mannitol.

[0029] Surfactant refers to pharmaceutically acceptable excipients used to protect the protein formulations against various stress conditions, like agitation, shearing, exposure to high temperature etc. The suitable surfactants include, polyoxyethylene-polyoxypropylene copolymer (e.g. Poloxamer).

[0030] Examples of salts include, but not limited to, sodium chloride, potassium chloride, magnesium chloride, sodium thiocyanate, ammonium thiocyanate, ammonium sulfate, ammonium chloride, calcium chloride, zinc chloride and / or sodium acetate.

[0031] Certain specific aspects and embodiments of the invention are more fully described by reference to the following examples. However, these examples should not be construed as limiting the scope of the invention in any manner.Detailed description of the embodiments

[0032] The present invention discloses a stable pharmaceutical formulation of a fusion protein comprising phosphate buffer, sucrose or mannitol, histidine and poloxamer.

[0033] In one embodiment, the invention discloses a stable pharmaceutical formulation of a CTLA4-Ig fusion protein comprising phosphate buffer, sucrose or mannitol, histidine and poloxamer.

[0034] In one embodiment, the invention discloses a stable pharmaceutical formulation of a CTLA4-Ig fusion protein comprising phosphate buffer, sucrose or mannitol, histidine and poloxamer.

[0035] In one embodiment, the invention discloses a stable pharmaceutical formulation of 125 mg / ml of CTLA4-Ig fusion protein, phosphate buffer, sucrose or mannitol, histidine and poloxamer and wherein the formulation has a pH of 6.0 to 8.0.

[0036] In one embodiment, the invention discloses a stable pharmaceutical formulation of a CTLA4-Ig fusion protein comprising phosphate buffer, sugar, histidine and poloxamer, and wherein the formulation does not require any other amino acids, that are commonly employed to stabilize a therapeutic protein formulation.

[0037] In any of the above embodiments, the sugar is sucrose or mannitol.

[0038] In any of the above said embodiments, the concentration of fusion protein in the formulation is about 125 mg / ml.

[0039] In any of the above mentioned embodiments of the invention, the pH of CTLA4-Ig fusion protein formulation is from 6.0-8.0, preferably 6.5 to 7.5.

[0040] In yet another embodiment of the invention, the buffer mentioned in the formulation is a phosphate buffer.

[0041] In any of the above said embodiment, the formulation optionally contains pharmaceutically acceptable excipients such as salts.

[0042] In an embodiment, the invention discloses a stable pharmaceutical formulation of CTLA4-Ig fusion protein comprising phosphate buffer, sucrose or mannitol, histidine and poloxamer, wherein the ratio of the fusion protein to sugar is 1: 0.8 or lower.

[0043] In the above mentioned embodiment, the ratio of the fusion protein to amino acid is 1: 0.1 or lower.

[0044] In an embodiment, the invention discloses a stable pharmaceutical formulation of CTLA4-Ig fusion protein comprising phosphate buffer, sucrose, histidine and poloxamer.

[0045] In another embodiment, the invention discloses a stable fusion protein formulation comprising CTLA4-Ig fusion protein molecule, phosphate buffer, mannitol, histidine and poloxamer.

[0046] In the above mentioned embodiments, the said stable formulation contains less than 10% of the protein in aggregate form when stored at 30 °C for at least two weeks.

[0047] In any of the above said embodiments, the formulation does not require any other amino acid / s, other than histidine.

[0048] In an embodiment, the invention discloses a stable pharmaceutical formulation of CTLA4-Ig fusion protein molecule comprising, citrate-phosphate buffer, mannitol, histidine and poloxamer.

[0049] In an embodiment, the invention discloses a stable pharmaceutical formulation of CTLA4-Ig fusion protein molecule comprising, 125 mg / ml of CTLA4-Ig fusion protein, phosphate buffer, 75 mg / ml of mannitol, 15 mg / ml of histidine and 8 mg / ml of poloxamer.

[0050] In an embodiment, the invention discloses a stable pharmaceutical formulation of CTLA4-Ig fusion protein molecule comprising, 125 mg / ml of CTLA4-Ig fusion protein, phosphate buffer, 85 mg / ml of mannitol, 10 mg / ml of histidine and 8 mg / ml of poloxamer.

[0051] In an embodiment, the invention discloses a stable pharmaceutical formulation of CTLA4-Ig fusion protein molecule comprising, 125 mg / ml of CTLA4-Ig fusion protein, phosphate buffer, 100 mg / ml of sucrose, 10 mg / ml of histidine and 8 mg / ml of poloxamer.

[0052] In an embodiment, the invention discloses a method of obtaining a stable formulation of CTLA4-Ig fusion protein comprising addition of buffer, sugar, histidine and poloxamer, and wherein histidine and sugar are also added to the pre-formulation process step of tangential flow filtration, performed as ultra-filtration [UF] and diafiltration [DF] and ultra-filtration, commonly performed for the concentration of the protein and buffer exchange.

[0053] In another embodiment, the invention discloses a method of increasing the stability of the CTLA4-Ig fusion protein composition, wherein the method comprises addition of histidine and sugar during the process step, in particular in the tangential flow filtration step [performed as ultrafiltration (UF) and diafiltration (DF) for product concentration and buffer exchange]. More particularly, the histidine and sugar are added to the buffer used in the diafiltration step.

[0054] In yet another embodiment, the invention discloses a method of increasing the stability of CTLA4- Ig fusion protein comprising steps of expression and purification of CTLA4- Ig fusion protein; followed by concentration and / or buffer exchange of the protein in phosphate buffer by UF - DF, wherein the buffer used in any of the UF-DF step includes histidine and sugar; and followed by formulation of the protein in phosphate buffer comprising histidine, sugar and poloxamer; wherein the stability of the protein is increased compared to the formulation of the protein that was processed by UF-DF steps without the inclusion of histidine and sugar in any of its buffer; and wherein the sugar is sucrose or mannitol.

[0055] In an embodiment, the invention discloses a method of preparing a stable high concentration CTLA4-Ig fusion protein formulation comprising; a) obtaining purified CTLA4-Ig fusion protein molecule from a chromatographic step b) subjecting CTLA4-Ig fusion protein obtained from step a) to ultrafiltration [UF] to concentrate the protein c) subjecting the concentrated CTLA4-Ig fusion protein obtained from step b) to diafiltration using a phosphate buffer comprising sugar and histidine and, d) subjecting the CTLA4-Ig fusion protein molecule from step c) to second ultrafiltration to obtain further and highly concentrated CTLA4-Ig fusion protein drug substance, wherein the concentration of formulation obtained in step d) is up to 200 mg / ml and is found to be stable as measured by the standard stability studies; wherein the sugar is sucrose or mannitol.

[0056] The stability of the protein molecule is found to be significantly increased in thermal and colloidal stability, when histidine and sugar components are added in the tangential flow filtration step. And addition of histidine and sugar in the UF or DF step, results in a stable product with % HMW being consistently less than 10, even after being subjected to accelerated stability studies.

[0057] In the above mentioned embodiment, the drug substance obtained from the above process is stable and drug product prepared from the drug substance is stable under accelerated stability conditions, wherein the concentration of the drug product is up to 140 mg / ml, preferably 130 mg / ml. The addition of histidine and sugar during DF step of TFF process helps in achieving stable and soluble higher concentrations of CTLA4-Ig fusion protein molecule (up to ~200 mg / ml) which in turn helps in preparation of desired concentration of drug product at commercial scale by simple dilution technique. This additionally saves time and resource.

[0058] In the above said embodiments, where histidine and sugar are also added to the pre-formulation process steps of UF or DF, the formulated protein contains less than 10 % of the protein in aggregate form, even when stored at 30 °C for at least two weeks.

[0059] In another embodiment, the invention discloses a stable fusion protein formulation comprising CTLA4-Ig fusion protein molecule, phosphate buffer, sucrose, histidine, sodium chloride and poloxamer.

[0060] In an embodiment, the invention discloses a method of reducing aggregation in CTLA4-Ig fusion protein formulation (upon storage) comprising addition of histidine and sugar to the fusion protein formulation, wherein the sugar is sucrose or mannitol.

[0061] In a further embodiment, the invention discloses a method of inhibiting fragmentation in CTLA4-Ig fusion protein formulation (upon storage) comprising addition of histidine and sugar to the formulation, wherein the sugar is sucrose or mannitol.

[0062] In another embodiment, the invention discloses a method to maintain colloidal stability of CTLA4-Ig fusion protein in a formulation comprising addition of histidine and sugar to the formulation containing the fusion protein, wherein the sugar is sucrose or mannitol. In the above said embodiment, the said stable formulation optionally contains an anti-oxidant.

[0063] In any of the above embodiments of the invention, the stable formulation is a liquid / aqueous formulation and is suitable for, and can be lyophilized as lyophilized powders. Further, the lyophilized formulation of CTLA4-Ig fusion protein can be reconstituted with appropriate diluent to achieve the liquid formulation suitable for administration.

[0064] In any of the above mentioned embodiments, the CTLA4-Ig fusion protein is abatacept or belatacept.

[0065] In any of the above said embodiments, the amino acid histidine added to the formulation, functions as a stabilizer and does not form part of a buffering agent.

[0066] In any of the above mentioned embodiments, the formulation of CTLA4-Ig fusion protein is a stable liquid (aqueous) formulation, which can be used for parenteral administration. Parenteral administration includes intravenous, subcutaneous, intra peritoneal, intramuscular· administration or any other route of delivery generally considered to be falling under the scope of parenteral administration and as is well known to a skilled person.

[0067] The disclosed formulations of the invention uses lesser amounts of sugar or sugar alcohol to stabilize the therapeutic fusion protein molecule. And the disclosed formulations of CTLA4-Ig fusion protein formulations comprising phosphate buffer, sucrose or mannitol, histidine and poloxamer are stable and can withstand multiple freeze thaw cycles and also agitation induced stress.

[0068] The disclosed formulation of the fusion protein, CTLA4-Ig is stabilized majorly by histidine and sugar combination and does not require any other amino acids Surprisingly, addition of any other amino acid or the combinations of amino acids to the formulation, indeed destabilizes the protein. CTLA4-Ig (eg., abatacept) being a fusion protein and dimeric in nature is a complex molecule, prone to aggregation and oxidation, is however unexpectedly stabilized only by the amino acid histidine (and sugar).EXAMPLES

[0069] CTLA4-Ig fusion protein molecule, abatacept, suitable for storage in the present pharmaceutical composition is produced by standard methods known in the art. For example, abatacept is prepared by recombinant expression of CTLA4 fused with CH2 and CH3 portion of human IgG in a mammalian host cell such as Chinese Hamster Ovary cells. Further, the expressed abatacept is harvested and the crude harvest is subjected to standard downstream process steps that include purification, filtration and optionally dilution or concentration steps. For example, the crude harvest of abatacept may be purified using standard chromatography techniques such as affinity chromatography, ion-exchange chromatography and combinations thereof. The purified abatacept solution can additionally be subjected to one or more filtration steps, and the solution obtained is subjected to further formulation studies.Example 1: Screening and selection of suitable buffer to formulate abatacept

[0070] To select suitable buffer / s for stabilizing abatacept, various buffers were prepared. 40 mg / ml of abatacept in phosphate buffer back ground obtained from downstream chromatographic process was buffer exchanged and diluted to 25 mg / ml in the respective different buffer back ground / s. Details of the formulations are given in Table 1.

[0071] All abatacept formulations were subjected for accelerated stability studies at 25 °C and 40 °C for four weeks. Post which, the samples were analyzed for high molecular weight (HMW) species and low molecular weight (LMW) species [results are shown Table 2 and 3] using size exclusion chromatography (SEC) and also checked for change in pH [Table 4] and visual inspection [Table 5]. Table 1: Compositions of various abatacept formulations in different buffers as per example 1Sample Name Composition Aba-IV-1Abatacept 25 mg / ml, 10 mM phosphate buffer, pH 7.2Aba- IV-2Abatacept 25 mg / ml, 20 mM histidine buffer, pH 7.1Aba- IV-3Abatacept 25 mg / ml, 20 mM citrate buffer, pH 7.1Aba- IV-4Abatacept 25 mg / ml, 20 mM histidine-phosphate buffer, pH 7.1Aba- IV-5Abatacept 25 mg / ml, 20 mM succinate buffer, pH 7.1Aba -IV-6Abatacept 25 mg / ml, 20 mM sodium-acetate buffer, pH 7.2 Table 2: SEC data of abatacept (25 mg / ml) formulations prepared as per example 1 Sample Name High Molecular Weight (HMW) Species T0 40 °C 25°C 0W 1W 2W 4W Δ 2W Δ 4W 2W 4W Δ 2W Δ 4W Aba-IV-126.734.937.440.410.713.729.129.72.43.0Aba-IV-226.275.977.755.851.529.628.228.62.12.4Aba-IV-327.341.741.240.413.913.132.532.55.25.2Aba-IV-427.064.150.942.823.915.830.230.93.23.9Aba-IV-527.351.443.039.015.711.729.329.12.01.8Aba - IV-626.835.237.335.410.58.628.729.81.93.0 W-indicates weeks, TO-indicates 'zero' time point, Δ (delta) indicates change in a value from zero time point to a specified time point Table 3: SEC data of abatacept 25 mg / ml) formulations prepared as per example 1 Sample Name Low Molecular Weight (LMW) Species T0 40 °C 25 °C 0W 1W 2W 4W Δ 2W Δ 4W 2W 4W Δ 2W Δ 4W Aba-IV-10.00.30.30.60.30.60.10.30.10.3Aba-IV-20.00.10.23.40.23.40.00.00.00.0Aba-IV-30.00.61.30.81.30.80.00.00.00.0Aba-IV-40.00.43.77.13.77.10.10.00.10.0Aba-IV-50.00.52.61.72.61.78.09.88.09.8Aba - IV-60.00.20.33.60.33.60.20.60.20.6 W-indicates weeks, TO-indicates 'zero' time point, Δ (delta) indicates change in a value from zero time point to a specified time point Table 4: pH of abatacept (25 mg / ml) formulations prepared as per example 1 at 40 °C and 25 °C Sample Name pH T0 40 °C 25 °C 0W 4W Δ pH 4W Δ pH Aba-IV-17.17.30.27.30.2Aba- IV-26.88.11.36.70.1Aba- IV-37.18.51.48.61.5Aba- IV-46.87.91.18.01.2Aba- IV-56.98.61.78.61.7Aba -IV-67.37.50.27.40.1 W-indicates weeks, TO-indicates 'zero' time point, Δ (delta) indicates change in a value from zero time point to a specified time point Table 5: Visual inspection data of abatacept (25 mg / ml) formulations prepared as per example 1 Visual Inspection Sample Name T0 40 °C 25 °C 0W 2W 4W 2W 4W Aba-IV-1ClearClearClearClearClearAba- IV-2ClearClearClearClearClearAba- IV-3ClearSlightly opalescentSlightly opalescentopalescentopalescentAba- IV-4ClearopalescentopalescentopalescentopalescentAba- IV-5ClearopalescentopalescentopalescentopalescentAba -IV-6ClearClearSlightly opalescentClearClear W-indicates weeks, TO-indicates 'zero' time point Example 2: High concentration abatacept (~125 mg / ml) formulations in presence of sugar(s) and amino acid(s)

[0072] Approximately 120 mg / ml of abatacept in phosphate buffer back ground obtained from tangential flow filtration (TFF) step of downstream process was buffer exchanged in the respective buffer back ground. Post which, various excipients such as sugars and amino acids were added to high concentration abatacept formulations in different combinations and concentrations. Details of the formulations are given in Table 6. FDA approved subcutaneous formulation of abatacept contains phosphate buffer, 170 mg / ml of sucrose and poloxamer. Hence, to maintain a reference standard, to ~ 120 mg / ml of in-house abatacept in phosphate buffer back ground, 170 mg / ml sucrose and 8 mg / ml of poloxamer were added to the formulation.

[0073] All high concentration abatacept formulations were subjected for accelerated stability studies at 30 °C for four weeks. Post which, the samples were analyzed for high molecular weight (HMW) species [results are shown Table 7] using size exclusion chromatography (SEC) and also checked for change in pH [Table 8] and visual inspection [Table 9]. Table 6: Compositions of various high concentration abatacept formulations (~ 120 mg / ml) prepared as per example 2Sample Name Composition Aba-RefAbatacept 120 mg / ml, 8 mM phosphate buffer, 170 mg / ml of sucrose, 8 mg / ml poloxamer pH 6.8-7.2Aba-SC-1Abatacept 120 mg / ml, 10 mM phosphate buffer, 125 mg / ml of sucrose, 5 mg / ml lysine, 10 mg / ml glycine, 10 mg / ml arginine, 10 mg / ml histidine, 8 mg / ml poloxamer, pH 7.2Aba-SC-2Abatacept 120 mg / ml, 10 mM phosphate buffer, 125 mg / ml of sucrose, 10 mg / ml glycine, 10 mg / ml arginine, 10 mg / ml histidine, 8 mg / ml poloxamer, pH 7.2Aba-SC-3Abatacept 120 mg / ml, 10 mM phosphate buffer, 125 mg / ml of sucrose, 10 mg / ml glycine, 10 mg / ml arginine, 8 mg / ml poloxamer, pH 7.2Aba-SC-4Abatacept 120 mg / ml, 10 mM phosphate buffer, 125 mg / ml of sucrose, 10 mg / ml histidine, 8 mg / ml poloxamer, pH 7.2Aba-SC-5Abatacept 120 mg / ml, 10 mM phosphate buffer, 125 mg / ml of mannitol, 5 mg / ml lysine, 10 mg / ml glycine, 10 mg / ml arginine, 10 mg / ml histidine, 8 mg / ml poloxamer, pH 7.2Aba-SC-6Abatacept 120 mg / ml, 10 mM phosphate buffer, 125 mg / ml of trehalose, 5 mg / ml lysine, 10 mg / ml glycine, 10 mg / ml arginine, 10 mg / ml histidine, 8 mg / ml poloxamer, pH 7.2Aba-SC-7Abatacept 120 mg / ml, 10 mM phosphate buffer, 125 mg / ml of sorbitol, 5 mg / ml lysine, 10 mg / ml glycine, 10 mg / ml arginine, 10 mg / ml histidine, 8 mg / ml poloxamer, pH 7.2Aba-SC-8Abatacept 120 mg / ml, 10 mM phosphate buffer, 115 mg / ml of sorbitol, 5 mg / ml lysine, 10 mg / ml glycine, 10 mg / ml arginine, 10 mg / ml histidine, 8 mg / ml poloxamer, pH 7.2Aba-SC-9Abatacept 120 mg / ml, 10 mM phosphate buffer, 115 mg / ml of mannitol, 5 mg / ml lysine, 10 mg / ml glycine, 10 mg / ml arginine, 10 mg / ml histidine, 8 mg / ml poloxamer, pH 7.2Aba-SC-10Abatacept 120 mg / ml, 10 mM phosphate buffer, 100 mg / ml of sucrose, 5 mg / ml lysine, 10 mg / ml glycine, 10 mg / ml arginine, 10 mg / ml histidine, 8 mg / ml poloxamer, pH 7.2Aba-SC-11Abatacept 120 mg / ml, 20 mM phosphate-citrate buffer, 125 mg / ml of sucrose, 5 mg / ml lysine, 10 mg / ml glycine, 10 mg / ml arginine, 10 mg / ml histidine, 8 mg / ml poloxamer, pH 7.2Aba-SC-12Abatacept 120 mg / ml, 20 mM phosphate-citrate buffer, 125 mg / ml of mannitol, 5 mg / ml lysine, 10 mg / ml glycine, 10 mg / ml arginine, 10 mg / ml histidine, 8 mg / ml poloxamer, pH 7.2 Table 7: SEC data of high concentration abatacept (~120 mg / ml) formulations prepared as per example 2 Sample Name High molecular weight (HMW) species at 30 °C 0W 1W 2W 4W Δ 2W Δ 4W Aba-Ref1.419.637.6NM36.2-Aba-SC-12.45.817.334.414.932.0Aba-SC-22.47.015.230.012.827.6Aba-SC-32.45.423.641.221.238.8Aba-SC-42.54.76.910.34.47.9Aba-SC-52.44.710.730.28.327.7Aba-SC-62.413.328.139.725.737.3Aba-SC-72.46.512.524.510.122.1Aba-SC-82.34.614.827.312.525.0Aba-SC-92.47.423.332.320.829.9Aba-SC-102.45.37.918.55.516.2Aba-SC-112.46.012.326.39.923.9Aba-SC-122.55.211.323.18.820.6 W-indicates weeks; NM-not measured since sample turned completely turbid, Δ (delta) indicates change in a value from zero time point to a specified time point Table 8: pH of high concentration abatacept formulations prepared as per example 2 at 30 °C Sample Name pH at 30 °C 0W 4W Δ pH Aba-Ref6.95.5-1.4Aba-SC-17.16.2-1.1Aba-SC-27.17.70.6Aba-SC-36.66.4-0.2Aba-SC-47.17.0-0.1Aba-SC-57.17.70.6Aba-SC-67.16.0-1.1Aba-SC-77.26.4-0.8Aba-SC-87.16.3-0.8Aba-SC-97.26.0-1.2Aba-SC-107.26.1-1.1Aba-SC-117.27.90.7Aba-SC-127.26.5-0.7 W-indicates weeks; Δ (delta) indicates change in a value from zero time point to a specified time point Table 9: Visual inspection data of abatacept (25 mg / ml) formulations prepared as per example 2 Sample Name Visual Inspection 30 °C 0W 2W 4W Aba-RefClearOpalescentTurbidAba-SC-1ClearOpalescentOpalescentAba-SC-2ClearOpalescentClearAba-SC-3ClearClearOpalescentAba-SC-4ClearOpalescentClearAba-SC-5ClearClearClearAba-SC-6ClearOpalescentOpalescentAba-SC-7ClearClearOpalescentAba-SC-8ClearOpalescentOpalescentAba-SC-9ClearOpalescentOpalescentAba-SC-10ClearClearOpalescentAba-SC-11ClearClearclearAba-SC-12ClearOpalescentOpalescent W-indicates weeks Example 3: High concentration abatacept (~125 mg / ml) formulations

[0074] Approximately 120 mg / ml of abatacept in phosphate buffer back ground obtained from tangential flow filtration (TFF) step of downstream process was buffer exchanged in the respective buffer back ground. Post which, various excipients such as sugars, amino acids and sodium chloride were added to high concentration abatacept formulations in different combinations and concentrations. Details of the formulations are given in Table 10. FDA approved subcutaneous formulation of abatacept contains phosphate buffer, 170 mg / ml of sucrose and poloxamer. Hence, to maintain a reference standard, to ~ 120 mg / ml of in-house abatacept in phosphate buffer back ground, 170 mg / ml sucrose and 8 mg / ml of poloxamer were added to the formulation.

[0075] All high concentration abatacept formulations were subjected for accelerated stability studies at 30 °C for two weeks. Post which, the samples were analyzed for high molecular weight (HMW) species and active dimer form [results are shown Table 11] using size exclusion chromatography (SEC) and also checked for change in pH [Table 12] and visual inspection [Table 13].

[0076] Light scattering of protein samples at 333 nm were also checked using nano drop and results are represented in Table 14. Table 10: Compositions of various high concentration abatacept formulations prepared as per example 3Sample Name Composition Aba-Ref120 mg / ml of Abatacept, phosphate buffer, 170 mg / ml of sucrose, 8 mg / ml Poloxamer pH 6.8-7.2Aba-SC-13120 mg / ml of Abatacept, phosphate buffer, 10 mg / ml histidine, 8 mg / ml poloxamer, pH 7.2Aba-SC-14120 mg / ml of Abatacept, phosphate buffer, 15 mg / ml histidine, 8 mg / ml poloxamer, pH 7.2Aba-SC-15120 mg / ml of Abatacept, phosphate buffer, 10 mg / ml arginine, 8 mg / ml poloxamer, pH 7.2Aba-SC-16120 mg / ml of Abatacept, phosphate buffer, 10 mg / ml histidine, 10 mg / ml arginine, 10 mM NaCl, 8 mg / ml poloxamer, pH 7.2Aba-SC-17120 mg / ml of Abatacept, phosphate buffer, 125 mg / ml of sucrose, 10 mg / ml histidine, 8 mg / ml poloxamer, pH 7.2Aba-SC-18120 mg / ml of Abatacept, phosphate buffer, 125 mg / ml of sucrose, 15 mg / ml histidine, 8 mg / ml poloxamer, pH 7.2Aba-SC-19120 mg / ml of Abatacept, phosphate buffer, 125 mg / ml of sucrose 10 mg / ml histidine, 10 mg / ml arginine, 10 mM NaCl, 8 mg / ml poloxamer, pH 7.2Aba-SC-20120 mg / ml of Abatacept, phosphate buffer, 125 mg / ml of sucrose, 10 mg / ml histidine, 10 mM NaCl, 8 mg / ml poloxamer, pH 7.2Aba-SC-21120 mg / ml of Abatacept, citrate-phosphate buffer, 125 mg / ml of mannitol, 10 mg / ml histidine, 8 mg / ml poloxamer, pH 7.2 Table 11: SEC data of dimer content of abatacept (~120 mg / ml) formulations prepared as per example 3 Sample Name Dimer content at 30 °C High molecular weight species at 30 °C Low molecular weight species at 30 °C 0W 1W 2W 0W 1W 2W 0W 1W 2W Aba-Ref97.892.988.72.27.111.00.00.00.3Aba-SC-1397.684.875.22.415.224.80.00.00.0Aba-SC-1497.785.668.22.314.431.70.00.00.2Aba-SC-1597.689.271.32.313.028.60.00.00.2Aba-SC-1697.683.074.82.417.025.20.00.00.0Aba-SC-1797.692.486.82.47.613.20.00.00.0Aba-SC-1897.792.688.22.37.411.80.00.00.0Aba-SC-1997.789.779.72.310.320.10.00.00.2Aba-SC-2097.891.685.12.28.414.90.00.00.0Aba-SC-2197.794.492.62.35.67.50.00.00.0 W-indicates weeks Table 12: pH of high concentration abatacept formulations prepared as per example 3 at 30 °C Sample Name pH at 30 °C 0W 1W 2W Δ pH at 2W Aba-Ref7.06.86.8-0.2Aba-SC-137.37.17.2-0.1Aba-SC-147.47.27.2-0.2Aba-SC-156.96.86.8-0.1Aba-SC-167.37.27.2-0.1Aba-SC-177.37.17.1-0.2Aba-SC-187.47.27.2-0.2Aba-SC-197.37.48.00.6Aba-SC-207.47.27.3-0.1Aba-SC-217.57.47.4-0.1 W-indicates weeks; Δ (delta) indicates change in a value from zero time point to a specified time point Table 13: Visual inspection data of high concentration abatacept formulations prepared as per example 3 at 30 °C Sample Name Visual Inspection 30 °C 0W 1W 2W Aba-RefClear, colorlessClear, colorlessClear, colorlessAba-SC-13Clear, colorlessClear, colorlessClear, colorlessAba-SC-14Clear, colorlessClear, colorlessClear, colorlessAba-SC-15Clear, colorlessClear, colorlessClear, colorlessAba-SC-16Clear, colorlessClear, colorlessClear, colorlessAba-SC-17Clear, colorlessClear, colorlessClear, colorlessAba-SC-18Clear, colorlessClear, colorlessClear, colorlessAba-SC-19Clear, colorlessClear, colorlessClear, colorlessAba-SC-20Clear, colorlessClear, colorlessClear, colorlessAba-SC-21Clear, colorlessClear, colorlessClear, colorless Table 14: Light scattering data at 333 nm (A333) of high concentration abatacept formulations prepared as per example 3 at 30 °C Sample Name Light scattering data at 30 °C 0W 1W 2W Aba-Ref0.10.10.2Aba-SC-130.00.20.4Aba-SC-140.00.20.1Aba-SC-150.00.10.0Aba-SC-160.10.10.0Aba-SC-170.00.10.1Aba-SC-180.00.10.3Aba-SC-190.00.10.3Aba-SC-200.10.10.0Aba-SC-210.10.10.0 Example 4: Addition of excipients during tangential Flow filtration (TFF) step for stability of high concentration CTLA4-Ig fusion proteins

[0077] In example 2 and 3, purified abatacept obtained from the downstream chromatographic step was further buffer exchanged into phosphate buffer and concentrated by tangential flow filtration (TFF), performed as a series of ultra-filtration, diafiltration and ultrafiltration steps. Post which, excipients were added to the formulation. However, differing from this conventional strategy, various sugars such as sucrose and mannitol and amino acid such as histidine and glycine were incorporated during the TFF itself (i.e., before the formulation step or before formulating the protein as a drug product). 8-15 mg / ml concentration of abatacept fusion protein in acetate buffer obtained from chromatographic step was subjected for ultrafiltration to concentrate up to 60 mg / ml. Post which, the samples were subjected for diafiltration wherein the diafiltration medium contained phosphate buffer (formulation buffer) with excipients such as sugars and amino acid(s), and in another separate experiment, the diafiltration medium without sugar and amino acid(s) in the phosphate buffer was experimented. Post diafiltration, the samples were subjected for second ultrafiltration to concentrate up to 180 mg / ml to 200 mg / ml. These high concentration samples were found to be stable without any visible particles / aggregates. The highly concentrated samples were further diluted to 125 mg / ml and some of the excipients such as sugars, surfactant and optionally amino acid such as glycine was added to prepare a final formulation. 8 mg / ml of poloxamer was added to all final formulations. Details of the formulations are given in Table 15. All the samples were subjected for accelerated stability studies at 30 °C for 2 weeks. The samples were analyzed for high molecular weight (HMW) species and active dimer form [results are shown Table 16] using size exclusion chromatography (SEC) and also checked for change in pH [Table 17] and visual inspection [Table 18]. Table 15: Compositions of various high concentration abatacept formulations prepared as per example 4Sample Name Formulation buffer composition during TFF Formulation composition after TFF Aba-SC-2220 mM phosphate buffer125 mg / ml of Abatacept, 20 mM phosphate buffer, 8 mg / ml poloxamer, pH 7.2Aba-SC-2320 mM phosphate buffer and 75 mg / ml Sucrose125 mg / ml of Abatacept, 20 mM phosphate buffer, 170 mg / ml sucrose, 8 mg / ml poloxamer, pH 7.2Aba-SC-2420 mM phosphate buffer and 170 mg / ml Sucrose125 mg / ml of Abatacept, 20 mM phosphate buffer, 170 mg / ml sucrose, 8 mg / ml poloxamer, pH 7.2Aba-SC-2520 mM phosphate buffer and 75 mg / ml Sucrose125 mg / ml of Abatacept, 20 mM phosphate buffer, 100 mg / ml sucrose, 10 mg / ml lysine, 0.58 mg / ml NaCl, 8 mg / ml poloxamer, pH 7.2Aba-SC-2620 mM phosphate buffer, 100 mg / ml sucrose, 10 mg / ml histidine125 mg / ml of Abatacept, 20 mM phosphate buffer, 100 mg / ml sucrose, 10 mg / ml histidine, 8 mg / ml poloxamer, pH 7.2Aba-SC-2720 mM phosphate buffer, 100 mg / ml sucrose, 10 mg / ml histidine, 10 mg / ml glycine125 mg / ml of Abatacept, 20 mM phosphate buffer, 100 mg / ml sucrose, 10 mg / ml histidine, 10 mg / ml glycine, 8 mg / ml poloxamer, pH 7.2Aba-SC-2820 mM phosphate buffer, 100 mg / ml sucrose, 10 mg / ml histidine, 10 mg / ml glycine125 mg / ml of Abatacept, 20 mM phosphate buffer, 100 mg / ml sucrose, 10 mg / ml histidine, 10 mg / ml glycine, 8 mg / ml poloxamer, 0.58 mg / ml NaCl, pH 7.2Aba-SC-2920 mM phosphate buffer, 75 mg / ml mannitol125 mg / ml of Abatacept, 20 mM phosphate buffer, 75 mg / ml mannitol, 6.6 mg / ml ammonium sulphate, 8 mg / ml poloxamer, pH 7.2Aba-SC-3020 mM phosphate buffer, 75 mg / ml mannitol125 mg / ml of Abatacept, 20 mM phosphate buffer, 75 mg / ml mannitol, 10 mg / ml histidine, 6.6 mg / ml ammonium sulphate, 8 mg / ml poloxamer, pH 7.2Aba-SC-3120 mM phosphate buffer, 75 mg / ml mannitol125 mg / ml of Abatacept, 20 mM phosphate buffer, 100 mg / ml mannitol, 15 mg / ml histidine, 8 mg / ml poloxamer, pH 7.2Aba-SC-3220 mM phosphate buffer, 75 mg / ml mannitol125 mg / ml of Abatacept, 20 mM phosphate buffer, 100 mg / ml mannitol, 10 mg / ml lysine, 8 mg / ml poloxamer, pH 7.2Aba-SC-3320 mM phosphate buffer, 75 mg / ml mannitol125 mg / ml of Abatacept, 20 mM phosphate buffer, 100 mg / ml mannitol, 10 mg / ml Glycine, 8 mg / ml poloxamer, pH 7.2Aba-SC-3420 mM phosphate buffer; 75 mg / ml mannitol, 10 mg / ml histidine125 mg / ml of Abatacept, 20 mM phosphate buffer, 75 mg / ml mannitol, 10 mg / ml histidine, 8 mg / ml poloxamer, pH 7.2Aba-SC-3520 mM phosphate buffer; 75 mg / ml mannitol, 15 mg / ml histidine125 mg / ml of Abatacept, 20 mM phosphate buffer, 75 mg / ml mannitol, 15 mg / ml histidine, 8 mg / ml poloxamer, pH 7.2Aba-SC-3620 mM phosphate buffer; 75 mg / ml mannitol, 10 mg / ml histidine125 mg / ml of Abatacept, 20 mM phosphate buffer, 75 mg / ml mannitol, 10 mg / ml histidine, 5 mg / ml glycine, 8 mg / ml poloxamer, pH 7.2Aba-SC-3720 mM phosphate buffer; 75 mg / ml mannitol, 15 mg / ml histidine125 mg / ml of Abatacept, 20 mM phosphate buffer, 75 mg / ml mannitol, 15 mg / ml histidine, 5 mg / ml glycine, 8 mg / ml poloxamer, pH 7.2Aba-SC-3820 mM phosphate buffer; 85 mg / ml mannitol, 10 mg / ml histidine125 mg / ml of Abatacept, 20 mM phosphate buffer, 85 mg / ml mannitol, 10 mg / ml histidine, 8 mg / ml poloxamer, pH 7.2Aba-SC-3920 mM phosphate buffer; 85 mg / ml mannitol, 10 mg / ml histidine125 mg / ml of Abatacept, 20 mM phosphate buffer, 85 mg / ml mannitol, 10 mg / ml histidine, 5 mg / ml glycine, 8 mg / ml poloxamer, pH 7.2Aba-SC-4020 mM phosphate buffer; 85 mg / ml mannitol, 15 mg / ml histidine125 mg / ml of Abatacept, 20 mM phosphate buffer, 85 mg / ml mannitol, 15 mg / ml histidine, 8 mg / ml poloxamer, pH 7.2Aba-SC-4120 mM phosphate buffer, 75 mg / ml mannitol, 10 mg / ml histidine125 mg / ml of Abatacept, 20 mM phosphate buffer, 75 mg / ml mannitol, 15 mg / ml histidine, 10 mg / ml proline, 8 mg / ml poloxamer, pH 7.2Aba-SC-4220 mM phosphate buffer, 75 mg / ml mannitol, 10 mg / ml histidine125 mg / ml of Abatacept, 20 mM phosphate buffer, 75 mg / ml mannitol, 10 mg / ml histidine, 10 mg / ml proline, 8 mg / ml poloxamer, pH 7.2 Table 16: SEC data of high concentration abatacept formulations prepared as per example 4 Sample Name Dimer content at 30 °C High molecular weight species at 30°C Low molecular weight species at 30°C 0W 2W 0W 2W 0W 2W Aba-SC-2298.659.71.437.602.7Aba-SC-2398.687.61.412.400Aba-SC-2498.994.31.15.700Aba-SC-2598.775.81.324.200Aba-SC-2698.991.81.18.200Aba-SC-2798.992.41.17.600Aba-SC-2898.892.21.27.800Aba-SC-2998.287.31.812.600.1Aba-SC-3098.389.21.710.600.1Aba-SC-3198.989.41.110.600Aba-SC-3298.984.31.115.700Aba-SC-3398.988.21.211.900Aba-SC-3498.891.21.28.800Aba-SC-3598.992.01.18.100Aba-SC-3698.891.71.28.300Aba-SC-3798.992.41.17.600Aba-SC-3898.991.71.18.300Aba-SC-3998.992.01.18.000Aba-SC-4098.892.31.27.700Aba-SC-4199.093.01.07.000Aba-SC-4299.087.51.012.500 W-indicates weeks Table 17:pH of high concentration abatacept formulations prepared as per example 4 at 30 °C Sample Name pH at 30 °C 0W 2W Δ pH Aba-SC-226.96.1-0.8Aba-SC-237.06.9-0.1Aba-SC-247.17.20.1Aba-SC-256.86.80Aba-SC-267.07.10.1Aba-SC-277.07.10.1Aba-SC-287.07.00Aba-SC-29NTNT-Aba-SC-30NTNT-Aba-SC-317.47.3-0.1Aba-SC-326.86.80Aba-SC-337.06.9-0.1Aba-SC-347.07.10.1Aba-SC-357.17.20.1Aba-SC-367.07.10.1Aba-SC-377.17.20.1Aba-SC-387.07.10.1Aba-SC-397.07.10.1Aba-SC-407.17.20.1Aba-SC-417.47.40Aba-SC-427.37.2-0.1 W-indicates weeks; NT-Not tested due to sample constrain; Δ (delta) indicates change in a value from zero time point to a specified time point Table 18: Visual inspection data of high concentration abatacept formulations prepared as per example 4 at 30 °C Sample Name Visual Inspection at 30 °C 0W 2W Aba-SC-22ClearTurbidAba-SC-23Clear, colorless, no visible particlesClear, colorless, no visible particlesAba-SC-24Clear, colorless, no visible particlesClear, colorless, no visible particlesAba-SC-25Clear, colorless, no visible particlesClear, colorless, no visible particlesAba-SC-26Clear, colorless, no visible particlesClear, colorless, no visible particlesAba-SC-27Clear, colorless, no visible particlesClear, colorless, no visible particlesAba-SC-28Clear, colorless, no visible particlesClear, colorless, no visible particlesAba-SC-29Clear, colorless, no visible particlesClear, colorless, no visible particlesAba-SC-30ClearOpalescentAba-SC-31Clear, colorless, no visible particlesClear, colorless, no visible particlesAba-SC-32Clear, colorless, no visible particlesClear, colorless, no visible particlesAba-SC-33Clear, colorless, no visible particlesClear, colorless, no visible particlesAba-SC-34Clear, colorless, no visible particlesClear, colorless, no visible particlesAba-SC-35Clear, colorless, no visible particlesClear, colorless, no visible particlesAba-SC-36Clear, colorless, no visible particlesClear, colorless, no visible particlesAba-SC-37Clear, colorless, no visible particlesClear, colorless, no visible particlesAba-SC-38Clear, colorless, no visible particlesClear, colorless, no visible particlesAba-SC-39Clear, colorless, no visible particlesClear, colorless, no visible particlesAba-SC-40Clear, colorless, no visible particlesClear, colorless, no visible particlesAba-SC-41Clear, colorless, no visible particlesClear, colorless, no visible particlesAba-SC-42Clear, colorless, no visible particlesClear, colorless, no visible particles W-indicates weeks; Viscosity of some of the abatacept formulations prepared as per example 4 were measured using m-VROC ®< viscometer. Results are given Table 19. Table 19: Viscosity of high concentration abatacept formulations Sample Name Viscosity (mPa / S) Aba-SC-2410.9Aba-SC-269.2Aba-SC-349.0Aba-SC-389.0

[0078] Alternatively, the amino acid methionine was added to the abatacept formulation of histidine and sugar combination and was evaluated for an effect on stability. The samples (with and without methionine in the formulation) were subjected for accelerated stability studies at 30 °C for one week. The samples were analyzed for high molecular weight (HMW) species and active dimer form [results are shown Table 20] using size exclusion chromatography (SEC). Table 20: SEC data of abatacept formulations prepared with and without methionineSample Name Composition High molecular weight species at 30 °C Dimer content at 30 °C '0' W'1' W'0' W'1' WAba-SC-38125 mg / ml of Abatacept, 20 mM phosphate buffer, 85 mg / ml mannitol, 10 mg / ml histidine, 8 mg / ml poloxamer, pH 7.21.35.098.795.0Aba-SC-43125 mg / ml of Abatacept, 20 mM phosphate buffer, 85 mg / ml mannitol, 10 mg / ml histidine, 10 mM methionine, 8 mg / ml poloxamer, pH 7.21.44.998.695.1W-indicates weeks Example 5: Long term stability data of high concentration abatacept formulations

[0079] From the above experiments, it is evident that addition of sugar and amino acid / (s) during TFF plays a significant role in stabilizing abatacept. Hence, further some of the formulations from the above experiment were further observed till 4 weeks at 30 °C. The samples were analyzed for high molecular weight (HMW) species and active dimer form [results are shown Table 21 using size exclusion chromatography (SEC). Table 21: SEC data of high concentration abatacept formulations prepared as per example 4 at 30 °C for four weeks.Sample Name Dimer content at 30 °C High molecular weight species at 30 °C Low molecular weight species at 30 °C 0W 4W 0W 4W 0W 4W Aba-SC-3498.885.91.214.100Aba-SC-3598.987.01.113.000Aba-SC-3698.886.71.213.300Aba-SC-3798.987.91.112.100Aba-SC-3898.986.71.113.300Aba-SC-3998.987.11.113.000Aba-SC-4098.887.61.212.400W-indicates weeks;

[0080] Abatacept formulations (A' (125 mg / ml abatacept, 100 mg / ml sucrose, 15 mg / ml histidine, 10 mg / ml glycine and 8 mg / ml polaxamer) and B' (125 mg / ml abatacept, 75 mg / ml mannitol, 15 mg / ml histidine, 10 mg / ml glycine and 8 mg / ml polaxamer ) were subjected to accelerated stability studies at 30 °C for four weeks, post which a CD28 receptor ligand based assay was performed to demonstrate and prove that the stable abatacept formulation is functionally active. The formulations were found to be functionally active and exhibited 90.7 % of potency (A') and 93.6% of potency (B').Example 6: Assessment of oxidation of high concentration abatacept formulations

[0081] Some of the abatacept fusion protein formulations prepared as per example 4 were subjected for mass spectrometry to understand the effective of excipients on oxidation sites of abatacept. Abatacept fusion protein contains seven methionine residues. A few of the Abatacept formulations containing sugar and amino acid / s from example 4, which were subjected for accelerated stability studies till four weeks at 30 °C for four weeks collected at '0' and four weeks' time point. The abatacpet formulations collected at '0' and '4' week time point at 30 °C were subjected for denaturation using a buffer (8.2 M guanidine hydrochloride, 1 mM EDTA and 0.1 M Tris, pH 7.5), and reduced using 10 mM Dithiothreitol and alkylated using iodoacetamide. The alkylated samples were subjected to PD-10 column to remove digestion buffer. The protein fractions obtained from PD-10 column were treated with trypsin and N-Glycanse at 37 °C for overnight. The peptide fragments obtained from the above step was subjected for mass spectrometry to understand the effect of excipients on oxidized sites of abatacept. The result of the study is given Table 22 and the % of oxidation demonstrates that the methionine residues are protected from oxidation Table 22: Percentage oxidation of methionine in abatacept formulations stored at 30 °CSample Name % Average oxidized methionine at T0 % Average oxidized methionine at 30 °C for 4 weeks Aba-SC-245.75.5Aba-SC-276.65.6Aba-SC-374.97.6T0-inidcates data point at '0' time point.

Claims

1. A stable aqueous formulation of CTLA4-Ig fusion protein comprising, 125 mg / ml CTLA4-Ig fusion protein, phosphate buffer, sucrose or mannitol, histidine and poloxamer, wherein the formulation has a pH of 6.0 to 8.0.

2. The formulation according to claim 1, which does not require any other amino acid(s) as stabilizer.

3. The formulation according to claim 1, wherein the ratio of CTLA4-Ig fusion protein to sugar is 1: 0.8 or lower, and ratio of the CTLA4-Ig fusion protein to amino acid is 1: 0.1 or lower.

4. The formulation according to claim 1, which is stable for at least two weeks at 30 °C, and contains less than 10% of the protein molecule in aggregate form.

5. A method of obtaining a stable formulation of CTLA4-Ig fusion protein comprising addition of phosphate buffer, sugar, histidine and poloxamer, and wherein the histidine and sugar are also added to the pre-formulation process steps of ultrafiltration and / or diafiltration steps containing the fusion protein and wherein the sugar is sucrose or mannitol.

6. A method of increasing stability of CTLA4-Ig fusion protein comprising steps of; expression and purification of CTLA4-Ig fusion protein; concentration and / or buffer exchange of the protein in phosphate buffer by ultrafiltration and diafiltration (UF-DF), wherein the buffer used in the ultrafiltration and / or diafiltration step(s) include histidine and sugar; followed by formulation of the protein in phosphate buffer comprising histidine, sugar and poloxamer; wherein the stability of the protein is increased compared to the formulation of the protein that was processed by UF-DF steps without the inclusion of histidine and sugar in its buffer and, wherein the sugar is sucrose or mannitol.