A stable pharmaceutical composition of fusion protein

Abstract

The present invention relates to a stable pharmaceutical composition of a pharmacologically active fusion protein, comprising a suitable buffering agent, stabilizer and surfactant wherein the composition essentially consists of a single sugar and is essentially free of mannitol. The present invention discloses a stable lyophilized composition of a Thrombopoietin Receptor Agonist (TPO-RA) fusion protein that maintains high purity of the monomer and low level of product-related impurities, even at elevated temperatures or during long-term storage, the formulation comprises a suitable buffering agent, a stabilizer and a surfactant. The use of specific buffer and excipients helps to ensure the stability of the peptibody during the freeze- drying (lyophilization) process and subsequent storage, thereby improving the product's shelf life and stability.

Description

[0001] TITLE: A STABLE PHARMACEUTICAL COMPOSITION OF FUSION PROTEIN

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a stable pharmaceutical composition of a pharmacologically active fusion protein, comprising a suitable buffering agent, stabilizer and surfactant wherein the composition essentially consists of a single sugar and is essentially free of mannitol. The formulation maintains the purity of the fusion protein of more than 85% at 25°C or at 40°C for a suitable period. More specifically, the present invention discloses a stable lyophilized composition of a Thrombopoietin Receptor Agonist (TPO-RA) fusion protein that maintains high purity of the monomer and low level of product-related impurities, even at elevated temperatures or during long-term storage, the formulation comprises a suitable buffering agent, a stabilizer and a surfactant wherein the composition essentially consists of single sugar and is essentially free of mannitol. The use of specific buffer and excipients helps to ensure the stability of the peptibody during the freeze-drying (lyophilization) process and subsequent storage, thereby improving the product's shelf life and stability.

[0004] BACKGROUND OF THE INVENTION

[0005] The development of fusion proteins by integrating two or more different protein domains to form one molecule using recombinant DNA technology significantly gained the advantage in recent years. To stabilize the fusion protein remains a challenge, the selection of suitable excipients is an important criterion to protect the fusion protein from chemical or physical degradation during processing and storage. It has been well recognised that there is no standard composition strategy which can work for all fusion proteins to provide stable and concentrated solution for pharmaceutical use. Moreover, in view of patient as well as physician compliance, auto-injector or pre-filled syringe is more in demand and there is always a need to prepare compositions comprising pharmacologically active fusion protein. Thrombotic thrombocytopenic purpura (TTP) is a disorder characterized by thrombotic microangiopathy, thrombocytopenia and microvascular thrombosis that can cause various degrees of tissue ischemia and infarction. Clinically, TTP patients are diagnosed by symptoms such as thrombocytopenia, schistocytes (fragments of erythrocytes) and elevated levels of lactate dehydrogenase (Moake J L. Thrombotic microangiopathies. N Engl J Med. 2002; 347:589-600. The present invention discloses a pharmaceutical composition of fusion protein using one sugar, free of mannitol, suitable buffer, surfactant, optionally chelating agent and optionally with suitable amino acid, the formulation maintains or improve purity and / or stability and provides low aggregation. The invention is also very useful for complex fusion protein for an example thrombopoietin receptor agonist (TPO-RA) which were formulated with two sugars sucrose and mannitol which may create operational challenges and make the formulation more complicated.

[0006] SUMMARY OF THE INVENTION

[0007] In an embodiment the invention provides a stable pharmaceutical composition comprising pharmacologically active Thrombopoietin Receptor Agonist (TPO-RA) fusion protein and a method for preparation of the same.

[0008] The present invention provides a stable pharmaceutical composition of a pharmacologically active fusion protein comprising a suitable buffering agent, stabilizing agent, and surfactant wherein the composition essentially consists of a single sugar and is essentially free of mannitol wherein the purity of the composition is more than 90% as determined by size exclusion high performance liquid chromatography (SEC-HPLC) after a freeze-thaw cycle; and wherein the composition maintains less than 0.5% high molecular weight (HMW) aggregates by weight after a freeze-thaw cycle.

[0009] In one embodiment, a stable pharmaceutical composition is disclosed. Said composition comprises: a. a purified pharmacologically active fusion protein Thrombopoietin Receptor agonist (TPO-RA); b. a buffering agent; c. a stabilizing agent ; d. a surfactant; wherein the stabilizing agent is sugar and optionally comprises amino acid; wherein the composition essentially consists of a single sugar; wherein the composition is free of mannitol; wherein the pharmacologically active fusion protein binds to the thrombopoietin receptor (c- Mpl); wherein the Thrombopoietin Receptor Agonist (TPO-RA) fusion protein present in the concentration selected from about 0.125mg / ml, or 0.25 mg / ml and 0.5 mg / ml; wherein the purity of the composition is more than 90% as determined by size exclusion high performance liquid chromatography (SEC-HPLC) after a freeze-thaw cycle; wherein the purity of the pharmaceutical composition under stress condition at 40°C on day 30 is greater than 85%; and / or wherein the purity of the pharmaceutical composition under accelerated condition at 25°C on day 30 is greater than 80%;and / or wherein the purity is measured by cation exchange high performance liquid chromatography (CEX-HPLC); wherein the purity of the pharmaceutical composition under stress condition at 40°C on day 21 is greater than 85%; and / or wherein the purity of the pharmaceutical composition under accelerated condition at 25°C on day 21 is greater than 80%; wherein the purity is measured by reversed-phase liquid chromatography (RPLC); and / or wherein the composition comprises less than 10% acidic variants as determined by cation exchange high performance liquid chromatography (CEX-HPLC).

[0010] In an embodiment, a stable pharmaceutical composition comprises: a. a pharmacologically active Thrombopoietin Receptor Agonist (TPO-RA)fusion protein; b. a buffering agent; c. a stabilizing agent; d. a surfactant; e. an amino acid ; and f. a chelating agent; wherein said Thrombopoietin Receptor Agonist (TPO-RA) fusion protein purified through a chromatography column; wherein the stabilizing agent is sugar; wherein the composition has a purity of greater than 80% as determined by reverse-phase high performance liquid chromatography (RPLC), and / or wherein the composition has a purity of greater than 70% as determined by cation exchange high performance liquid chromatography (CEX-HPLC);and / or wherein the purity of the pharmaceutical composition under stress condition at 40°C on day 30 is greater than 85%; and / or wherein the purity of the pharmaceutical composition under accelerated condition at 25°C on day 30 is greater than 80%; and / or wherein the purity is measured by cation exchange high performance liquid chromatography (CEX-HPLC); wherein the purity of the pharmaceutical composition under stress condition at 40°C on day 21 is greater than 85%; and / or wherein the purity of the pharmaceutical composition under accelerated condition at 25°C on day 21 is greater than 80%; wherein the purity is measured by reversed-phase liquid chromatography (RPLC); and / or wherein the composition comprises less than 10% acidic variants as determined by cation exchange high performance liquid chromatography (CEX-HPLC); and / or wherein the purity of the composition under stress condition at 40°C is greater than 90% as determined by size exclusion high performance liquid chromatography (SEC-HPLC); and / or wherein the purity of the composition under accelerated condition at 25°C is greater than 90% as determined by size exclusion high performance liquid chromatography (SEC-HPLC).

[0011] In an embodiment, the purity of the composition is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 99.9% as determined by size exclusion high performance liquid chromatography (SEC-HPLC) after a freeze-thaw cycle. In one embodiment, the composition maintains less than 0.5% high molecular weight (HMW) aggregates by weight after a freezethaw cycle.

[0012] In an embodiment, the composition is selected from 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% ,98%, and 99% as determined by reverse-phase high performance liquid chromatography (RPLC).

[0013] In an embodiment, the purity of the composition is selected from 71%, 72%, 73%, 74% 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% ,98%, and 99% as determined by cation exchange high performance liquid chromatography (CEX-HPLC).

[0014] In an embodiment, the purity of the composition under the stress condition at 40°C on day 30 is selected from 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% ,98%, and 99% as determined by cation exchange high performance liquid chromatography (CEX- HPLC).

[0015] In an embodiment, the purity of the composition under the accelerated condition at 25°C on day 30 is selected from 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% ,98%, and 99% as determined by cation exchange high performance liquid chromatography (CEX-HPLC).

[0016] In an embodiment, the purity of the composition under the stress condition at 40°C on day 21 is selected from 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% ,98%, and 99% as determined by reversed-phase liquid chromatography (RPLC).

[0017] In an embodiment, the purity of the composition under the accelerated condition at 25°C on day 21 is selected from 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% ,98%, and 99% as determined by reversed-phase liquid chromatography (RPLC).

[0018] In one embodiment, the composition comprises acidic variants about 9 % upon 30 days at 40°C. In another embodiment, the composition comprises acidic about 8% upon 30 days at 40°C. In another embodiment, the composition comprises acidic about 7% upon 30 days at 40°C.

[0019] In one embodiment, the composition comprises acidic about 8 % upon 30 days at 25°C. In another embodiment, the composition comprises acidic about 7 % upon 30 days at 25°C.

[0020] In one embodiment, the purity of the composition under stress condition at 40°C is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97% ,98%, and 99% as determined by size exclusion high performance liquid chromatography (SEC-HPLC).

[0021] In one embodiment, the purity of the composition under stress condition at 25°C is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97% ,98%, and 99% as determined by size exclusion high performance liquid chromatography (SEC-HPLC).

[0022] In an embodiment, the composition maintains less than 0.5% high molecular weight (HMW) aggregates by weight after a freeze-thaw cycle. In an embodiment, the sugar is suitable to provide stability to the Thrombopoietin Receptor Agonist (TPO-RA) fusion protein and improves cake appearance of a lyophilized cake.

[0023] In an embodiment, the sugar is present in an amount in the range of about 130 mM to about 280 mM ; wherein the sugar is present in an amount in the range of about 50 mg / ml to about 105 mg / ml. In one embodiment, the sugar is present in an amount of 185mM.

[0024] In an embodiment, the composition exhibits a thermal stability characterized by one or more characteristics selected from denaturation of the Thrombopoietin Receptor Agonist (TPO-RA) fusion protein; wherein the temperature for denaturation ranges from about 50°C to about 80°C as measured by differential scanning fluorometry (DSF).

[0025] In one embodiment, the composition exhibits a thermal stability, having an onset temperature of about 50°C, a first melting temperature (Tml) of about 60°C, and a second melting temperature (Tm2) of about 77°C.

[0026] In an embodiment, the composition exhibits a thermal stability characterized by one or more characteristics selected from transition temperature for the Thrombopoietin Receptor Agonist (TPO-RA) fusion protein; wherein the transition temperature for Thrombopoietin Receptor Agonist (TPO-RA) fusion protein is in range of about 50°C to about 90°C as measured by differential scanning calorimetry (DSC).

[0027] In another embodiment, the composition exhibits a thermal stability characterized by differential scanning calorimetry (DSC), having a first thermal transition (Tl) of about 55°C and a second thermal transition (T2) of about 80°C.

[0028] In one embodiment, the pharmacologically active Thrombopoietin Receptor Agonist (TPO- RA) fusion protein is Romiplostim.

[0029] In an embodiment, the buffering agent is present in amount ranging from about 7mM to about 15mM. In one embodiment, buffering agent is present in an amount of lOmM. In an embodiment, the surfactant is present in an amount ranging from about 0.008mM to about 0.4 mM. In one embodiment, the surfactant is present in an amount of 0.03mM.

[0030] In an embodiment, the chelating agent is present in an amount ranging from about O.OOlmM to about 0.255mM. In one embodiment, the chelating agent is present in an amount of 0.002mM.

[0031] In an embodiment, the amino acid is present in an amount ranging from about 5mM to about 200mM. In another embodiment, the amino acid is present in an amount ranging from about 40mM to about lOOmM. In one embodiment, the amino acid is present in an amount of 47mM.

[0032] In an embodiment, the buffering agent is present in an amount ranging from about 0.83mg / ml to about 1.78mg / ml. In one embodiment, the buffering agent is present in an amount of 1.18 mg / ml.

[0033] In an embodiment, the pH of the buffer is about 4 to about 6. In one embodiment, the pH of the buffer is 5.

[0034] In an embodiment, the sugar is present in an amount ranging from about 50mg / ml to about 105mg / ml. In another embodiment, the sugar is present in an amount ranging from about 60mg / ml to 90mg / ml. In one embodiment, the sugar is present in an amount of 50mg / ml. In one embodiment, the sugar is present in an amount of 70mg / ml. In one embodiment, the sugar is present in an amount of 90mg / ml.

[0035] In an embodiment, the surfactant is present in an amount ranging from about O.Olmg / ml to about 0.5mg / ml. In another embodiment, the surfactant is present in an amount ranging from about 0.02mg / ml to about 0.04mg / ml. In one embodiment, the surfactant is present in an amount of 0.04 mg / ml.

[0036] In an embodiment, the chelating agent is present in an amount ranging from about 0.0004mg / ml to about O.lmg / ml. In one embodiment, the chelating agent is present in an amount of O.OOlmg / mL. In an embodiment, the amino acid is present in an amount ranging from about Img / ml to about 75mg / ml. In another embodiment, the amino acid is present in an amount ranging from about 5mg / ml to about 50mg / ml. In another embodiment, the amino acid is present in an amount ranging from about 9mg / ml to about 20mg / ml. In one embodiment, the amino acid is present in an amount lOmg / ml.

[0037] In an embodiment, the buffering agent is selected from sodium succinate, succinic acid, arginine acetate, citrate, acetate sodium phosphate, and combination thereof. In one embodiment, the buffering agent is sodium succinate.

[0038] In an embodiment, the single sugar is selected from trehalose, xylitol, maltose, arginine, raffinose. In one embodiment, the single sugar is trehalose.

[0039] In an embodiment, the surfactant is selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, pol oxamer 188 and combination thereof. In one embodiment, the surfactant is polysorbate 20.

[0040] In an embodiment, the chelating agent is selected from diethylenetriaminepentaacetic acid (DTP A), ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(P-aminoethyl ether)- N,N,N',N' -tetraacetic acid (EGTA), citric acid, gluconic acid, and glutathione. In one embodiment, the chelating agent is diethylenetriaminepentaacetic acid (DTP A).

[0041] In an embodiment, the amino acid is selected from arginine, lysine, methionine, glycine, proline, asparagine, phenylalanine, aspartic acid, glutamic acid and suitable salt thereof. In one embodiment, the amino acid is arginine HC1.

[0042] In one embodiment, a stable pharmaceutical composition comprises: a. 0.5mg / mL of pharmacologically active fusion protein binding to the thrombopoietin receptor (c-Mpl); b. at least 7mM to 15mM of the buffering agent; c. at least 130mM to about 280mM of the stabilizing agent; d. at least 0.008mM to 0.4 mM of the surfactant; e. at least O.OOlmM to 0.255mM of chelating agent; f. at least 5mM to 200mM of amino acid; and g. optionally comprises of about O.OOOlmM to about 300mM of an antioxidant.

[0043] In an embodiment, the antioxidant is present in an amount ranging from about 0.5mg / ml to about 3mg / ml.

[0044] In an embodiment, the antioxidant is selected from methionine, N-acetylcysteine, ascorbic acid, pyruvic acid, benzoic acid, methyl paraben, ethyl paraben, propyl paraben, butyl paraben, m- cresol, benzyl alcohol, phenoxyethanol, benzalkonium chloride, phenol, and combination thereof. In one embodiment, the antioxidant is methionine.

[0045] In an embodiment, the composition has the pH of about 4.5 to about 6.5. In one embodiment, the composition has the pH of 5±0.2.

[0046] In an exemplary embodiment, the composition is administered subcutaneously; and / or wherein the composition is administered subcutaneously through device selected from syringe, autoinjector, on body injector, pre filed syringe, and / or pen.

[0047] In an embodiment, a stable pharmaceutical composition, comprises: a. 0.5 mg / ml Romiplostim; b. 1.18mg / ml of sodium succinate; c. 70mg / ml of trehalose; d. 0.04mg / ml of polysorbate 20; e. 0.001 mg / ml of di ethylenetriaminepentaacetic acid (DTP A); f. 10 mg / ml of Arg. HC1; wherein the composition has the pH between 4.5 to 5.5; wherein the composition essentially consists of single sugar; wherein the composition of free of mannitol; wherein the composition is suitable for subcutaneous administration; wherein the composition has a purity of greater than 80% as determined by reverse-phase high performance liquid chromatography (RPLC), and / or wherein the composition has a purity of greater than 70% as determined by cation exchange high performance liquid chromatography (CEX-HPLC); wherein the purity of the pharmaceutical composition under stress condition at 40°C on day 30 is greater than 85%; and / or wherein the purity of the pharmaceutical composition under accelerated condition at 25°C on day 30 is greater than 80%; wherein the purity is measured by cation exchange high performance liquid chromatography (CEX-HPLC); wherein the purity of the pharmaceutical composition under stress condition at 40°C on day 21 is greater than 85%; and / or wherein the purity of the pharmaceutical composition under accelerated condition at 25°C on day 21 is greater than 80%; wherein the purity is measured by reversed-phase liquid chromatography (RPLC); and / or wherein the composition comprises less than 10% acidic variants as determined by cation exchange high performance liquid chromatography (CEX-HPLC); and / or wherein the purity of the composition under stress condition at 40°C is greater than 90% as determined by size exclusion high performance liquid chromatography (SEC-HPLC); and / or wherein the purity of the composition under accelerated condition at 25°C is greater than 90% as determined by size exclusion high performance liquid chromatography (SEC-HPLC).

[0048] In one embodiment, the stable pharmaceutical composition is lyophilized. In another embodiment, the stable pharmaceutical composition is a liquid composition.

[0049] In an embodiment, a method of preparing the stable pharmaceutical composition comprising a pharmacologically active TPO-RA fusion protein is disclosed. The method for preparing said comprises: a) preparing a bulk liquid solution comprising the TPO-RA fusion protein and pharmaceutical excipients; b) filling the bulk liquid composition into vials for lyophilization; c) subjecting the vials to a lyophilization process, wherein the lyophilization method comprises the steps as described in Table A.

[0050] In an embodiment, a method of preparing the stable pharmaceutical composition comprising a pharmacologically active TPO-RA fusion protein is disclosed. The method for preparing said comprises: a) preparing a bulk liquid solution comprising the TPO-RA fusion protein and pharmaceutical excipients; b) filling the bulk liquid composition into vials for lyophilization; c) subjecting the vials to a lyophilization process, wherein the lyophilization method comprises the steps as described in Table Al.

[0051] In an embodiment, a method for preparing a lyophilized pharmaceutical composition comprising a pharmacologically active TPO-RA fusion protein, the method comprising : a) preparing a bulk liquid solution comprising the TPO-RA fusion protein and one or more pharmaceutical excipients; b) filling the bulk liquid composition into vials for lyophilization; c) subjecting the vials to a lyophilization method, wherein the lyophilization method comprises the steps: i. performing a loading of said vials at a temperature ranging from about 20°C to about 5 °C for a period of about 25 minutes to about 55 minutes; ii. performing cooling of said vial comprising liquid composition in a lyophilization chamber to a temperature in a range of about from 0°C to about -60°C with holding; wherein the holding is performed at temperature ranging from 0°C to about -60°C for a period in a range of about 1.5 hours to about 20 hours; iii. performing an annealing step with hold at a temperature -13°C to -20°C over a period in range of about 3 hours to about 10 hours; iv. performing a primary drying; wherein the primary drying comprises heating the chamber to a temperature in a range of about -60°C to about 0°C and holding at said temperature at a pressure in a range of about 10 mTorr to about 150 mTorr for a period in a range of about 0.05 hours to about 50 hours to produce a primary dried composition; and v. performing a secondary drying , wherein the secondary drying comprises heating the chamber to a temperature in a range of about 0°C to about 30°C and holding at said temperature at a pressure in a range of about 10 mTorr to about 100 mTorr for a period in a range of about 0.5 hours to about 30 hours to produce the lyophilized cake suitable for pharmaceutical use. In one embodiment, the temperature before holding in step (ii) in step (iv), and in step (v) gradually decreases at a rate of about 0.1°C / min to about 1.0 °C / min. In another embodiment, the temperature before holding in step (ii) in step (iv), and in step (v) gradually decreases at a rate of more than about 0.2°C / min to about 0.8 C / min. In another embodiment, the temperature before holding in step (ii) in step (iv), and in step (v) gradually decreases at a rate of more than about 0.3°C / min to about 0.6 C / min.

[0052] In one embodiment, the holding in step (ii) is performed at temperature from about -45° C to about -55° C.

[0053] In one embodiment, the holding in step (iii) is performed at temperature below -13°C over a period of less than about 6 hours.

[0054] In one embodiment, the holding in step (iv) is performed at temperature from about -55° C to about -25°C.

[0055] In one embodiment, the bulk liquid is present in a suitable concentration in said vials. In another embodiment, the bulk liquid concentration in the vial is selected from about 0.125mg / ml, or 0.25 mg / ml and 0.5 mg / ml.

[0056] In one embodiment the invention analyses the purity, thermal stability and stability of the resulting composition.

[0057] BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure : 1 Graphical representation of % Purity for Stability under stress condition 40 °C as analysed by CEX-HPLC for present composition Cl

[0059] Figure: 1 A Graphical representation of % total charged variants under stress condition (40°C) through CEX-HPLC for present composition Cl

[0060] Figure :2 Graphical representation of % Purity for Stability under stress condition 40 °C as analysed by RPLC for present composition Cl

[0061] Figure :3 Graphical representation of % Purity for Stability under accelerated condition 25°C as analysed by CEX-HPLC for present composition Cl Figure :3 A Graphical representation of % acidic variants under accelerated condition 25°C as analysed by CEX-HPLC for present composition Cl

[0062] Figure :4 Graphical representation of % Purity for Stability under accelerated condition 25°C as analysed by RPLC for present composition Cl

[0063] Figure :5 Graphical representation of %Purity under stress condition (40°C) through CEX-HPLC

[0064] Figure: 5 A Graphical representation of % acidic charged variants under stress condition

[0065] (40°C) through CEX-HPLC for different compositions

[0066] Figure :6 Graphical representation of %Purity under stress condition (40°C) through RP LC

[0067] Figure :7 Graphical representation of %Purity under accelerated condition (25°C) through CEX-HPLC

[0068] Figure :7A Graphical representation of % acidic charged variants under accelerated condition (25 °C) through CEX-HPLC

[0069] Figure :8 Graphical representation of %Purity under accelerated condition (25°C) through RPLC

[0070] DESCRIPTION OF INVENTION:

[0071] The terms used in this specification generally have their ordinary meanings in the art, within the context of the invention, and in the specific context where each term is used. Certain terms that are used to describe the invention are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner regarding the description of the invention. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification including examples of any terms discussed herein is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified term. The invention is not limited to the various embodiments given in this specification. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the case of conflict, the present document, including definitions will control. As used herein, the following terms shall have the meanings as indicated below unless otherwise specified or required by context:

[0072] As used throughout the specification and in the appended claims, the singular forms “a,” “an,” and “the” include the plural reference unless the context clearly dictates otherwise. The phrase “consists essentially of,” or variations such as “consist essentially of’ or “consisting essentially of,” as used throughout the specification and claims, indicate the inclusion of any recited elements or group of elements, and the optional inclusion of other elements of similar or different nature than the recited elements, that do not materially change the basic or novel properties of the specified dosage regimen, method, or formulation. As a non-limiting example, a binding compound that consists essentially of a recited amino acid sequence may also include one or more amino acids, including substitutions of one or more amino acid residues, that do not materially affect the properties of the binding compound. “Comprising” or variations such as “comprise”, “comprises” or “comprised of’ or “comprising of’ are used throughout the specification and claims in an inclusive sense, i.e., to specify the presence of the stated features but not to preclude the presence or addition of further features that may materially enhance the operation or utility of any of the embodiments of the invention, unless the context requires otherwise due to express language or necessary implication.

[0073] The term “about”, as used herein, is intended to refer to ranges of approximately 5% greater than or less than the referenced value. In certain circumstances, one of skill in the art will recognize that, due to the nature of the referenced value, the term “about” can mean more or less than a 5% deviation from that value.

[0074] The term “and / or” used herein is referred as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0075] The use of the alternative (e.g., “or”) should be understood to mean either one, both, and any combination thereof of the alternatives. As used herein, the indefinite articles “a” or “an” should be understood to refer to “one or more” of any recited or enumerated component.

[0076] The term “fusion protein” refers to proteins created through the joining of two or more genes that originally coded for separate proteins. The fusion proteins are made using recombinant DNA techniques. Fusion protein consisting of receptor including but not limited to selected from Thrombopoietin Receptor Agonist (TPO-RA), CTLA4, GLP-1, TNFR, VEGF, HER-2, PCSK9 fused with constant region of immunoglobulin selected from IgGl, IgG2, IgG3 and IgG4. The term “peptibody” refers to a type of engineered therapeutic protein that combines a biologically active peptide with the fragment crystallizable (Fc) domain of an antibody. This design is used to extend the peptide's short half-life and improve its therapeutic effectiveness. The term peptibody and fusion protein may be used interchangeably in the specification.

[0077] The term “Thrombopoietin Receptor Agonist” or “TPO-RA” or “TPO mimetic molecule” or “Peptibody” are used interchangeably and refer to a protein molecule that comprises at least a polypeptide having a thrombopoietin receptor domain or portion thereof and an immunoglobulin constant region or portion thereof. In an embodiment, the TPO-RA is Romiplostim. Romiplostim (Nplate) is a thrombopoietin receptor agonist (TPO-RA). A member of the TPO mimetic class, is an Fc-peptide fusion protein (peptibody). The peptibody molecule contains two identical single-chain subunits, each consisting of human immunoglobulin IgGl Fc domain, covalently linked at the C-terminus to a peptide containing two thrombopoietin receptor-binding domains.

[0078] The term used “Size variants” refers to LMW, HMW or aggregates.

[0079] The term used “low molecular weight” or “LMW” which is a protein backbone-truncated fragments & considered as product-related impurities that contribute to the size heterogeneity of protein molecule. LMW species often have low or substantially reduced activity relative to the monomeric form of the protein molecule and can lead to immunogenicity or potentially impact pharmacokinetic properties in vivo. As a result, LMW species are considered critical quality attributes that are routinely monitored during drug development and as part of release testing of purified drug product during manufacturing.

[0080] The term used “High Molecular Weight” or “HMW” is product-related impurities that contribute to the size heterogeneity of protein molecule. The formation of HMW species within a therapeutic protein drug product as a result of protein aggregation can potentially compromise both drug efficacy and safety (e.g., eliciting unwanted immunogenic response). HMW is considered critical quality attributes that are routinely monitored during drug development and as part of release testing of purified drug product during manufacturing.

[0081] The term used “Aggregates” are classified based on types of interactions and solubility. Soluble aggregates are invisible particles and cannot be removed with a filter. Insoluble aggregates can be removed by filtration and are often visible to the human eye. Both types of aggregates cause problems in biopharma development. Covalent aggregates arise from the formation of a covalent bond between multiple monomers of a given peptide. Disulfide bond formation of free thiols is a common mechanism for covalent aggregation. Oxidation of tyrosine residues can lead to formation of tyrosine which often results in aggregation. Reversible protein aggregation typically results from weaker protein interactions they include dimers, trimers, multimers among others.

[0082] The term used “Acidic Variant”, or “Acidic species” and “AV” refer to the variants of a protein, which are characterized by an overall acidic charge. Acidic variants are formed through Chemical and enzymatic modifications such as deamidation and sialylation, respectively, result in an increase in the net negative charge on the protein molecule and cause a decrease in pl values, thereby leading to formation of acidic variants. C-terminal lysine cleavage results in the loss of net positive charge and leads to acidic variant formation. Another mechanism for generating acidic variants is the formation of various types of covalent adducts, e.g. glycation, where glucose or lactose can react with the primary amine of a lysine residue during manufacturing in glucose-rich culture media or during storage if a reducing sugar is present in the formulation. MAbs. 2010 November-December; 2(6): 613-624.

[0083] The term “composition” as used herein with reference to a formulation, e.g., lyophilised vial and / or an aqueous formulation, that it is useful for treating a disease or disorder. In an embodiment, the pharmaceutical formulation refers to preparations which are in such a form as to permit the biological activity of the active ingredients to be effective and therefore may be administered to a subject for therapeutic use.

[0084] The term “Stable pharmaceutical Composition” or “Stable Pharmaceutical Formulation” or “pharmaceutical composition” or “composition” or “ formulation” used interchangeably used herein refers to the protein formulation, essentially retains its physical stability and / or chemical stability and / or biological activity upon storage. Various analytical techniques for measuring protein stability are available in the art for example. In one embodiment, the stability of the protein is determined according to the percentage of monomer protein in the solution, with a low percentage of degraded (e.g., fragmented) and / or aggregated protein. An example, a "stable" formulation may be one that comprises less than about 10% and preferably less than about 5% of the protein is present as an aggregate in the formulation, “stable” formulation is one in which the protein therein essentially retains its physical and chemical stability and integrity upon storage. Various analytical techniques for measuring protein stability are available in the art. Stability can be measured at a selected temperature for a selected period. The term “lyophilization” used herein refers to refers to a dehydration process performed under reduced pressure and temperature to preserve a material. The process generally includes, but is not limited to, the steps of: freezing the material to a temperature below its eutectic or glass transition point; primary drying, where water is removed via sublimation of the ice under a vacuum; and secondary drying, where residual, non-frozen bound water is removed via desorption to achieve a target moisture content, typically less than 4% (w / w). This term is intended to encompass any variant of this process that achieves a stable, dry solid state, including processes that modify temperature or pressure protocols. In lyophilization (freeze- drying), the ramp rate is the controlled speed (e.g., °C / minute) at which the shelf temperature (Ts) is increased or decreased during freezing or drying, critically impacting ice crystal size, cake structure, and drying time; faster rates can sometimes lead to smaller, uniform ice crystals (good for stability) or even collapse (bad), while slower rates allow larger crystals, but can be less efficient, with the optimal rate depending heavily on the product's formulation and the freeze-dryer's capabilities. The temperature can be gradually increase or decrease at controlled rate.

[0085] The term "Lyoprotectant" used herein refers to a molecule added to the formulation combines with a protein of interest, significantly prevents or reduces chemical and / or physical instability of the protein upon lyophilization and subsequent storage. Examples include lyoprotectants such as sugars, amino acids, salts and polyols.

[0086] The term “lyophilized cake” used herein refers to the appearance of the cake formed at the end of lyophilization process that is intact, white to off white in color and is stable.

[0087] It should be understood that when describing a range of values in various embodiments below, the characteristic being described could be an individual value found within the range. For example, "a pH from about pH 4 to about pH 7," could be, but is not limited to, pH 4, 4.2, 4.6, 5.1, 5.5 etc. and any value in between such values. Additionally, "a pH from about pH 4 to about pH 7," should not be construed to mean that the pH of a formulation in question varies 3 pH units in the range from pH 4 to pH 7 during storage, but rather a value may be picked in that range for the pH of the solution, and the pH remains buffered at about that pH.

[0088] When the term "about" is used, it means the recited number plus or minus 5%, 10%, 15% or more of that recited number. The actual variation intended is determinable from the context. The term "Reconstituted composition / formulation” used herein refers to a solution prepared by dissolving a lyophilized protein formulation in a diluent such that the protein is dispersed in the reconstituted formulation. The reconstituted formulation in suitable for parenteral administration or subcutaneous administration.

[0089] The term "Bulking agent" used herein refers to a compound which adds mass to the lyophilized mixture and contributes to the physical structure of the lyophilized cake (e.g. facilitates the production of an essentially uniform lyophilized cake which maintains an open pore structure). Exemplary bulking agents include glycine, polyethylene glycol and sorbitol.

[0090] The term “Surfactant” used herein is a surface-active molecule containing both a hydrophobic portion (e.g., alkyl chain) and a hydrophilic portion (e.g., carboxyl and carboxylate groups). Surfactant may be added to the formulations of the invention. Surfactants suitable for use in the formulations of the present invention include, but are not limited to, polysorbates (e.g., polysorbates 20 or 80); poloxamers (e.g., pol oxamer 188).

[0091] The term 'chelating agent' (or 'chelator' or 'sequestering agent') refers to a substance that possesses two or more electron-donating atoms that form coordinated bonds with a single metal ion. This term is an open-ended term and includes, but is not limited to, a compound, typically an organic compound, that forms stable, water-soluble complexes with metal ions, thereby removing those ions from solution or masking their chemical activity. Chelating agents suitable for use in present compositions include, but are not limited to ethylenediaminetetraacetic acid and its salts (e.g., EDTA) disodium, calcium disodium, citric acid and its salts (e.g., sodium citrate), tartaric acid, gluconic acid, sorbitol, (DTP A) diethylenetriaminepentaacetic acid, ethylene glycol tetraacetic acid, HEDP (1 -hydroxy ethylidene 1,1-diphosphonic acid), nitrilotriacetic acid, phosphonates, polyphosphates, and combinations thereof. The term encompasses the agent capable of forming a stable coordination complex with a metal ion.

[0092] The term “Viscosity” refers to the resistance of a liquid formulation to flow, such as when injected through a syringe needle during administration to a patient. Viscosity of a protein solution depends on the nature of the individual and protein-protein interaction (PPI). Both of the individual characteristics, such as particle size and shape, as well as the pair interactions can be influenced by components in the formulation. As a result, it is often desirable to reduce viscosity values so that formulations are suitable for a particular application or process (i.e., injection). Hence, it is desirable to keep viscosity low in formulation as it increases glide force and break loose force of syringe. Furthermore, it is known that viscosity causes severe pain to patient during injection. In addition, high viscosity causes loss of product as viscous protein tends to bind to container closure. The term “Osmolality” refers to a measure of solute concentration, defined as the number of moles of solute per kg of solution. A desired level of osmolality can be achieved by the addition of one or more stabilizer such as a sugar or polyol including sorbitol, mannitol, dextrose, raffinose, trehalose, maltose, mannose.

[0093] The term “sugar” as used herein refers to carbohydrates such as monosaccharides, disaccharides, and polysaccharides. Examples of sugars include, trehalose, maltose, raffinose dextrose, maltose and polyols such as mannitol, xylitol and sorbitol. The terms 'a single sugar' or 'the single sugar' as used herein refer to a composition comprising one and only one type of saccharide compound, to the exclusion of other saccharide compounds, which acts as the stabilizing agent for the fusion protein formulation.

[0094] The term “fermentor” and understood by a person skilled in the art as interchangeable with 'bioreactor,' refers to an industrial vessel configured to provide a controlled environment for the cultivation of microorganisms (e.g., Escherichia. Coli (E.coli)\ thereby enabling the production of a target product, such as a fusion protein.

[0095] The term “cultured cells” as used herein refers to the microorganisms grown within the controlled environment of the fermentor. As known to a person skilled in the art, this term is used to describe a population of living cells maintained in a controlled, artificial environment (a culture medium) under conditions that promote their proliferation and the expression of the target protein. The phrase "cultured cells" is used broadly within the art to encompass both microbial cells (like bacteria and yeast used in fermentation) and mammalian cells (like CHO or HEK cells used in bioreactors) that are being grown to produce biological products.

[0096] The term “inclusion bodies” as used herein refers to intracellular, insoluble aggregates of overexpressed recombinant proteins that accumulate within microbial expression system, during heterologous expression. These aggregates are composed primarily of misfolded or partially folded polypeptides that lack biological activity and require solubilization and subsequent refolding to yield the functional protein.

[0097] The term “buffer exchange” refers to the process of substituting the solution surrounding a protein with a different buffer system. This is typically achieved by diafiltration or repeated concentration and dilution cycles to prepare the protein for downstream purification, refolding, or formulation. The “solubilization buffer” as used herein is a chaotropic solubilization buffer comprising at least one chaotropic agent, at least one chelating agent, at least one buffering agent / solvent and combination thereof, as known to the skilled person.

[0098] The “refolding buffer” as used herein comprises at least one buffering agent, at least one stabilizer, at least one denaturant, at least one additive, at least one reducing agent and combination thereof, as known to the skilled person.

[0099] The term “EQBT’as used herein refers to equilibration bufferl. The EQB I comprise at least one buffering agent and at least one ionic salt, as known to skilled person.

[0100] The term “drug substance” or “DS” refers to the purified, concentrated, and formulated recombinant protein that constitutes the active pharmaceutical ingredient (API), suitable for sterile filtration, bulk storage, and further processing into a finished drug product.

[0101] As used herein, the terms "Ultrafiltration" or "UF" refer to a pressure-driven membrane separation process that separates components based on molecular weight and size using a semi- permeable membrane. The membrane typically retains macromolecules, such as the fusion protein of interest, while allowing the solvent and small solute molecules (salts, small impurities) to pass through as permeate. UF is typically employed to increase the concentration of the target protein solution by removing volume, a process often referred to as concentration.

[0102] As used herein, the term "Diafiltration" or "DF" refers to a specialized application of filtration used to remove, replace, or lower the concentration of small molecular weight components (e.g., salts, solvents, existing buffer ions) from a solution containing the large target molecules (e.g., the fusion protein). This process involves continuously or sequentially adding a new solvent or buffer (diafiltration buffer) to the protein solution (retentate) at approximately the same rate as the permeate is removed, thus exchanging the buffer while maintaining a relatively constant volume of the retained protein solution.

[0103] The skilled person recognizes that both UF and DF are typically performed using Tangential Flow Filtration (TFF) systems, where the fluid flows tangentially across the membrane surface to minimize membrane fouling and maximize efficiency. A standard UF / DF sequence involves an initial concentration step (UF), followed by a buffer exchange step (DF), and optionally a final concentration step (UF). The specific operational parameters, such as membrane molecular weight cut-off (MWCO), transmembrane pressure (TMP), and number of diavolumes (DV) exchanged, are well-known to the skilled artisan and can be optimized through routine experimentation for a specific fusion protein product.

[0104] The term “eutectic temperature” used herein is the lowest possible melting point for a specific mixture of two or more substances. At this precise temperature and specific composition (known as the eutectic point or eutectic composition), the mixture melts or solidifies completely and sharply, behaving like a pure substance rather than a mixture

[0105] The term 'size exclusion high performance liquid chromatography' or 'SEC-HPLC as used herein refers to a mode of chromatography used to separate molecules (typically proteins or peptides) in solution based on their hydrodynamic size (size and shape). The separation is achieved by the differential exclusion of solute molecules from pores within the stationary phase of a chromatography column. The term is an open-ended term and includes, but is not limited to, methods utilizing various column chemistries, mobile phases, flow rates, and detection methods (e.g., UV absorbance detection, light scattering, refractive index detection), provided that the method effectively separates species based on size or molecular weight. SEC- HPLC is typically used to determine the proportion of aggregates, fragments, monomers, and other size-variant impurities within a composition

[0106] The term 'cation exchange high performance liquid chromatography' or 'CEX-HPLC as used herein refers to a mode of chromatography used to separate molecules (typically proteins or peptides) in solution based on differences in their net surface electrostatic charge at a given pH. The separation is achieved by differential binding of solute molecules to a negatively charged stationary phase within a chromatography column, followed by elution using changes in mobile phase ionic strength or pH. The term is an open-ended term and includes, but is not limited to, methods utilizing various column chemistries, buffers (e.g., acetate, phosphate, histidine), salt gradients (e.g., NaCl), flow rates, and detection methods (e.g., UV absorbance detection). CEX-HPLC is typically used to determine the proportion of acidic, basic, or neutral charged variants within a composition.

[0107] The term “bulk liquid” used herein refers to the solution at various intermediate stages of manufacturing, purification, and potentially the initial reconstitution step, before it is dispensed into individual vials and lyophilized for stability and storage.

[0108] The term 'differential scanning fluorimetry' or 'DSF' (also known as the thermal shift assay or (TSA) as used herein refers to a biophysical technique used to measure the thermal stability of a molecule, typically a protein, in solution. The method assesses stability by monitoring changes in the intrinsic or extrinsic fluorescence of the molecule as the temperature is systematically increased. The term is an open-ended term and includes, but is not limited to, methods utilizing various extrinsic fluorescent dyes (e.g., SYPRO Orange), different instrumentation platforms (e.g., qPCR machines, dedicated plate readers), varying heating rates, buffer conditions, and sample concentrations, provided that the method effectively yields a melting temperature (Tm) or an onset temperature (To) value. DSF is typically used to assess the binding of ligands or excipients to a target molecule, or to determine optimal formulation conditions that enhance stability. The first melting temperature (Tml) as used herein refers to the melting temperature at which the fusion protein firsts start to denature, and the second melting temperature (Tm2) is the melting temperature at which the fusion protein further denatures, higher the melting temperature more thermostable protein.

[0109] The term 'differential scanning calorimetry' or 'DSC as used herein refers to a thermo analytical technique in which the difference in the amount of heat required to increase the temperature of a sample and an inert reference is measured as a function of temperature. Both the sample and reference are maintained at nearly the same temperature throughout the experiment. The term is an open-ended term and includes, but is not limited to, methods utilizing various instrument designs (e.g., power-compensation DSC, heat-flux DSC), varying heating or cooling rates, sample pans, purge gases, and processing conditions, provided that the method effectively measures a thermal transition (Thermal transition I and thermal transition II). DSC is typically used to assess the thermal properties, purity, and stability of a material.

[0110] It is understood that the skilled artisan can adjust the specific ranges for onset temperature, first melting temperature (Tml), second melting temperature (Tm2), thermal transition I and thermal transition II, based on routine optimization and the specific formulation parameters. Any such variations in range or value are within the contemplation of this disclosure and the scope of the appended claims.

[0111] The term "Pharmaceutical use" as used herein generally refers to the application of a substance as a medicine for the diagnosis, prevention, treatment, or cure of diseases in humans or animals. In the specific context of a TPO-RA (thrombopoietin receptor agonist) protein such as romiplostim (Nplate), its pharmaceutical use is precisely defined as a targeted therapeutic intervention for the treatment of thrombocytopenia (low blood platelet counts), particularly chronic immune thrombocytopenia (ITP) in patients who have responded inadequately to conventional therapies. This use involves administering the protein to stimulate the TPO receptors on bone marrow cells, thereby increasing the production of platelets to normal levels, reducing the risk of clinically relevant bleeding events, and improving patient outcomes.

[0112] The present invention disclosed herein provides a stable pharmaceutical composition formulated to address the need for enhanced stability of therapeutic proteins. The composition comprises an effective amount of a therapeutic protein (e.g., (TPO-RA fusion protein), in combination with a suitable buffer, a suitable stabilizer, at least one chelating agent, an amino acid, and a suitable surfactant. In specific embodiments, the composition disclosed herein exhibit advantageous viscosity profiles and maintain the protein's integrity by preventing the formation of aggregates.

[0113] The stable pharmaceutical formulation of the present invention is mainly useful for the treatment of but not limited to patients with immune thrombocytopenia (ITP), and patients acutely exposed to myelosuppressive doses of radiation. In an embodiment, the pharmacology active mimetic class of fusion protein increases platelet production through binding and activation of the TPO receptor, a mechanism analogous to endogenous TPO for the treatment of thrombocytopenia.

[0114] An embodiment of the present invention provides a stable pharmaceutical composition that is stable for at least two weeks to two years. In an embodiment, the present invention is stable even at the stress conditions. The stable pharmaceutical composition of the present invention comprises: a. a pharmacologically active fusion protein; b. a suitable buffering agent; c. a suitable stabilizing agent.

[0115] In an embodiment, the stable pharmaceutical composition comprises: a. at least 0.5 mg / ml pharmacologically active Thrombopoietin Receptor Agonist (TPO- RA) fusion protein; b. a suitable buffering agent; c. a suitable stabilizing agent; d. a suitable surfactant.

[0116] In an embodiment, the stable pharmaceutical composition comprises: a. at least 0.5 mg / ml pharmacologically active Thrombopoietin Receptor Agonist (TPO- RA) fusion protein; b. a suitable buffering agent; c. a suitable stabilizing agent; d. a suitable surfactant; e. a suitable chelating agent; and f. a suitable amino acid.

[0117] The formulation of the present invention provides stability of fusion protein for at least two weeks. In an embodiment, the formulation is stable for at least 1 months, 3 months, 6 months, 10 months, 1 year or at least 2 years .

[0118] In certain embodiment, the formulations of the present invention are suitable for subcutaneous administration.

[0119] In certain embodiment, the present invention is stable at room temperature. In certain embodiment, the novel formulations of the present invention are stable at 2°C to 8°C.

[0120] In certain embodiment, the novel formulations of the present invention are stable at 40°C.

[0121] The formulation provides stability of fusion protein for at least two weeks to one month.

[0122] In an embodiment, the pharmacologically active fusion protein binds to the thrombopoietin receptor (c-Mpl). The pharmacologically active fusion protein is a Thrombopoietin Receptor Agonist (TPO-RA) fusion protein. The concentration of the Thrombopoietin Receptor Agonist (TPO-RA) fusion protein in the present composition is selected from about 0.125mg / ml, or 0.25 mg / ml and 0.5 mg / ml.

[0123] In an embodiment, the pharmacological active Thrombopoietin Receptor Agonist (TPO-RA) fusion protein is present in concentration at least 125 mcg / 0.25ml, 250mcg / 0.5ml, 500 mcg / ml. In certain embodiment, the pharmacological fusion protein is present in concentration is at least 0.125 mg / ml. In certain embodiment, the pharmacological fusion protein is present in concentration is selected from 0.125 mg / ml, 0.25 mg / ml, and 0.5 mg / ml. In certain embodiment, the pharmacological fusion protein is present in 125 mcg, 250 mcg or 500 mcg as a lyophilized powder in single-dose vials.

[0124] In an embodiment, the concentration of the Thrombopoietin Receptor Agonist (TPO-RA) fusion protein in the present composition is preferably 0.5 mg / ml. In certain embodiment, the Thrombopoietin Receptor Agonist (TPO-RA) fusion protein is Romiplostim.

[0125] In an embodiment, the composition comprises of a suitable buffering agent to maintain the pH in range of 4.5-5.5, preferably 5, providing long term stability to the Thrombopoietin Receptor Agonist (TPO-RA) fusion protein. The suitable buffering agent includes but does not limit to sodium succinate, succinic acid, arginine acetate, citrate, acetate, sodium phosphate, and combinations thereof.

[0126] In an embodiment, the buffering agent is present in the composition at a concentration sufficient to provide adequate buffering capacity. This concentration effectively stabilizes the Thrombopoietin Receptor Agonist (TPO-RA) fusion protein under extreme storage conditions as well and during administration.

[0127] In an embodiment, the buffer is concentration is in range from about ImM to about 200mM. In an embodiment, the buffer is concentration in the range of about ImM, about 2mM, about 3mM, about 4mM, about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about lOmM, about l lmM, about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 17mM, about 18mM, about 19mM, about 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM to 60mM, 60mM to 70mM, 70mM to 80mM, 80mM to 90mM, 90mM to lOOmM, lOOmM to HOmM, HOmM to 120mM, 120mM to 130mM, 130mM to 140mM, 140mM to 150mM, 150mM to 160mM,160mM to 170mM, 170mM to 180mM, 180mM to 190mM, and 200mM.

[0128] In certain embodiment, the buffer concentration is at least 0.8mg / ml, 1 mg / ml. In certain embodiment, the buffer concentration is in the range of 1 mg / ml to 20 mg / ml. In certain embodiment, the buffer concentration is in the range of 1 mg / ml to 10 mg / ml. In certain embodiment, the buffer concentration is in the range of 1 mg / ml to 5 mg / ml. In certain embodiment, the buffer concentration selected from about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, about 10 mg / ml, about 11 mg / ml, about 12 mg / ml, about 13 mg / ml, about 14 mg / ml, about 15 mg / ml, about 16 mg / ml, about 17 mg / ml, about 18 mg / ml, about 19 mg / ml, and about 20 mg / ml.

[0129] In specific embodiments, the concentration of the buffering agent is maintained in the range of at least 7 mM to 15 mM, corresponding to a concentration range of approximately 0.83 mg / mL to about 2.0 mg / mL. In an embodiment, the concentration of the buffering agent is lOmM.

[0130] In an embodiment, the concentration of the buffering agent is 1.18mg / ml.

[0131] In an embodiment, the composition comprises of at least one stabilizing agent, protecting the Thrombopoietin Receptor Agonist (TPO-RA) fusion protein degradation pathways, including aggregation and denaturation, thereby ensuring long-term stability and efficacy. The suitable stabilizing agents include sugars and polyols selected from the group consisting of trehalose, xylitol, maltose, arginine, raffinose, and combinations thereof.

[0132] In an embodiment, the stabilizing agent is present in a concentration sufficient to confer the desired stability. In specific embodiments, the concentration of the stabilizing agent is maintained in the range of at least 130mM to about 280mM, corresponding to a range of approximately 50 mg / mL to about 105 mg / mL. This concentration range is optimized to effectively stabilize the fusion protein under various storage and stress conditions.

[0133] In certain embodiment, the concentration of sugar at least about Img / ml. In certain embodiment, the concentration of sugar and / or polyol are selected from about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, about 10 mg / ml, about l lmg / ml, about 12mg / ml, about 13mg / ml, about 14mg / ml, about 15mg / ml, about 16mg / ml, about 17mg / ml, about 18mg / ml, about 19mg / ml, about 20mg / ml, about 21mg / ml, about 22mg / ml, about 23mg / ml, about 24mg / ml, about 25mg / ml, about 26mg / ml, about 27mg / ml, about 28mg / ml, about 29mg / ml, about 30mg / ml, about 31mg / ml, about 32mg / ml, about 33mg / ml, about 34mg / ml, about 35mg / ml, about 36mg / ml, about 37mg / ml, about 38mg / ml, about 39mg / ml, about 40mg / ml, about 41mg / ml, about 42mg / ml, about 43mg / ml, about 44mg / ml, about 45mg / ml, about 46mg / ml, about 47mg / ml, about 48mg / ml, about 49mg / ml, about 50mg / ml, about 51mg / ml, about 52mg / ml, about 53 mg / ml, about 54mg / ml, about 55 mg / ml, about 56mg / ml, about 57mg / ml, about 58mg / ml, about 59mg / ml, about 60mg / ml, about 61mg / ml, about 62mg / ml, about 63mg / ml, about 64mg / ml, about 65mg / ml, about 66mg / ml, about 67mg / ml, about 68mg / ml about 69mg / ml, about 70mg / ml, about 71mg / ml, about 72mg / ml, about 73mg / ml, about 74mg / ml, about 75mg / ml, about 76mg / ml, about 77mg / ml, about 78mg / ml, about 79mg / ml, about 80mg / ml, about 81mg / ml, about 82mg / ml, about 83mg / ml, about 84mg / ml, about 85mg / ml, about 86mg / ml, about 87mg / ml, about 88mg / ml, about 89mg / ml, about 90mg / ml, about 91mg / ml, about 92mg / ml, about 93mg / ml, about 94mg / ml, about 95mg / ml, about 96mg / ml, about 97mg / ml, about 98mg / ml, about 99mg / ml, about lOOmg / ml, about lOlmg / ml, about 102mg / ml, about 103mg / ml, about 104mg / ml, and about 105mg / ml.

[0134] In an embodiment, the sugar concentration is in range from about lOmM to about 300mM. In an embodiment, the sugar concentration selected from about lOmM, about 20mM, about 30mM, about 40mM, about 50mM, about 57mM, about 58mM, about 59mM, about 60mM, about 70mM, about 80mM, about 90mM, about lOOmM, about HOmM, about 120mM, about 130mM, about 140mM, about 150mM, about 160mM, about 170mM, about 180mM, about 190mM, about 200mM, about 210mM, about 220mM, about 230mM, about 240mM about 250mM, about 275mM, about 280mM, and about 300mM.

[0135] In certain embodiment, the concentration of sugar is from about 130mM to about 280mM.

[0136] In an embodiment, the concentration of the stabilizing agent is 50 mg / mL.

[0137] In an embodiment, the concentration of the stabilizing agent is 70 mg / mL.

[0138] In another embodiment, the concentration of the stabilizing agent is 90 mg / mL.

[0139] In one embodiment, the stabilizing agent in the present composition is trehalose.

[0140] The present application applicant found that succinate has suitable buffering property for fusion protein and provides better stability in combination with trehalose and surfactant.

[0141] In an embodiment, the composition comprises of a suitable surfactant to minimize the physical degradation of the fusion protein, specifically preventing surface-induced aggregation and denaturation during manufacturing, shipping, and storage. The suitable surfactants are selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, pol oxamer 188, and combinations thereof.

[0142] In an embodiment, the surfactant is present in an amount sufficient to provide adequate stabilization. In specific embodiments, the concentration of the surfactant is maintained in the range of 0.008 mM to 0.4 mM, corresponding to about O.Olmg / ml to about 0.5mg / ml. This concentration range is optimized to effectively stabilize the fusion protein formulation against agitation and interfacial stresses.

[0143] In an embodiment, the concentration of the surfactant is 0.04 mg / mL.

[0144] In an embodiment, the surfactant in the present composition is polysorbate 20.

[0145] In an embodiment, the surfactant in the present composition is poloxamer 188. In an embodiment, the present composition comprises a suitable chelating agent. The suitable chelating agents are selected from the group consisting of diethylenetriaminepentaacetic acid (DTP A), ethylenediaminetetraacetic acid (EDTA), citric acid, gluconic acid, glutathione, and combinations thereof.

[0146] In an embodiment, the chelating agent is present in a concentration sufficient to sequester trace metal ions and confer the desired stability. In specific embodiments, the concentration of the chelating agent is maintained in the range of about 0.001 mM to about 0.255 mM, corresponding to a range of approximately 0.0004 mg / mL to about 0.1 mg / mL. This low concentration range is effective in preventing metal -catalyzed degradation within the formulation.

[0147] In an embodiment, the concentration of the chelating agent is O.OOlmg / mL.

[0148] In an embodiment, the chelating agent in the present composition is diethylenetriaminepentaacetic acid (DTP A).

[0149] In an embodiment, the present invention comprises of at least an amino acid. The suitable amino acids are selected from arginine, lysine, methionine, glycine, proline, asparagine, phenylalanine, aspartic acid, glutamic acid & suitable salt thereof.

[0150] In an embodiment, the amino acid is present in a concentration ranging from about ImM to 200mM. In an embodiment, the concentration of antioxidant ranges from about 0.0001 mM, or 0.001 mM, or 0.01 mM, to about 100 mM, or up to about 200 mM, or up to about 300 mM.

[0151] In another embodiment, the amino acid is present in the concentration of at least 1 mg / ml. In certain embodiment, the amino acid present in the range of 1 mg / ml to 75 mg / ml. In certain embodiment, the amino acid present in the range of 5 mg / ml to 50 mg / ml. In certain embodiment, the amino acid present in range of 9 mg / ml to 50 mg / ml. In certain embodiment, the amino acid present in range of 9 mg / ml to 20 mg / ml.

[0152] In an embodiment, formulation optionally comprise antioxidant selected from methionine, N- acetylcysteine, ascorbic acid, pyruvic acid, benzoic acid, methyl paraben, ethyl paraben, propyl paraben, butyl paraben, m-cresol, benzyl alcohol, phenoxyethanol, benzalkonium chloride, or phenol. In an embodiment, the concentration of antioxidant ranges from about 0.0001 mM, or 0.001 mM, or 0.01 mM, to about 100 mM, or up to about 200 mM, or up to about 300 mM. In certain embodiment, the novel formulations of the present invention have pH 3.0 to pH 7.5. In certain embodiment, the novel formulations of the present invention have pH 3.5 to pH 7.0. In an embodiment, the novel formulations of the present invention have pH 3.0, pH 3.5, pH 4.0, pH 4.5, pH 5.0, pH 6.0, pH 6.5, pH 7.0, pH 7.5.

[0153] In preferred embodiment the pH is 5±0.2.

[0154] In certain embodiment, the novel formulations have viscosity of at least 1 cps. In certain embodiment, the formulation has viscosity of 5 cps to 15 cps. In certain embodiment, the formulation has viscosity of 7 cps to 15 cps. In certain embodiment, the formulation has viscosity of 9 cps to 15 cps.

[0155] In one aspect of such embodiment the viscosity is selected from leps, 2cps, 3cps, 4cps, 5cps, 6cps, 7cps, 8cps, 9cps, lOcps, Heps, 12cps, 13cps, 14cps and 15cps.

[0156] In certain embodiment, the formulation has viscosity of 1 cps to 9 cps.

[0157] In certain aspect the present invention discloses a desirable viscosity about leps to about 9cps when formulated thrombopoietin receptor agonist (TPO-RA) fusion protein in succinate buffer with trehalose and surfactant.

[0158] In certain aspect the present invention discloses a desirable viscosity about leps to about 9cps when formulated thrombopoietin receptor agonist (TPO-RA) fusion protein in succinate buffer with trehalose and / or mannitol and surfactant.

[0159] In certain embodiment the stable pharmaceutical novel liquid formulations have osmolality selected from 250 to 500 mOsm / kg. In an embodiment, the osmolality of a stable pharmaceutical formulation is selected from about 200 mOsm / kg, about 250 mOsm / kg, about 290 mOsm / kg, about 300 mOsm / kg, about 310 mOsm / kg, about 320 mOsm / kg, about 330 mOsm / kg, about 340 mOsm / kg, about 350 mOsm / kg, about 360 mOsm / kg, about 370 mOsm / kg, about 380 mOsm / kg, about 390 mOsm / kg, about 400 mOsm / kg, about 410 mOsm / kg, about 420 mOsm / kg, about 430 mOsm / kg, about 440 mOsm / kg, about 450 mOsm / kg, and about 500 mOsm / kg.

[0160] In certain aspect, the present invention discloses a desirable viscosity about 250mOsm / Kg to about 350 mOsm / Kg when formulated thrombopoietin receptor agonist (TPO-RA) fusion protein in succinate buffer with trehalose and surfactant at pH 5.0. In certain aspect, the present invention discloses a desirable viscosity about 250mOsm / Kg to about 350 mOsm / Kg when formulated thrombopoietin receptor agonist (TPO-RA) fusion protein in succinate buffer with trehalose and / or mannitol and surfactant at pH 5.0.

[0161] In certain aspect, the present invention discloses a desirable viscosity about 250mOsm / Kg to about 350 mOsm / Kg when formulated thrombopoietin receptor agonist (TPO-RA) fusion protein at pH 5.0.

[0162] In certain aspect, the present invention discloses a desirable viscosity about 250mOsm / Kg to about 350 mOsm / Kg when formulated thrombopoietin receptor agonist (TPO-RA) fusion protein in succinate buffer at pH 5.0.

[0163] In certain embodiment, the formulation has high monomer and low aggregation or HMWs and LMWs.

[0164] In certain embodiment, the formulation comprises HMW below 10% or below 9% or below 8% or below 7% or below 6% or below 5% or below 4% or below 3% or below 2% or below 1% or below 0.5% or below 0.4% or below 0.3% or below 0.2% or below 0.1%.

[0165] In certain embodiment, the formulation comprises LMW below 10% or below 9% or below 8% or below 7% or below 6% or below 5% or below 4% or below 3% or below 2% or below 1% or below 0.5% or below 0.4% or below 0.3% or below 0.2% or below 0.1%.

[0166] In certain embodiment, the formulation comprises charge variants (acidic) below 10% or below 9% or below 8% or below 7% or below 6% or below 5% or below 4% or below 3% or below 2% or below 1% or below 0.5% or below 0.4% or below 0.3% or below 0.2% or below 0.1%.

[0167] In certain embodiment, the formulation comprises monomer more than 90%. In certain embodiment the formulation provides monomer more than 95%, 96%, 97%, 98%, or 99%.

[0168] In an embodiment, the process of preparing said composition is disclosed. The process comprises steps of preparing a bulk liquid solution comprising the TPO-RA fusion protein and one or more pharmaceutical excipients; filling the bulk liquid composition into vials for lyophilization; subjecting the vials to a lyophilization process, wherein the lyophilization method comprises the steps as described in Table A.

[0169] In an embodiment, the lyophilization process to produce the present lyophilized cake is disclosed. The lyophilization process of the present invention comprises: a) freezing and annealing step, where the formulated composition is subjected to cooling in a lyophilization chamber containing the liquid composition of present invention to a temperature ranging from about 0° C to about -60° C to produce a frozen composition, and holding the lyophilization chamber at a temperature ranging from about 0° C to about -60° C, for a time period of about 1.5 hours to about 20 hours. This is followed by annealing step at temperature below -13°C, where the temperature in the annealing step is ramped down at a temperature below -13°C for about one hour, and is maintained at said temperature below -13°C for about less than 6 hours. b) primary drying step, where the lyophilization chamber is heated to a temperature ranging from about -60° C to about 0° C. The temperature in the lyophilized chamber is hold at about -60° C to about 0° C with pressure ranging from about 10 mTorr to about 150 mTorr for a time period of about 0.05 hours to about 50 hours, to produce a primary dried formulation. c) Secondary drying, where the lyophilization chamber is heated to a temperature ranging from about 0° C to about 30° C. The temperature in the lyophilized chamber is hold at about 0° C to about 30° C with pressure ranging from about 10 mTorr to about 100 mTorr for a time period of about 0.5 hours to about 30 hours to produce the present lyophilized formulation.

[0170] While the present invention has been described in detail with reference to specific embodiments thereof, it will be apparent to those skilled in the art that various changes and modifications can be made, and equivalents employed, without departing from the scope of the appended claims. The examples provided herein are intended solely to illustrate the invention and are not meant to be restrictive or limiting of the invention's full scope.

[0171] A fusion protein molecule capable to bind TPO RA fusion protein, expressed in bacterial cell line Escherichia. Coli (E. coli) is cultured in a fermentor, as per the process known to the skilled person and the protein is harvested from the cultured cells. The harvested protein is further purified as shown in below example:

[0172] Example 01: Recovery and Purification of fusion protein from Inclusion Bodies The fusion protein of interest that is Thrombopoietin Receptor Agonist TPO-RA, protein expressed in Escherichia coli as inclusion bodies (IBs) was recovered and purified using the process described herein.

[0173] The isolated IBs were suspended in a solubilization buffer containing 50mM Tris,7M GuHCl, 2mM EDTA, pH 8.0. The suspension was incubated at 25 °C with gentle agitation for 2 hours until complete solubilization was achieved.

[0174] The denatured protein solution was refolded by gradual dilution into refolding buffer containing 50 mM Tris-HCl, 0.4 M arginine, 2% sorbitol, 2 mM cysteine, and 0.2 mM cystine, pH 8.0. The solution was incubated at 4 °C for 18 hours with controlled mixing to allow proper folding. The refolded solution was concentrated to ~5 mg / mL using a 10 kDa MWCO ultrafiltration membrane and buffer-exchanged into 20 mM Tris-HCl, pH 6.5. A controlled pH shift was applied, resulting in selective precipitation of misfolded proteins. The mixture was clarified by depth filtration followed by sterile 0.22 pm filtration. The clarified protein was loaded onto an SP Sepharose FF column equilibrated with EQB I buffer, pH 5.0. The protein was eluted using acetate buffer and NaCl gradient from 125mM to 225mM for first Cation- Exchange I (CEX I). This was followed by Anion-Exchange (AEX) where the eluate was passed through a Q membrane operated in flow-through mode at pH 6.5 and conductivity 8.5 mS / cm, effectively removing host cell DNA and endotoxins. The protein was further polished on an SP Sepharose HP column equilibrated with EQB I buffer, pH 5.2, and eluted using an elution buffer gradient (90M to 150mM) for last Cation-Exchange II (CEX II).

[0175] The purified protein was concentrated to 2 mg / mL and buffer-exchanged into a formulation containing 10 mM Tris-HCl, pH 7.0.

[0176] The drug substance was sterile filtered filled into vials, and stored at -20 °C. The final product, Romiplostim demonstrated high purity and retained bioactivity as confirmed by in vitro assays.

[0177] Example 02: Process for preparing the stable TPO-RA fusion protein liquid formulation A solution containing the TPO-RA fusion protein as obtained from example 01, Romiplostim is subjected to a buffer exchange process using tangential flow filtration (TFF), specifically ultrafiltration / diafiltration (UF / DF). The diafiltration is performed using a 10 mM sodium succinate buffer adjusted to a pH 5. The retentate collected from filtering from 0.22-micron filter yields the protein at a target concentration of approximately 0.5 mg / mL. The protein concentrate (retentate) is then formulated into the final bulk solution. This step involves the addition and dissolution of a stabilizing agent (e.g., trehalose) and a surfactant (polysorbate 20). Further a chelating agent such as DTPA and amino acid Arginine HC1 were added to the mixture to achieve the desired concentrations specified for the final composition (Cl). The prepared formulation bulk is aseptically filled into vials. The vials are subsequently loaded into a lyophilizer (freeze-dryer) to remove the solvent (water) through a controlled freeze-drying cycle, resulting in a stable lyophilized cake.

[0178] Example 03: Process of preparing lyophilized formulation

[0179] A method for preparing a lyophilized pharmaceutical composition comprising a pharmacologically active Romiplostim and excipients, wherein the method comprising : a) Preparing a bulk liquid composition comprising Romiplostim and one or more excipients ; b) filling the bulk liquid composition into vials for lyophilization; c) subjecting the vials to a lyophilization, wherein the lyophilization method comprises the steps: i. performing a loading of said vials at a temperature ranging from about 20°C to about 5°C for a period of about 25 minutes to about 55 minutes; ii. performing cooling of said vial comprising liquid composition in a lyophilization chamber to a temperature in a range of about from 0°C to about - 60°C with holding; wherein the holding is performed at temperature ranging from 0°C to about -60°C for a period in a range of about 1.5 hours to about 20 hours; iii. performing an annealing step with hold at a temperature -13 to -20 °C over a period of less than about 8 hours; iv. performing a primary drying; wherein the primary drying comprises heating the chamber to a temperature in a range of about -60°C to about 0°C and holding at said temperature at a pressure in a range of about 10 mTorr to about 150 mTorr for a period in a range of about 0.05 hours to about 50 hours to produce a primary dried composition; and v. performing a secondary drying, wherein the secondary drying comprises heating the chamber to a temperature in a range of about 0°C to about 30°C and holding at said temperature at a pressure in a range of about 10 mTorr to about 100 mTorr for a period in a range of about 0.5 hours to about 30 hours to produce the lyophilized cake suitable for pharmaceutical use as in Table A R: Ramp and H: Hold

[0180] Table A: Steps of improved lyophilized cycle

[0181] Example 03-A

[0182] Filled vials which consists of non- siliconized 5 ml USP type I glass vials were filled with 1.24 mL of Formulation 2 solution and stoppered with a Fluro Tec® polymer coated stopper. For cycle development and analytical control purposes, formulation buffer was used throughout, referred to as S5T70AD (lOmM Sodium Succinate buffer + 70 mg / mL trehalose + 10 mg / mL Arg. HC1 + 0.001 mg / mL DTPA + 0.04 mg / mL P20). Sample vials containing Romiplostim drug product were also formulated in S5T70AD, with a protein concentration of 0.5 mg / mL.

[0183] For 0.25 mg / mL protein concentration , the 5 ml USP type I glass vials were filled with 0.76mL of solution whereas for 0.125 mg / mg protein concentration the 2 ml USP type I glass vials were filled with 0.48mL.

[0184] Improved Lyophilization Cycle

[0185] A liquid protein formulation was prepared as described above and introduced into a lyophilization chamber. The chamber was cooled from a loading temperature at about 5° C to about -50° C with annealing step. The annealing step was performed at about -15 °C with ramp rate of 0.6 °C / min. and hold for a time of about 5 hour. The holding of a chamber temperature was about -50 °C for about 6 hours. Primary drying occurred at a temperature of about -50°C to about -30 °C and at vacuum pressure 50 mTorr for a time period of about 22 to about 24 hour. Secondary drying occurred at 25° C after ramping up at a rate of 0.4° C / min and at vacuum pressure of 50 mTorr for time period of about 15 hour to about 17 hour.

[0186] To enable vial stoppering, the temperature of the chamber was lowered to 2-8°C, and the lyophilization chamber was aerated with nitrogen from about 200 Torr to about 500 Torr. The vial containing the lyophilized protein formulation was removed from the lyophilization chamber and stored at 2-8° C until further processing and analysis. The overall cycle time using the inventive cycle lasted for 57 hrs (precisely 56.58, 2 to 2.5 days) compared with a cycle time of more than 90 hrs using previous cycling conditions.

[0187] Table Al: Improved Lyophilization cycle for preparation of Lyophilized formulation comprising S5T70AD.

[0188] With a final time of about 56.58 hours, the improved lyophilization cycle of present invention represents an approximately 37.13% decrease in time compared to reference lyophilization cycles. Example 04:

[0189] The purified DS of fusion protein TPO is further formulated to prepare drug product.

[0190] Following compositions / formulations are prepared

[0191] Following compositions were prepared: Composition 1(C1)

[0192] Table 01: Composition 1(C1

[0193] Composition 2 (C2)

[0194] Table 02: Composition 2 (C2)

[0195] Composition 3 (C3) Table 03: Composition 3 (C3)

[0196] Composition 4 (C4) Table 04: Composition 4 (C4)

[0197] Composition 5 (C5)

[0198] Table 05: Composition 5 (C5)

[0199] Composition 6 (C6)

[0200] Table 06: Composition 6 (C6) Composition 7 (C7)

[0201] Table 07: Composition 7 (C7)

[0202] Composition 8 (C8)

[0203] Table 08: Composition 8 (C8)

[0204] Control formulation (CF1)

[0205] Table 09: Control formulation (CF1)

[0206] Control formulation 2 (CF2)

[0207] Table 10: Control formulation 2 (CF2)

[0208] Stability Analysis

[0209] The resulting compositions were analysed for thermal stability using Differential Scanning Fluorimetry (DSF), differential scanning calorimetry (DSC). The compositions were further analysed for stability under stress conditions at varied temperatures of 25°C and at 40°C. The compositions were also analysed for purity and charged variants. The results demonstrated that these compositions exhibited greater thermal stability, higher stability in stress conditions compared to the control formulation, indicating a robust and stable final product.

[0210] Example: 05

[0211] The composition of the present invention (Cl) was analysed for purity along side the control formulation (CF1). The composition of the present invention (Cl) was compared with the control comprising the present fusion protein along with the excipients of the conventional composition such as histidine buffer, sucrose, and mannitol. Said compositions were analysed for their purity through RPLC and CEX- HPLC. The results of the same are demonstrated in Table 11 and Table 12.

[0212] Table 11: % Purity by RPLC

[0213] Table 12: %Charged variant Purity by CEX-HPLC

[0214] As shown in Table 11 and Table 12, the present invention pertains to a stabilized protein formulation, wherein the control formulation utilizes excipients comprising histidine buffer, mannitol, and sucrose in combination with the TPO-RA. In contrast, the formulation (Cl) as disclosed in present specification replaces the histidine buffer with sodium succinate, and stabilizers as trehalose. Furthermore, the protein TPO-RA used in formulation (Cl) was eluted from the chromatographic column using acetate buffer along with NaCl as shown in example 2. Analytical assessment using RPLC revealed that purity of the protein improves upon formulation preparation (Cl) and comparatively higher purity was observed in the present formulation Cl. This is due to disrupting protein -protein interactions and solubilization of the oxidative impurities in the sodium succinate buffer of the present invention. The present formulation demonstrated improved purity, of 94.35% (CEX HPLC) in the purified protein, and 96.2% (CEX HPLC) in the liquid formulation. Protein formulations prepared with sodium succinate buffer demonstrated significantly higher purity percentages compared to formulations prepared with histidine buffer. Thus, it is evident that sodium succinate buffer in the present composition Cl stabilizes the protein TPO-RA.

[0215] Example: 06

[0216] The compositions of the present invention were analysed for stability under stress conditions through CEX-HPLC and RPLC under 40°C for at least 30 days and 21 days respectively. The results of the same are herein under:

[0217] Table: 13 Stability under stress condition 40 °C as analysed by CEX-HPLC

[0218] Table: 13A Total charged variants under stress condition 40 °C as analysed by CEX-HPLC

[0219] As evident from table 13 and figure 01 the control formulation (CF1) exhibited significant degradation under accelerated stress conditions for 40 °C 30 days, with initial purity declining from 86.10% to 78.30%. In contrast, the present composition Cl demonstrated a superior charged variant stability, maintaining a high initial purity 96.00% and only experiencing a minor decrease to 91.00% under the same conditions as measured by CEX-HPLC, thereby highlighting the superior stability profile conferred by the components of the present invention. As shown in table 13 A, the total charged variants as analysed under stress condition at 40°C by CEX-HPLC, the present composition Cl shows considerably less than 10% of total charged variants.

[0220] Table: 14 Stability under stress condition 40 °C as analysed by RPLC

[0221] As evident from table 14 and figure 02 the control formulation (CF1) exhibited significant degradation under stress conditions for 40 °C 21 days, with initial purity declining from 85.7% to 80.85%. In contrast, the present composition Cl demonstrated a superior stability, maintaining a high initial purity 83.01% and only experiencing a minor decrease to 82.54% under the same conditions as measured by RPLC, thereby highlighting the superior stability profile conferred by the components of the present invention.

[0222] Example 07

[0223] The compositions of the present invention were analysed for stability under stress conditions through CEX-HPLC and RPLC under 25°C for at least 30 days and 21 days respectively. The results of the same are herein under: Table 15: Stability under accelerated condition 25°C as analysed by CEX-HPLC

[0224] Table: 15A Total charged variants under accelerated condition 25°C as analysed by CEXHPLC

[0225] As evident from table 15 and figure 03, the control formulation (CF1) exhibited significant degradation under accelerated conditions for 25°C 30 days, with initial purity declining from 86.1% to 79.4%. In contrast, the present composition Cl demonstrated a superior stability, maintaining a high initial purity 96.0% and only experiencing a minor decrease to 91.1% under the same conditions as measured by CEX-HPLC, thereby highlighting the superior stability profile conferred by the components of the present invention. As shown in table 15 A, the total charged variants as analysed under accelerated condition 25°C by CEX-HPLC, the present composition Cl shows considerably less than 10% of total charged variants.

[0226] As evident from table 16 and figure 04, the control formulation (CF1) exhibited significant degradation under accelerated conditions for 25°C 21 days, with initial purity declining from 85.70% to 83.70%. In contrast, the present composition Cl demonstrated a superior stability, maintaining a high initial purity 83.01% and remains constant up to 21 days under the same conditions as measured by RPLC, thereby highlighting the superior stability profile conferred by the components of the present invention. Formulation discloses slow degradation compared to formulation with sucrose and mannitol.

[0227] Example 08: The stability profile of the present fusion protein was evaluated under accelerated stress conditions (40°C) through CEX-HPLC. The results of the same are herein under:

[0228] Table 17: Purity of fusion protein under stress condition (40°C) through CEX-HPLC

[0229] Table 17A: Acidic charged variants under stress condition (40°C) through CEX-HPLC

[0230] As shown in table 17 and figure 05, the control formulation, containing standard excipients, exhibited a notably less purity of 78.30% over the study duration of 30 days. Composition 2

[0231] (C2), incorporating varying concentrations of trehalose (90 mg / mL), demonstrated higher purity than the control formulation. Composition 3 (C3), utilizing a combination of trehalose and mannitol with the present fusion protein, resulted in a purity of 85.80% by day 30 under stress conditions. In contrast, Composition 1 (Cl) of the present invention maintained a purity of 91.00% on day 30 under the same stress condition, as measured by CEX-HPLC. This result highlights the superior stability profile conferred by the specific components of the present invention, specifically utilizing a single sugar and being substantially free of mannitol. As shown in table 17 A, the total charged variants as analysed under stress condition at 40°C by CEX-HPLC, the present composition Cl shows considerably less than 10% of acidic variants.

[0232] Example 09:

[0233] The stability profile of the present fusion protein was evaluated under accelerated stress conditions (40°C) through RPLC. The results of the same are herein under:

[0234] Table 18 Purity of fusion protein under stress condition (40°C) through RPLC

[0235] As shown in table 18 and figure 06, the control formulation, containing standard excipients, exhibited a notably less purity of 80.85% over the study duration of 21 days. Also, the rate of protein degradation in control is 34% higher compared to present composition 1 Cl with degradation rate of 3%. Composition 3 (C3), utilizing a combination of sucrose and mannitol with the present fusion protein, resulted in a purity of 82.24% by day 21 under stress conditions. In contrast, Composition 1 (Cl) and composition 2 (C2) of the present invention maintained a purity of 82.54% and 82.97% on day 21 under the same stress condition, as measured by RPLC. This result highlights the superior stability profile conferred by the specific components of the present invention, specifically utilizing a single sugar and being substantially free of mannitol.

[0236] Example 10:

[0237] The stability profile of the present fusion protein was evaluated under accelerated stress conditions (25°C) through CEX-HPLC. The results of the same are herein under: Table 19 Purity of fusion protein under accelerated condition (25°C) through CEX-HPLC

[0238] Table 19A Acidic Charged Variants under accelerated condition (25°C) through CEX-HPLC

[0239] As shown in table 19 and figure 07, the control formulation, containing standard excipients, exhibited a notably less purity of 79.40% over the study duration of 30 days. Composition 2 (C2), incorporating varying concentrations of trehalose (90 mg / mL), demonstrated higher purity than the control formulation. Composition 3 (C3), utilizing a combination of sucrose and mannitol with the present fusion protein, resulted in a purity of 88.70% by day 30 under stress conditions. In contrast, Composition 1 (Cl) of the present invention maintained a purity of 91.10% on day 30 under the same stress condition, as measured by CEX-HPLC. This result highlights the superior stability profile conferred by the specific components of the present invention, specifically utilizing a single sugar and being substantially free of mannitol. As shown in table 19A, the acidic as analysed under accelerated condition at 25°C by CEX-HPLC, the present composition Cl shows considerably less than 10% of acidic variants. The present invention addresses the need for a simplified, yet highly effective, protein stabilization system that avoids the drawbacks associated with conventional multi-component formulations or less stable sugars. By utilizing trehalose as the singular sugar excipient, the inventive composition leverages its unique properties, such as a strong preferential exclusion effect and the highest glass transition temperature (eutectic temperature) among common disaccharides (e.g., sucrose, mannitol, sorbitol). This choice minimizes molecular mobility and enhances long-term stability in a manner that is superior to merely combining known stabilizers. Specifically, trehalose avoids the hydrolytic instability of sucrose under acidic conditions (remaining >99% intact at pH 5) and circumvents the crystallization issues of mannitol during lyophilization. The resultant composition provides unexpected superior stabilization in the final solid state by reducing molecular mobility within the matrix, positioning it as an advantageous and efficient choice for robust, post-lyophilization acidic formulations using only a single sugar component.

[0240] Example 11:

[0241] The stability profile of the present fusion protein was evaluated under accelerated stress conditions (25°C) through RPLC. The results of the same are herein under:

[0242] Table 20 Purity of fusion protein under stress condition (25°C) through RPLC

[0243] As shown in table 20 and figure 08, the control formulation, containing standard excipients, exhibited a notably less purity of 83.70% over the study duration of 21 days. Also, the rate of protein degradation in control is 14% higher compared to present composition 1 Cl that has significant constant purity till day 21. Composition 3 (C3), utilizing a combination of sucrose and mannitol with the present fusion protein, resulted in a purity of 83.9% by day 21 under stress conditions. In contrast, Composition 1 (Cl) of the present invention maintained a purity of 84.02% on day 21 under the same accelerated condition, as measured by RPLC. This result highlights the superior stability profile conferred by the specific components of the present invention, specifically utilizing a single sugar and being substantially free of mannitol.

[0244] Example 12:

[0245] The purity and stability of the fusion protein were analysed across different compositions following a freeze-thaw cycle treatment. The resulting data were quantified and assessed using Size Exclusion Chromatography -High-Performance Liquid Chromatography (SEC-HPLC).

[0246] As shown in Table 21, the present composition-maintained stability even after undergoing a freeze-thaw cycles. Furthermore, the levels of High Molecular Weight Species (HMWs) in the inventive compositions were relatively lower than those observed in the control formulation, indicating enhanced stability performance. The composition of the present invention maintains less than 0.5% aggregates by weight after freeze-thaw cycle. In one embodiment, the composition of the present invention maintains less than 0.4% aggregates by weight after freeze-thaw cycle. In another embodiment, the composition of the present invention maintains less than 0.3% aggregates by weight after freeze-thaw cycle. In another embodiment, the composition of the present invention maintains less than 0.2% aggregates by weight after freeze-thaw cycle. In another embodiment, the composition of the present invention maintains less than 0.1% aggregates by weight after freeze-thaw cycle.

[0247] Example 13:

[0248] The thermal stability profiles of the various compositions were analysed using Differential Scanning Fluorimetry (DSF) yielding the following results:

[0249] As detailed in table 22, the thermal stability of the inventive composition is substantially higher than that of the control formulation. The onset temperature for the control formulation (CF2) was measured at 43.41 °C. In contrast, compositions C6 and C7 of the present invention exhibited a higher onset, indicating increased resistance to the initiation of protein denaturation. Furthermore, both the first melting temperature Tml and the second melting temperature Tm2 of the present composition were substantially higher than those of the control formulation, thereby confirming the enhanced thermal stability conferred by the inventive formulation components.

[0250] Example 14:

[0251] The thermal stability profiles of the various compositions were analysed using Differential Scanning Calorimetry (DSC), yielding the following results:

[0252] Table 23: Thermal Stability using Differential Scanning Calorimetry (DSC)

[0253] As shown in Table 23, the present composition Cl exhibits greater thermostability compared to the control formulation. This enhanced stability is evidenced by a higher thermal transition temperature, thereby demonstrating a superior stability profile for composition Cl.

[0254] Example 15:

[0255] The compositions were further analysed for purity at 40°C and 25°C through size exclusion chromatography (SEC-HPLC). The results of the same are as under:

[0256] Table 24: % purity by SEC-HPLC

[0257] Table 24 shows the results of stability and purity analysis for various pharmaceutical compositions containing a Thrombopoietin Receptor Agonist (TPO-RA) fusion protein, specifically Romiplostim, under different temperature and time conditions. The table compares the percentage purity of the fusion protein in different compositions after storage at 40°C and 25°C for up to 30 days, using Size Exclusion Chromatography (SEC-HPLC) as the analytical method. The data demonstrates that the tested formulations, especially those using sodium succinate buffer and trehalose as the single sugar (and free of mannitol), are highly effective at maintaining the purity and stability of Romiplostim even under accelerated stress conditions (high temperature and prolonged storage).

[0258] While the invention has been described in detail with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof. Accordingly, the invention is not to be limited as by the aforementioned illustrative examples, but is to be understood in broad terms as defined by the language of the appended claims.

Claims

Claims1. A stable pharmaceutical composition, comprising: a. a purified pharmacologically active fusion protein Thrombopoietin Receptor agonist (TPO-RA); b. a buffering agent; c. a stabilizing agent; d. a surfactant; wherein the stabilizing agent is sugar and optionally comprising amino acid; wherein the composition essentially consists of a single sugar; wherein the composition is free of mannitol; wherein the pharmacologically active fusion protein binds to the thrombopoietin receptor (c-Mpl); wherein the Thrombopoietin Receptor Agonist (TPO-RA) fusion protein present in the concentration selected from about 0.125mg / ml, 0.25 mg / ml and 0.5 mg / ml; wherein the purity of the composition is more than 90% as determined by size exclusion high performance liquid chromatography (SEC-HPLC) after a freeze-thaw cycle; and / or wherein the purity of the pharmaceutical composition under stress condition at 40°C on day 30 is greater than 85%; and / or wherein the purity of the pharmaceutical composition under accelerated condition at 25°C on day 30 is greater than 80%;and / or wherein the purity is measured by cation exchange high performance liquid chromatography (CEX-HPLC); wherein the purity of the pharmaceutical composition under stress condition at 40°C on day 21 is greater than 85%; and / or wherein the purity of the pharmaceutical composition under accelerated condition at 25°C on day 21 is greater than 80%; wherein the purity is measured by reversed-phase liquid chromatography (RPLC); and / or wherein the composition comprises less than 10% acidic variants as determined by cation exchange high performance liquid chromatography (CEX-HPLC).

2. A stable pharmaceutical composition, comprising: a. a pharmacologically active Thrombopoietin Receptor Agonist (TPO-RA)fusion protein;b. a buffering agent; c. a stabilizing agent; d. a surfactant; e. an amino acid ; and f. a chelating agent; wherein said Thrombopoietin Receptor Agonist (TPO-RA) fusion protein purified through a chromatography column; wherein the stabilizing agent is sugar; wherein the composition has a purity of greater than 80% as determined by reversephase high performance liquid chromatography (RPLC), and / or wherein the composition has a purity of greater than 70% as determined by cation exchange high performance liquid chromatography (CEX-HPLC); and / or wherein the purity of the pharmaceutical composition under stress condition at 40°C on day 30 is greater than 85%; and / or wherein the purity of the pharmaceutical composition under accelerated condition at 25°C on day 30 is greater than 80%; and / or wherein the purity is measured by cation exchange high performance liquid chromatography (CEX-HPLC); wherein the purity of the pharmaceutical composition under stress condition at 40°C on day 21 is greater than 85%; and / or wherein the purity of the pharmaceutical composition under accelerated condition at 25°C on day 21 is greater than 80%; wherein the purity is measured by reversed-phase liquid chromatography (RPLC); and / or wherein the composition comprises less than 10% acidic variants as determined by cation exchange high performance liquid chromatography (CEX-HPLC); and / or wherein the purity of the composition under stress condition at 40°C is greater than 90% as determined by size exclusion high performance liquid chromatography (SEC- HPLC); and / or wherein the purity of the composition under accelerated condition at 25°C is greater than 90% as determined by size exclusion high performance liquid chromatography (SEC-HPLC).

3. The composition as claimed in claim 1, wherein the purity of the composition is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 99.9% asdetermined by size exclusion high performance liquid chromatography (SEC-HPLC) after a freeze-thaw cycle.

4. The composition as claimed in claim 1 and 2, wherein the purity of the composition is selected from 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% ,98%, and 99% as determined by reverse-phase high performance liquid chromatography (RPLC).

5. The composition as claimed in claim 1 and 2, wherein the purity of the composition is selected from 71%, 72%, 73%, 74% 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% ,98%, and 99% as determined by cation exchange high performance liquid chromatography (CEX-HPLC).

6. The composition as claimed in claim 1 and 2, wherein the purity of the composition under the stress condition at 40°C on day 30 is selected from 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% ,98%, and 99% as determined by cation exchange high performance liquid chromatography (CEX-HPLC).

7. The composition as claimed in claim 1 and 2, wherein the purity of the composition under the accelerated condition at 25°C on day 30 is selected from 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% ,98%, and 99% as determined by cation exchange high performance liquid chromatography (CEX-HPLC).

8. The composition as claimed in claim 1 and 2, wherein the purity of the composition under the stress condition at 40°C on day 21 is selected from 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% ,98%, and 99% as determined by reversed-phase liquid chromatography (RPLC).

9. The composition as claimed in claim 1 and 2, wherein the purity of the composition under the accelerated condition at 25°C on day 21 is selected from 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% ,98%, and 99% as determined by reversed-phase liquid chromatography (RPLC).

10. The composition as claimed in claim 1 and 2, wherein the composition comprises acidic variants about 9 % upon 30 days at 40°C.

11. The composition as claimed in claim 1 and 2, wherein the composition comprises acidic about 8% upon 30 days at 40°C.

12. The composition as claimed in claim 1 and 2, wherein the composition comprises acidic about 7% upon 30 days at 40°C.

13. The composition as claimed in claim 1 and 2, wherein the composition comprises acidic about 8 % upon 30 days at 25 °C.

14. The composition as claimed in claim 1 and 2, wherein the composition comprises acidic about 7 % upon 30 days at 25 °C.

15. The composition as claimed in claim 1 and 2, wherein the purity of the composition under stress condition at 40°C is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97% ,98%, and 99% as determined by size exclusion high performance liquid chromatography (SEC-HPLC).

16. The composition as claimed in claim 1 and 2, wherein the purity of the composition under stress condition at 25°C is selected from 91%, 92%, 93%, 94%, 95%, 96%, 97% ,98%, and 99% as determined by size exclusion high performance liquid chromatography (SEC-HPLC).

17. The composition as claimed in claim 1 and claim 2, wherein the composition maintains less than 0.5% high molecular weight (HMW) aggregates by weight after a freeze-thaw cycle.

18. The composition according to claim 1 and 2, wherein the sugar is suitable to provide stability to the Thrombopoietin Receptor Agonist (TPO-RA) fusion protein and improves cake appearance of a lyophilized cake.

19. The composition according to claims 1 and 2, wherein the sugar is present in an amount in the range of about 130 mM to about 280 mM ; wherein the sugar is present in an amount in the range of about 50 mg / ml to about 105 mg / ml.

20. The composition according to claim 19, wherein the sugar is present in an amount of 185mM.

21. The composition according to claim 1 and 2, wherein the composition exhibits a thermal stability characterized by one or more characteristics selected from denaturation temperature of the Thrombopoietin Receptor Agonist (TPO-RA) fusion protein; wherein the denaturation temperature ranges from about 50°C to about 80°C as measured by differential scanning fluorometry (DSF).

22. The composition according to claim 21, wherein the composition exhibits a thermal stability having an onset temperature of about 50°C, a first melting temperature (Tml) of about 60°C, and a second melting temperature (Tm2) of about 77°C.

23. The composition according to claim 1 and claim 2, wherein the composition exhibits a thermal stability characterized by one or more characteristics selected from transition temperature for the Thrombopoietin Receptor Agonist (TPO-RA) fusion protein; wherein the transition temperature for Thrombopoietin Receptor Agonist (TPO-RA) fusion protein is in range of about 50°C to about 90°C as measured by differential scanning calorimetry (DSC).

24. The composition according to claim 23, wherein the composition exhibits a thermal stability characterized by differential scanning calorimetry (DSC), having a first thermal transition (Tl) of about 55 °C and a second thermal transition (T2) of about 80°C.

25. The composition according to claim 1 and claim 2, wherein the pharmacologically active Thrombopoietin Receptor Agonist (TPO-RA) fusion protein is Romiplostim.

26. The composition according to claim 1 and claim 2, wherein the buffering agent is present in amount ranging from about 7mM to about 15mM.

27. The composition according to claim 26, wherein the buffering agent is present in an amount of lOmM.

28. The composition according to claim 1 and claim 2, wherein the surfactant is present in an amount in the range of about 0.008mM to about 0.4 mM.

29. The composition according to claim 28, wherein the surfactant is present in an amount of 0.03mM.

30. The composition according to claim 2, wherein the chelating agent is present in an amount in the range of about O.OOlmM to about 0.255mM.

31. The composition according to claim 30, wherein the chelating agent is present in an amount of 0.002mM.

32. The composition according to claim 1 and claim 2, wherein the amino acid is present in an amount in the range of about 5mM to about 200mM.

33. The composition according to claim 32, wherein the amino acid is present in an amount of about 40mM to about lOOmM.

34. The composition according to claim 32, wherein the amino acid is present in an amount of 47mM.

35. The composition according to claim 1 and 2, wherein the buffering agent is present in an amount of about 0.83mg / ml to about 1.78mg / ml.

36. The composition according to claim 35 wherein the buffering agent is present in an amount of 1.18 mg / ml.

37. The composition according to claim 35, wherein the pH of the buffer is about 4 to about 6.

38. The composition according to claim 37, wherein the pH of the buffer is 5.

39. The composition according to claim 1 and 2, wherein the sugar is present in an amount of about 50mg / ml to about 105mg / ml.

40. The composition according to claim 39, wherein the sugar is present in an amount of about 60mg / ml to 90mg / ml.

41. The composition according to claim 39, wherein the sugar is present in an amount of 50mg / ml.

42. The composition according to claim 39, wherein the sugar is present in an amount of 70mg / ml.

43. The composition according to claim 39, wherein the sugar is present in an amount of 90mg / ml.

44. The composition according to claim 1 and 2, wherein the surfactant is present in an amount ranging from about O.Olmg / ml to about 0.5mg / ml.

45. The composition according to claim 44, wherein the surfactant is present in an amount ranging from about 0.02mg / ml to about 0.04mg / ml.

46. The composition according to claim 44, wherein the surfactant is present in an amount 0.04 mg / ml.

47. The composition according to claim 2, wherein the chelating agent is present in an amount ranging from about 0.0004mg / ml to about O.lmg / ml.

48. The composition according to claim 47, wherein the chelating agent is present in an amount of O.OOlmg / ml.

49. The composition according to claim 1 and claim 2, wherein the amino acid is present in an amount ranging from about 1 mg / ml to about 75mg / ml.

50. The composition according to claim 49, wherein the amino acid is present in an amount ranging from about 5mg / ml to about 50mg / ml.

51. The composition according to claim 49, wherein the amino acid is present in an amount ranging from about 9mg / ml to about 20mg / ml.

52. The composition according to claim 49, wherein the amino acid is present in an amount lOmg / ml.

53. The composition according to claim 1 and 2, wherein the buffering agent is selected from sodium succinate, succinic acid, arginine acetate, citrate, acetate sodiumphosphate, and combination thereof; and / or wherein the buffering agent is sodium succinate.

54. The composition according to claim 1 and 2, wherein the single sugar is selected from trehalose, xylitol, maltose, arginine, raffinose; and / or wherein the single sugar is trehalose.

55. The composition according to claim 1 and 2, wherein the surfactant is selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, pol oxamer 188 and combination thereof; and / or wherein the surfactant is polysorbate 20.

56. The composition according to claim 2, wherein the chelating agent is selected from diethylenetriaminepentaacetic acid (DTP A), ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(P-aminoethyl ether) -N,N,N',N' -tetraacetic acid (EGTA), citric acid, gluconic acid, and glutathione; and / or wherein the chelating agent is diethylenetriaminepentaacetic acid (DTP A).

57. The composition according to claim 1 and claim 2, wherein the amino acid is selected from arginine, lysine, methionine, glycine, proline, asparagine, phenylalanine, aspartic acid, glutamic acid and suitable salt thereof; and / or wherein the amino acid is arginine HC1.

58. A stable pharmaceutical composition, comprising: a. 0.5mg / mL of pharmacologically active fusion protein binding to the thrombopoietin receptor (c-Mpl); b. at least 7mM to 15mM of the buffering agent; c. at least 130mM to about 280mM of the stabilizing agent; d. at least 0.008mM to 0.4 mM of the surfactant; e. at least O.OOlmM to 0.255mM of chelating agent; f. at least 5mM to 200mM of amino acid; and g. optionally comprises of about O.OOOlmM to about 300mM of an antioxidant.

59. The composition according to claim 58, wherein the antioxidant is present in an amount ranging from about 0.5mg / ml to about 3mg / ml.

60. The composition according to claim 58, wherein the antioxidant is selected from methionine, N-acetylcysteine, ascorbic acid, pyruvic acid, benzoic acid, methyl paraben, ethyl paraben, propyl paraben, butyl paraben, m-cresol, benzyl alcohol, phenoxyethanol, benzalkonium chloride, phenol, and combination thereof; and / or wherein the antioxidant is methionine.

61. The composition according to any of the preceding claims, wherein the composition has the pH of about 4.5 to about 6.5.

62. The composition according to any of the preceding claims, wherein the composition has the pH of 5±0.2.

63. The composition according to any of the preceding claims, wherein the composition is administered subcutaneously; and / or wherein the composition is administered subcutaneously through device selected from syringe, autoinjector, on body injector (OBI), pre filed syringe, and pen.

64. A stable pharmaceutical composition, comprising: a. 0.5 mg / ml Romiplostim; b. 1.18mg / ml of sodium succinate; c. 70mg / ml of trehalose; d. 0.04mg / ml of polysorbate 20; e. 0.001 mg / ml of di ethylenetriaminepentaacetic acid (DTP A); f. 10 mg / ml of Arg. HC1; wherein the composition has the pH between 4.5 to 5.5; wherein the composition essentially consists of single sugar; wherein the composition of free of mannitol; wherein the composition is suitable for subcutaneous administration; wherein the composition has a purity of greater than 80% as determined by reversephase high performance liquid chromatography (RPLC), and / or wherein the composition has a purity of greater than 70% as determined by cation exchange high performance liquid chromatography (CEX-HPLC); wherein the purity of the pharmaceutical composition under stress condition at 40°C on day 30 is greater than 85%; and / or wherein the purity of the pharmaceutical composition under accelerated condition at 25°C on day 30 is greater than 80%; wherein the purity is measured by cation exchange high performance liquid chromatography (CEX-HPLC); wherein the purity of the pharmaceutical composition under stress condition at 40°C on day 21 is greater than 85%; and / or wherein the purity of the pharmaceutical composition under accelerated condition at 25°C on day 21 is greater than 80%;wherein the purity is measured by reversed-phase liquid chromatography (RPLC); and / or wherein the composition comprises less than 10% acidic variants as determined by cation exchange high performance liquid chromatography (CEX-HPLC); and / or wherein the purity of the composition under stress condition at 40°C is greater than 90% as determined by size exclusion high performance liquid chromatography (SEC- HPLC); and / or wherein the purity of the composition under accelerated condition at 25°C is greater than 90% as determined by size exclusion high performance liquid chromatography (SEC-HPLC).

65. The composition according to any preceding claim, wherein the composition is lyophilized composition.

66. The composition according to any preceding claim, wherein the composition is liquid composition.

67. A method for preparing a stable lyophilized pharmaceutical composition comprising a pharmacologically active TPO-RA fusion protein, the method comprising : a) preparing a bulk liquid solution comprising the TPO-RA fusion protein and one or more pharmaceutical excipients; b) filling the bulk liquid composition into vials for lyophilization; c) subjecting the vials to a lyophilization process, wherein the lyophilization method comprises the steps as described in Table A.

68. A method for preparing a stable lyophilized pharmaceutical composition comprising a pharmacologically active TPO-RA fusion protein, the method comprising : a) preparing a bulk liquid solution comprising the TPO-RA fusion protein and one or more pharmaceutical excipients; b) filling the bulk liquid composition into vials for lyophilization; c) subjecting the vials to a lyophilization process, wherein the lyophilization method comprises the steps as described in Table Al.

69. A method for preparing a lyophilized pharmaceutical composition comprising a pharmacologically active TPO-RA fusion protein, the method comprising : a) preparing a bulk liquid solution comprising the TPO-RA fusion protein and one or more pharmaceutical excipients; b) filling the bulk liquid composition into vials for lyophilization;c) subjecting the vials to a lyophilization method, wherein the lyophilization method comprises the steps: i. performing a loading of said vials at a temperature ranging from about 20°C to about 5 °C for a period of about 25 minutes to about 55 minutes; ii. performing cooling of said vial comprising liquid composition in a lyophilization chamber to a temperature in a range of about from 0°C to about -60°C with holding; wherein the holding is performed at temperature ranging from 0°C to about -60°C for a period in a range of about 1.5 hours to about 20 hours; iii. performing an annealing step with hold at a temperature -13°C to -20°C over a period in range of about 3 hours to about 10 hours; iv. performing a primary drying; wherein the primary drying comprises heating the chamber to a temperature in a range of about -60°C to about 0°C and holding at said temperature at a pressure in a range of about 10 mTorr to about 150 mTorr for a period in a range of about 0.05 hours to about 50 hours to produce a primary dried composition; and v. performing a secondary drying , wherein the secondary drying comprises heating the chamber to a temperature in a range of about 0°C to about 30°C and holding at said temperature at a pressure in a range of about 10 mTorr to about 100 mTorr for a period in a range of about 0.5 hours to about 30 hours to produce the lyophilized cake suitable for pharmaceutical use.

70. The method according to claim 69, wherein the temperature before holding in step (ii) in step (iv), and in step (v) gradually decreases at a rate of about 0.1°C / min to about 1.0 °C / min.

71. The method according to claim 69, wherein the temperature before holding in step (ii) in step (iv), and in step (v) gradually decreases at a rate of more than about 0.2°C / min to about 0.8 C / min.

72. The method according to claim 69, wherein the temperature before holding in step (ii) in step (iv), and in step (v) gradually decreases at a rate of more than about 0.3°C / min to about 0.6 C / min.

73. The method according to claim 69, wherein the holding in step (ii) is performed at temperature from about -45° C to about -55° C.

74. The method according to claim 69, wherein the holding in step (iii) is performed at temperature below -13°C over a period of less than about 6 hours.

75. The method according to claim 69, wherein the holding in step (iv) is performed at temperature from about -55° C to about -25°C.

76. The method according to claim 68 and claim 69, wherein the bulk liquid is present in a suitable concentration in said vials.

77. The method according to claim 68 and claim 69, wherein the bulk liquid concentration in the vial is selected from about 0.125mg / ml, or 0.25 mg / ml and 0.5 mg / ml.

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