Title of Invention	PHARMACEUTICAL FORMULATION OF INTERFERON CONJUGATE
Abstract	The present invention provides stable pharmaceutical formulations of pegylated interferons comprising pegylated interferon alfa conjugates and phannaceutically acceptable excipients. The formulations of the invention are in the form of lyophilized powder.

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FORM 2 THE PATENTS ACT, 1970 (39 of 1970) & The Patents Rules, 2003 COMPLETE SPECIFICATION (See section 10 and rule 13) PHARMACEUTICAL FORMULATIONS OF INTERFERON CONJUGATES Intas Biopharmaceuticals Limited An Indian company having its registered office at Plot No: 423/P/A/GIDC Sarkhej-Bavla Highway Moraiya, Tal.: Sanand Ahmedabad - 382 210 Gujarat, India The following specification particularly describes the invention and the manner in which it is to be performed. PHARMACEUTICAL FORMULATIONS OF INTERFERON CONJUGATES RELATED APPLICATIONS This application takes priority to 2077/MUM/2008 filed December 1, 2008, titled 'Pharmaceutical Compositions of Interferon Conjugates' and is incorporated herein at its entirety. FIELD OF INVENTION The invention relates to formulations of pegylated interferons. In particular, the invention provides pharmaceutical formulations of pegylated interferons comprising pegylated interferon alfa conjugates and pharmaceutically acceptable excipients wherein the formulations is a lyophilized powder. BACKGROUND OF THE INVENTION Interferon belongs to a group of cytokines which are classified in accordance with their cellular origin and the type of receptors to which they bind. There are three classes of interferons, namely alfa, beta and the gamma. With respect to interferon alfa, there are two subtypes, namely interferon alfa 2a and interferon alfa 2b which differ only by one amino acid residue. In interferon alfa 2a, residue number 23 is lysine, whereas the same residue in interferon alfa 2b is an arginine. Two commercially available products exist namely peg-interferon alfa 2 a marketed by Hoffmann La Roche under the tradename Pegasys®, and peg-interferon alfa 213 marketed by Schering Plough Corporation under the tradename peg-Intron A®. Peg-interferon alfa is used for a number of indications, including hepatitis B and C and various cancer conditions such as renal cell carcinoma, AIDS-related Kaposi's sarcoma, chronic and acute hepatitis B and C and chronic and acute hepatitis non-A. Recombinant proteins may be limited in their effective functions by factors such as immunogenecity, solubility, stability and short pharmacological half-life. While using these proteins it is also important to achieve therapeutically stable levels of the proteins in patient's blood. Some of these problems have been circumvented, very early on, by conjugating the proteins with low immunogenecity to polymers such as polyethylene glycol {See US4I79337, US4766106, US4917888). Conjugation of proteins to polymers has also been effectively used to enhance the solubility of proteins {See US4766106, US4917888). US4902502 has shown that recombinant proteins conjugated with polyethylene glycol (PEG) and other polymers leads to increased half-life and reduced immunogenicity of the proteins thus making them effective therapeutics. Lack of stability and degradation of the protein are a few other problems confronted by researchers while working with protein therapeutics. It is important that protein-polymer conjugates can be stable for longer periods of time to withstand the manufacturing and storage conditions. Protein conjugates are often lyophilized to prevent them from degradation. Lyophilization or freeze-drying is a process wherein water is sublimed from the frozen composition thus making the protein in dried state stable for longer periods. Protein conjugates are often formulated with additional excipients which serve to protect the active ingredient during the lyophilization process. Cryoprotectants and stabilizers have been particularly used in lyophilized formulations to render protection and stability to the protein of interest. US5762923 discloses an aqueous interferon solution containing (a) an interferon-alfa; (b) a non-ionic detergent; (c) a buffer for adjusting pH to 4.5-5.5; (d) benzyl alcohol; and optionally (e) an isotonizing agent. Indian patent IN207233 describes a lyophilized formulation containing (a) a Peg-interferon (b) cryoprotectant (c) a buffer (d) a stabilizer wherein the cryoprotectant is sucrose at a concentration of 20-100mg/ml and the stabilizer is polysorbate. WO2008107908 describes a lyophilized product of pegylated interferon alfa conjugates containing (a) PEG-interferon-alfa (b) a cryoprotectant (c) a non-ionic detergent, (c) a buffer for adjusting 4.0-7.0 (d) glycine. Herein, the cryoprotectant used is raffinose. US6180096 discloses a formulation of Pegylated interferon alfa conjugates containing a buffer, stabilizer, a cryoprotectant and a solvent wherein the buffer is sodium phosphate, stabilizer is poly(oxy-l,2-ethanediyl) and cryoprotectant is sucrose. It is however seen that at lower concentrations of sucrose, the stability of the formulation is compromised due to cake defect. WO2006020720 provides a stable pegylated interferon formulation comprising PEG interferon, a cryoprotectant, a buffer and a stabilizer wherein the trehalose as a cryoprotectant comprises at least 60% by weight of the formulation. The preferred amount of trehalose in the above formulation is 90 - 100%. The formulation, however, categorically requires 60% or more of trehalose to achieve a stable interferon formulation. The present invention provides a formulation of stable pegylated interferon with trehalose up to a concentration of 50% of the cryoprotectant, buffer and a surfactant. One or more disaccharide (ex: sucrose) or sugar alcohol (ex: mannitol), is used as additional cryoprotectant. The present formulation of pegylated interferon is found to withstand the lyophilization process. Use of carbohydrates and sugar alcohols as pharmaceutical excipients in formulation of proteins is known in the art. Pharmaceutically acceptable excipients include fructose, maltose, galactose, glucose, mannose, sorbose, lactose, sucrose, trehalose, cellobiose, raffinose, melezitose, maltodextrans, dextrans, starches, mannitol, xylitol, lactitol, glucitol, pyranosyl sorbitol, myoinositol and combinations thereof. WO2005079755 discloses IL-13 antagonist formulation comprising one or more carbohydrates including cellobiose. Similarly, WO2007044069 describes parathyroid hormone preparation comprising one or more polyol selected from sucrose, mannitol, sorbitol, lactose, L-arabinose, D- erythrose, D-ribose, D-xylose, D-mannose, trehalose, D-galactose, lactulose, cellobiose, gentibiose, glycerin and polyethylene glycol. Use of mannitol, lactose, sorbitol, xylitol, sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose, gentiobiose, isomaltose, arabinose, glucosamine, fructose or the combinations thereof as stabilizer in formulating recombinant von Willebrand Factor has been provided in WO2009086400. The present invention also provides a formulation of stable pegylated interferon which comprises cellobiose as cryoprotectant, a buffer and a stabilizer. It is for the first time that pegylated interferon has been formulated using cellobiose as a cryoprotectant to render stability to the lyophilized compound. SUMMARY OF THE INVENTION The present invention provides a novel formulation of pegylated interferon comprising pegylated interferon, a cryoprotectant, a buffer and a stabilizer at a pH range between 4.0 to 8.0. In one aspect, the invention provides Formulation 1 comprising pegylated interferon, a cryoprotectant, a buffer and a stabilizer wherein the cryoprotectant comprises up to 50% by weight of the cryoprotectant. Additional cryoprotectant of Formulation 1 is a disaccharide or a sugar alcohol. Disaccharide of the Formulation 1 is selected from the group consisting of sucrose, cellobiose and maltose. Sugar alcohol of the Formulation 1 is mannitol or sorbitol. In another aspect the buffer of Formulation 1 is selected from the group consisting of histidine, histidine hydrochloride, sodium mono/dibasic phosphate, sodium citrate/citric acid and sodium acetate/acetic acid. In yet another aspect, the Formulation 1 comprises the stabilizer which is a poloxamer or a polysorbate. The stabilizers may also be selected from the group consisting of tween, glycerol, dimethylsulfoxide, polyvinyl alcohol (PVA) and polyethylene glycol (PEG. The stabilizer that is used for Formulation 1 also encompasses amino acid selected from the group consisting of histidine, arginine and lysine.The most preferred poloxamer is poloxamer 188 and the preferred polysorbate is polysorbate 20 or polysorbate 80. The invention further provides a pharmaceutical formulation comprising about 0.02 to 2 mg/ml of pegylated interferon alpha conjugate, about 5 mg/ml to 18 mg/ml of trehalose, about 5 mg/ml to 18 mg/ml of sucrose, about 0.5 mg/ml to 30 mg/ml of histidine and about 0.1 mg/ml to 1.0 mg/ml of poloxamer 188 and the pH of the formulation is maintained between 4.0 to 8.0. The invention also provides Formulation 2 comprising pegylated interferon, a cryoprotectant, a buffer and a stabilizer wherein the cryoprotectant is cellobiose. Formulation 2 of the invention comprises buffer selected from the group consisting of monobasic/dibasic phosphate, histidine, histidine hydrochloride, sodium citrate/citric acid and sodium acetate/acetic acid. The preferred buffer is monobasic sodium phosphate and dibasic sodium phosphate. In one aspect, Formulation 2 comprises stabilizer which is a poloxamer or a polysorbate. In another aspect, Formulation 2 comprises stabilizer selected from the group consisting of polyvinyl alcohol, polyethylene glycol and amino acids. The amino acid of Formulation 2 is selected from the group consisting of histidine, arginine and lysine. The invention further provides a pharmaceutical formulation comprising about 0.02 to 2.0 mg/ml of pegylated interferon alpha conjugate, up to 120 mg/ml of cellobiose, preferably in the range of 60 mg/ml to 120 mg/ml and 5 mM to 150 raM of phosphate buffer and about 0.1 mg/ml to 1.0 mg/ml of poloxamer 188 and the pH of the formulation is maintained between 4.0 to 8.0. The invention provides formulations for pegylated interferons, preferably pegylated interferon alfa and most preferably pegylated interferon alfa 2a and 2b. The formulations of the present invention are most stable as lyophilized powder. The invention also encompasses a process of lyophilization of the formulation of claim 1 or claim 15, wherein the said process comprises the steps of (a) formulating the ingredients of claim 1 or claim 15 in a solution (b) aliquoting the solution in vials (c) placing the vials in lyophilizer chamber at ambient pressure and between -50°C and 4°C (d) subjecting the loaded vials to freezing at ambient pressure for 2 to 12 hours at a temperature between -50°C and 40°C (e) drying the frozen solution in two steps under vacuum at a pressure of between 300 and 50 millibar: (i) performing the primary drying step at a temperature between -40°C and-15°C; (ii) for 15 to 45 hours (ii) performing the secondary drying step at a temperature between 15°C and 35°C for 4 to 12 hours; (e) unloading the vials containing the final lyophilized powder at ambient pressure and room temperature. BRIEF DESCRIPTION OF DRAWINGS Fig. 1. Formulation 1 loaded under non-reducing conditions. Fig. 2. Formulation 1 loaded under reducing conditions. Fig. 3. Graphical representation of Stress Study data for Formulation 1. Fig. 4. Cake appearance of Formulation 1 with 15mg/ml of sucrose.. Fig. 5. Cake appearance of Formulation 1 with 5mg/ml of trehalose and 15mg/ml of sucrose. Fig. 6. Formulation 2 loaded under non-reducing conditions. Fig. 7. Formulaiton 2 loaded under reducing conditions. Fig. 8. Graphical representation of accelerated Study data for Formulation 2. DETAILED DESCRIPTION OF THE INVENTION The present invention provides novel formulations of pegylated interferons that are stable during long-term storage at room temperature, as well as withstand the process for preparing the lyophilized formulations. This section presents a detailed description of these formulations, their preparations and their applications. The formulations of the invention encompass pegylated interferons in general and pegylated interferon alfa conjugates in particular. Pegylated interferon alfa conjugates are interferon alfa molecules covalently attached to a polymer molecule, for example, polyethylene glycol (PEG). Commercially available pegylated interferon alfa conjugates include interferon alfa 2a (Pegasys Hoffman La-Roche), interferon alfa 2b (Intron, Schering-Plough) and interferon alfa 2c (Berofor Alfa, Boehringer Ingelheim). 40KDa-branched-(Lysine)-mPEG-HNS PEG is used in PEGylated Interferon alfa 2a. The PEG used in 40KDa-branched-(Lysine)-rnPEG-Interferon alfa 2a consists of two monomethoxy peg chains, each with an average molecular weight of 20 kDa each. The carboxy group of the lysine linker is activated to a N-hydroxysuccinimidyl ester that can then form stable amide bonds with amino groups located on lysine residues in the protein. The two monomethoxy PEG chains are joined via hydrolytically stable urethane bonds to a lysine linker molecule, one at the lysine € amino group and another at the lysine a-amino group. 12KDa-PEG Interferon alfa 2b is a covalent conjugate of iFN-alfa 2b linked to a single chain 12 kDa PEG molecule. 12KDa-PEG Interferon alfa 2b is synthesized by the reaction of purified recombinant IFN alfa 2b with an electrophilic derivative of PEG, succinimidyl carbonate PEG (SC-PEG), in 100 raM sodium phosphate (pH 6.6). 12kDa-PEG-SC is linear mono-methoxy poly ethelyne glycol is converted into its N-succinimide carbonate (SC) derivative. This form of the polymer reacts readily with amino groups of proteins in aqueous buffer. The crude product is fractionated by ion-exchange chromatography to yield predominantly mono-pegylated final product. The formulations of the present invention are particularly useful in protecting pegylated interferons during the process of lyophilization and storage conditions. The pegylated interferon of the invention is preferably pegylated interferon alfa. Further, the pegylated interferon alfa may be pegylated interferon alfa 2a, 2b or 2c. The pegylated interferon alfa conjugates are found at a concentration of 0.02 to 2 mg/ml, preferably 0.1 to 0.5 mg/ml in the formulations of the invention. The pegylated interferon alfa formulations of the present invention comprise a pegylated interferon alfa, a cryoprotectant or a combination of cryoprotectants, buffer and a stabilizer. The cryoprotectant of the invention protects the pegylated interferon from damage, adsorption and loss from vacuum utilized in lyophilization. The cryoprotectant also serves to stabilize the pegylated interferon during the freeze-drying process and in the resulting powder, and as a bulking agent to form an easily reconstitutible cake. The amount of cryoprotectant used is typically based on the total weight of the formulation. In the total weight of the formulation, the cryoprotectant constitutes about 90% to 100% by total weight of the formulation. The present invention provides two formulations of pegylated interferon alfa. In one embodiment, the first formulation (hitherto referred to as Formulation 1) comprises pegylated interferon alfa, a buffer, a stabilizer and trehalose making up to 50% of the cryoprotectant. Trehalose is a disaccharide containing two glucose molecules bound in a a,a-l, 1 linkage. Any form of trehalose is suitable for use in preparing the formulations of the present invention. A preferred form of trehalose is trehalose dihydrate. Additional cryoprotectants of the aforesaid formulation are carbohydrates such as saccharides and sugar alcohols. Saccharides of the formulation are selected from the group consisting of sucrose, cellobiose and maltose. The invention also allows use of sugar alcohol as an additional cryoprotectant in Formulation One, The sugar alcohol of the invention is preferably mannitol or sorbitol. Based on the amount of trehalose present in Formulation 1, the saccharide or the sugar alcohol make up the rest of the total cryoprotectant of the invention. For example, if trehalose constitutes 25%, 30%, 40% or 50%, saccharide or sugar alcohol maybe respectively in the amount of 65%-70%, 60%-65%, 50%-55% or 40%-45%. Preferably, Formulation I of the invention has sucrose as the additional cryoprotectant. Trehalose comprises up to 50% of the total weight of the cryoprotectant in the formulation. The cryoprotectant of Formulation 1 comprises trehalose in a percentage range between 0.05% and 50% by weight, for example, 15%, 25%, 45% or 50%. Preferably, trehalose comprises 25% or 45% of the total weight of the cryoprotectant. More preferably, trehalose constitutes up to 50% of the cryoprotectant, by weight. In one preferred embodiment, the formulations prepared by lyophilizing a solution containing 5 mg (20 %) trehalose dihydrate and 19 mg (80 %) sucrose as the cryoprotectants. In another embodiment, the solution comprises 15 mg (50 %) trehalose dihydrate and 15 mg (50 %) sucrose as the cryoprotectant. In yet another embodiment, the cryoprotectant consists of trehalose dihydrate, which is present in the solution at 18 mg (50 %) and 18 mg (50 %) sucrose as the cryoprotectant, Formulation 1 of the invention also contains a buffer for maintaining the pH of the formulation in the range of 4.0 to 8.0. The preferred pH range is 5.5 to 7.5 and the most preferred pH range is 6.5 to 7.3. In one embodiment, the preferred buffer comprises histidine or histidine hydrochloride in the formulation at a concentration of up to 300 mM, preferably at 10 mM concentration. Other suitable buffer systems to maintain the desired pH range that is encompassed by the present invention include sodium mono/dibasic phosphate, sodium citrate/citric acid and sodium acetate/acetic acid. Phosphate buffer is used in the range of up to 150 mM, preferably at 10 mM concentration. Citrate and acetate buffer are used in the range up to 100 mM, preferably 10 mM concentration. In another embodiment, Formulation 1 of the pegylated interferon alfa comprises stabilizer for preventing adsorption of the pegylated-interferon to the stainless steel and glass surfaces of the equipment and containers used to lyophilize and store the formulations. Stabilizers can stabilize the pegylated-interferon conjugates by minimizing its exposure to air-water and ice-water interfaces during lyophilization and storage. As one example, Poloxamers and Polysorbates are useful as stabilizing agents wherein a preferred poloxamer is poloxamer 188 and preferred Polysorbates are polysorbate 20 and 80. When Poloxamer 188 is utilized, the preferred concentration is 0.005 to 0.01 % (w/v). Other suitable stabilizers are surface active agents such as tween, glycerol, dimethylsulfoxide, polyvinyl alcohol (PVA) and polyethylene glycol (PEG). In addition to the said stabilizers there are a few amino acids like histidine, arginine or lysine that are used as stabilizing agents. The most preferred stabilizer in the aforesaid formulation is histidine. The second formulation (hitherto referred to as Formulation 2) of the invention comprises pegylated interferon alfa, a buffer, a stabilizer and cellobiose as a cryoprotectant. Cellobiose is a disaccharide with the formula [HOCH2CHO (CHOH)3]20. The molecule is derived from the condensation of two glucose molecules linked in a Ji (1? 4) bond. It can be hydrolyzed by bacteria or cationic ion exchange resins to give glucose. Cellobiose has eight free alcohol (COH) groups and three ether linkages, which give rise to strong -inter- and intra-molecular hydrogen bonds. Any form of cellobiose is suitable for use in preparing the formulations of the present invention. Cellobiose is used at a concentration of up to 120 mg/ml, more preferably in the range of about 60 mg/ml to 120 mg/ml. In one embodiment, Formulation 2 of the invention contains a buffer for maintaining the pH of the formulation in a range of 4.0 to 8.0. The preferred pH range is 5.5 to 7.5 and the most preferred pH range is 6.5 to 7.3. A preferred buffer comprises sodium mono/dibasic phosphate in the formulation being up to 0.1 molar. Other suitable buffer systems for maintaining the desired pH range of the above formulation includes histidine or histidine hydrochloride, sodium citrate/citric acid and sodium acetate/acetic acid. The stabilizer used in Formulation 2 performs identical function as detailed for Formulation 1. In a preferred embodiment, Formulation 2 comprises stabilizers selected from the group consisting of poloxamers and polysorbates. Preferred poloxamer is poloxamer 188 and preferred polysorbates are polysorbate 20 and 80. The stabilizers, poloxamer or polysorbate are used at a concentration of up to 0.1%, preferably at 0.05%. Other suitable stabilizers are surface active agents such as polyvinyl alcohol (PVA) and polyethylene glycol (PEG), which may be used for the lyophilized formulation. In addition to the said stabilizers there are few amino acids like histidine, arginine or lysine are also used as a stabilizing agent and wherein the most preferred one is histidine. Formulations 1 and 2 of the present invention are stable in lyophilized form and thus the invention encompasses lyophilized formulations. Both formulations are reconstituted with water for injection with or without sodium chloride, mannitol or sorbitol which increases the osmolality. To prepare the formulations of the present invention, the pegylated interferon, cryoprotectant, buffer and stabilizer are dissolved in water for injection in an amount selected to achieve concentrations of the solid ingredients that are suitable for lyophilization. As used herein, the term "Water for Injection" means sterile, purified water that meets regulatory standards for particulates, dissolved solids, organics, inorganics, microbial and endotoxin contaminants. Once all the formulation ingredients are in solution, the solution is aliquoted into glass containers suitable for use in a lyophilizer and for storage of the resulting lyophilized powder. Preferred container is a vial. As used herein, the term "Vial" refers to a small glass container with a flat or slightly concaved bottom, short neck and flat flange designed for stoppering. Vials are placed directly on the lyophilizer chamber trays or shelves for direct heat transfer. Glass containers containing a pegylated interferon formulation of the invention are subjected to lyophilization under conditions appropriate to produce a lyophilized powder with moisture content less than 3%, and more preferably less than 2%. The lyophilization conditions are also chosen to achieve acceptable levels of cake defects while maintaining the desired low moisture content. As used herein, the phrase "cake defect" refers to cake that has one or more physical defects, such as collapse, shrinkage, liddedness, or meltback. Collapsed cakes, which are usually due to excessive warming of the formulation during freeze-drying, are associated with loss of product elegance and poor stability. Shrinkage, which is caused by an inefficient freeze-drying cycle, may be a sign of partial or micro collapse and may result in poor stability of the pegylated interferon upon storage. Liddedness is a physical defect in which the top of the cake forms a thin film or crust, separate from the bulk of the cake. Meltback refers to a common form of cake collapse that is primarily due to incomplete sublimation (change from solid to vapor state) of the formulation. Meltback is associated with a change in the physical form of the pegylated interferon or moisture pocket(s), which may result in instability and depegylation of the pegylated interferon. As per the formulations of the present invention, the percentage of these type of cake defects in the lyophilized powder is less than 20%, more preferably less than 10%, still more preferably less than 2%, and most preferably less than 1%. In one embodiment, the amount of unpegylated interferon in the lyophilized powder, after storage at 25°C for 6 months, is less than 10%, preferably less than 8%, and most preferably less than 6%. The use of trehalose in formulation 1 or cellobiose in formulation 2 of the present invention permit aggressive lyophilization conditions to achieve lyophilized powders having the desired low moisture content and minimal cake defects. Loading the glass containers into the lyophilizer may be performed at ambient pressure between -50°C and 4°C. The loaded containers maybe subjected to freezing at ambient pressure for 2 to 12 hours at a temperature between -50°C and -40°C. The frozen solution is dried in two steps under vacuum at a pressure of between 300 and 50 millibar (mbar), the primary drying step maybe performed for 15 to 45 hrs at a temperature between -40°C and -15°C while secondary drying step may be performed for 4 to 12 hrs. at a temperature between 15°C to 35°C. Unloading the glass containers containing the lyophilized powder maybe performed at ambient pressure and room temperature or below. The invention contemplates that variations of these lyophilization conditions will produce lyophilized powder having the desired characteristics. The skilled artisan can readily design and test alternate lyophilization processes based on considerations known in the art. The preferred lyophilization cycle for Formulation 1 and Formulation 2 requires less than 48 and 50 hours respectively, for completion, including time required to change the temperature of the chamber between steps as applicable. Pegylated interferon formulations prepared according to the preceding description are stable during the process of lyophilization as well as upon storage at room temperature. The following examples provide illustrative embodiments of the invention. A person skilled in the art will readily recognize the various modifications and variations that may be performed without altering the scope of the present invention. Such modifications and variations are encompassed within the scope of the invention and the examples do not in any way limit the scope of the invention. EXAMPLES The examples provide a description of the formulations of the present invention and protection of pegylated interferon alfa conjugate during lyophilization and storage. The formulation of pegylated interferon alfa conjugate is lyophilized and stored as a dry powder. The study design for the Formulations 1 and 2 are as follows: Example 1. Experiments pertaining to Formulation 1 Compostion of Formulation 1: Formulation 1 as provided in Table 1 consists of 0.1 mg/ml of pegylated interferon alfa 2b (PEG-Interferon aifa-2b), 15mg/ml of sucrose, 5mg/ml of trehalose, 0.5mg/ml of poloxamer 188 and L55mg/ml of Histidine. Study Design: A fixed number of vials of lyophilized Formulation 1 were charged at 40°C (stress condition as per ICH guideline No.Q5C) for 23 days and at 2-8°C (Real-time condition) for a period of 2 months. The samples from stress condition were withdrawn at each time point (as mentioned in Table 3) and were reconstituted with water for injection for analysis. The reconstituted solution is analyzed for purity by Size Exclusion Chromatography (SE-HPLC). The samples form real-time condition were withdrawn (as mentioned in Table 2) and reconstituted with water for injection for analysis. The reconstituted solution was analyzed for purity by SDS-PAGE (data for initial time point is represented in the Fig. 1 and 2) and Size Exclusion Chromatography (SE-HPLC). In order to determine the stability of the product at 40°C, the retained amount of mono pegylated-interferon alfa 2b was plotted against time (Fig. 3) for which the formulation was stored at stress conditions (40°C). Table 1. Composition of Formulation 1 for lyophilization and storage The above formulation was lyophilized, reconstituted and analyzed for purity by SDS-PAGE as shown in Fig.l and Fig. 2. • Purity of Formulation 1 by SDS-PAGE (Initial Point) The results of the SDS-PAGE (Fig. 1 and 2) show that no new high or low molecular weight impurities were generated after lyophilization. • Real Time study data (2 - 8°C 2 Months) for Formulation 1 Table 2. Effect of real-time storage on PEG Interferon Formulation 1 analyzed by SE-HPLC. • Stress study data (40°C, 23 days) for Formulation-1 Table 3. Effect of stress on retention of monopegylated interferon alfa 2b analyzed by SE-HPLC The results of the real-time study data (Table 2) show that the total protein mass content is relatively stable over a period of 2 months. Additionally, the change in degree of the monopegylated alfa 2b (i.e., degradation to free interferon and polymer or creation of dipegylated interferon) is negligible. The results of the stress data (Table 3) show that Formulation 1 is relatively stable under stress conditions (40°C) for 23 days. Results of Cake defects: In the present invention it is observed that addition or presence of smaller amounts of trehalose along with sucrose provides elegant cake appearance and with less than 1% cake defects. Fig. 4 shows cake defects when only sucrose at concentration of 15 mg/ml is present in the formulation. Fig. 5 represents Formulation 1 containing 5mg/ml of trehalose and 15mg/ml of sucrose and there is no observable cake defect. Example 2. Experiments pertaining to Formulation 2 Compostion of Formulation 2: Formulation 2 as provided in Table 4 consists of 0.1 mg/ml of pegylated interferon alfa 2b (PEG-Interferon alfa-2b), 80mg/ml of cellobiose, 5mg/ml of trehalose, 0.5mg/ml ofpoloxamer 188 and 1.5mg/ml of each sodium phosphate dibasic anhydrous and sodium phosphate monobasic dihydrate. Study Design: The study design was identical to that carried out with Formulation 1 given in detail in Example 1. Purity and stability under room temperature and stress conditions were evaluated as described. The above formulation was lyophilized, reconstituted and analyzed for purity by SDS-PAGE as shown in Fig. 6 and Fig. 7 below. Table 4. Composition of Formulation 2 for lyophilization and storage • Purity by SDS-PAGE (lnitial point) The results of the SDS-PAGE gels (Fig. 6 and 7) show that no new high or low molecular weight impurities were generated after iyophilization. • Accelerated study data (25°C, 6 Months) for Formulation 2 Table 5. Effect of accelerated condition on PEG-Interferon formulation. • Stress study data (40°C. 15 days) for Formulation 2 Table 6. Effect of stress on % monopegylated interferon alfa-2b The results of the accelerated study data (Table 5) show that the total protein mass content is relatively stable over the period of 6 months. Additionally, the change in degree of the monopegylated alfa-2b (i.e., degradation to free interferon and polymer or creation of dipegylated interferon) is not significant. The results of the stress data (Table 6) show that the formulation-2 is relatively stable under stress conditions (40°C) for 15 days. Claims We claim: 1. A pharmaceutical formulation comprising pegylatecl interferon, a cryoprotectant, a buffer and a stabilizer wherein trehalose comprises up to 50% by weight of the cryoprotectant. 2. The formulation of claim 1 further comprising additional cryoprotectant selected from the group consisting of disaccharide and sugar alcohol. 3. The formulation of claim 2 wherein the disaccharide is selected from the group consisting of sucrose, cellobiose and maltose. 4. The formulation of claim 2 wherein the sugar alcohol is mannitol or sorbitol. 5. The formulation of claim 1 wherein the buffer is selected from the group consisting of histidine, histidine hydrochloride, sodium mono/dibasic phosphate, sodium citrate/citric acid and sodium acetate/acetic acid. 6. The formulation of claim 1 wherein the buffer is histidine or histidine hydrochloride. 7. The formulation of claim 1 wherein the stabilizer is a poloxamer or a polysorbate. 8. The formulation of claim 7 wherein the poloxamer is poloxamer 188. 9. The formulation of claim 7 wherein the polysorbate is polysorbate 20 or polysorbate 80. 10. The formulation of claim 1 wherein the stabilizer is an amino acid selected from the group consisting of histidine, arginine and lysine. 11. The formulation of claim 1 wherein the pH is maintained between 4.0 to 8.0. 12. The formulation of claim 1 wherein the pegylated interferon is interferon alpha covalently attached to linear or branched polyethylene glycol. 13. The formulation of claim 1 which is in the form of a lyophilized powder. 14. A pharmaceutical formulation comprising about 0.02 to 2 mg/ml of pegylated interferon alpha conjugate, about 5 mg/ml to 18 mg/ml of trehalose, about 5 mg/ml to 18 mg/ml of sucrose, about 0.5 mg/ml to 30 mg/ml of histidine and about 0.1 mg/ml to 1.0 mg/ml of poloxamer 188 and the pH of the formulation is maintained between 4.0 to 8.0. 15. A pharmaceutical formulation comprising pegylated interferon, a cryoprotectant, a buffer and a stabilizer wherein the cryoprotectant is cellobiose. 16. The formulation of claim 15 wherein the buffer is selected from the group consisting of monobasic/dibasic phosphate, histidine, histidine hydrochloride, sodium citrate/citric acid and sodium acetate/acetic acid. 17. The formulation of claim 15 wherein the buffer is monobasic sodium phosphate and dibasic sodium phosphate. 18. The formulation of claim 15 wherein the stabilizer is a poloxamer or a polysorbate. 19. The formulation of claim 18 wherein the poloxamer is poloxamer 188. 20. The formulation of claim 18 wherein the polysorbate is polysorbate 20 or polysorbate 80. 21. The formulation of claim 15 wherein the stabilizer is selected from the group consisting of polyvinyl alcohol, polyethylene glycol and amino acids. 22. The formulation of claim 21 wherein the amino acid is selected from the group consisting of histidine, arginine and lysine. 23. The formulation of claim 15 wherein the pH is maintained between 4.0 and 8.0. 24. The formulation of claim 15 wherein the pegylated interferon is interferon alpha covalently attached to linear or branched polyethylene glycol. 25. The formulation of claim 15 which is in the form of a lyophilized powder. 26. A pharmaceutical formulation comprising about 0.02 to 2.0 mg/ml of pegylated interferon alpha conjugate, about 60 mg/ml to 120 mg/ml of cellobiose, about 5 mM to 150 mM of phosphate buffer and about 0.1 mg/ml to 1.0 mg/ml of poloxamer 188 and the pH of the formulation is maintained between 4.0 to 8.0. 27. A process of lyophilization of the formulation of claim 1 or claim 15, wherein the said process comprises the steps of (a) formulating the ingredients of claim 1 or claim 15 in a solution (b) aliquoting the solution in vials (c) placing the vials in lyophilizer chamber at ambient pressure and between -50°C and 4°C (d) subjecting the loaded vials to freezing at ambient pressure for 2 to 12 hours at a temperature between -50°C and 40°C (e) drying the frozen solution in two steps under vacuum at a pressure of between 300 and 50 millibar: (i) performing the primary drying step at a temperature between -40°C and-15°C; (ii) for 15 to 45 hours (ii) performing the secondary drying step at a temperature between 15°C and 35°C for 4 to 12 hours; (e) unloading the vials containing the final lyophili^ed powder at ambient pressure and room temperature.

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2077-mum-2008-abstract.doc

2077-mum-2008-abstract.pdf

2077-MUM-2008-CLAIMS(30-11-2009).pdf

2077-MUM-2008-CLAIMS(AMENDED)-(21-5-2012).pdf

2077-MUM-2008-CLAIMS(AMENDED)-(6-3-2012).pdf

2077-MUM-2008-CLAIMS(MARKED COPY)-(6-3-2012).pdf

2077-mum-2008-claims.doc

2077-MUM-2008-CORRESPONDENCE(11-5-2012).pdf

2077-MUM-2008-CORRESPONDENCE(24-9-2009).pdf

2077-MUM-2008-CORRESPONDENCE(30-11-2009).pdf

2077-MUM-2008-CORRESPONDENCE(7-5-2010).pdf

2077-MUM-2008-CORRESPONDENCE(8-2-2010).pdf

2077-mum-2008-correspondence(ipo)-(11-1-2010).pdf

2077-mum-2008-correspondence.pdf

2077-MUM-2008-DESCRIPTION(COMPLETE)-(30-11-2009).pdf

2077-mum-2008-description(provisional).doc

2077-mum-2008-description(provisional).pdf

2077-MUM-2008-DRAWING(30-11-2009).pdf

2077-MUM-2008-DRAWING(6-3-2012).pdf

2077-mum-2008-drawing.pdf

2077-MUM-2008-FORM 1(30-11-2009).pdf

2077-MUM-2008-FORM 1(7-5-2010).pdf

2077-mum-2008-form 1.pdf

2077-MUM-2008-FORM 13(11-5-2012).pdf

2077-mum-2008-form 13(24-9-2009).pdf

2077-mum-2008-form 13(8-2-2010).pdf

2077-MUM-2008-FORM 18(30-11-2009).pdf

2077-mum-2008-form 2(30-11-2009).pdf

2077-MUM-2008-FORM 2(TITLE PAGE)-(30-11-2009).pdf

2077-mum-2008-form 2(title page).pdf

2077-mum-2008-form 2.doc

2077-mum-2008-form 2.pdf

2077-MUM-2008-FORM 26(24-9-2009).pdf

2077-MUM-2008-FORM 26(30-11-2009).pdf

2077-MUM-2008-FORM 3(30-11-2009).pdf

2077-MUM-2008-FORM 5(30-11-2009).pdf

2077-MUM-2008-FORM 5(7-5-2010).pdf

2077-MUM-2008-FORM 9(30-11-2009).pdf

2077-MUM-2008-RECEIPT(24-9-2009).pdf

2077-MUM-2008-REPLY TO EXAMINATION REPORT(6-3-2012).pdf

2077-MUM-2008-REPLY TO HEARING(21-5-2012).pdf

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