FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION (See section 10 and rule 13)
A LYOPHILIZED FORMULATION OF PEG-INTERFERON ALPHA 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 describes the invention.

FIELD OF THE INVENTION
The invention relates to the formulations of pegylated interferons. In particular, the invention provides pharmaceutical formulations of pegylated interferons comprising pegylated interferon and pharmaceutically acceptable excipients wherein the formulation is a lyophilized powder.
BACKGROUND OF THE INVENTION
Various natural and recombinant proteins have pharmaceutical utility. Once they have been purified, separated, and formulated, they can be parenterally administrated for various therapeutic applications. However, parenterally administered proteins may be immunogenic, may be relatively water insoluble, and may have a short pharmacological half life. Consequently, it can be difficult to achieve therapeutically useful blood levels of the proteins in patients.
These problems can be overcome by conjugating the proteins to polymers, such as polyethylene glycol. While preparation of protein-polymer conjugates is beneficial, they cannot be used in a practical manner unless they can be stored for an extended period of time during manufacturing and distribution to health care providers, even in frozen solutions.
So, there was a need for a formulation suitable to protect protein polymer conjugates, and in particular pegylated interferon alpha conjugates, from damage during lyophilization. Such formulation allowed pegylated interferon alpha polymer conjugates to maintain their biological activity, physical stability and chemical stability over extended periods of time. Two commercially available products exist namely peg-interferon alpha 2a marketed by Hoffmann La Roche under the tradename Pegasys®, and peg-interferon alpha 2b marketed by Schering Plough Corporation under the tradename PEG-Intron®.
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 should be stable for a prolonged period 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-alpha; (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 disclosed about the invention relates to a lyophilized powder produced by lyophilization of an aqueous formulation that prevent loss and damage of PEG- interferon alpha conjugates during and following lyophilization. The formulations protect PEG-interferon alpha conjugates from loss and degradation during the lyophilization process, as well as degradation during subsequent storage. The formulations are suitable for protection of PEG-interferon alpha conjugates from various types of degradation, including, but not limited to loss of biological activity and changes in the degree and/or nature of conjugation. A preferred PEG-interferon alpha conjugate protectable in the formulations is an interferon alpha-2b-polyethylene glycol (12,000) conjugate.
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-1,2-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.
WO2006/020720 describes about a lyophilized formulations of pegylated interferon which are prepared using trehalose as a cryoprotectant. The formulations have a low moisture content, which helps stabilize the pegylated interferon during storage of the formulations at room temperature. In addition, methods for preparing these formulations are provided.
Indian patent application 412/MUM/2007 describes about' the invention relates to novel lyophilized and stabilized formulations of PEG-interferon alpha conjugates and the process for their preparation. The novel formulation of PEG-interferon alpha conjugates reported by the inventors of this application requires shorter lyophilization cycle and are more cost competitive.

Aqueous formulations have been found to be inappropriate for some pegylated interferon alpha conjugates as they are unable to prevent the breakdown of the conjugate to release free interferon alpha. While lyophilized formulations are in general better protective of pegylated interferon alpha conjugates than aqueous formulations. Furthermore, some of the manufacturing processes for some lyophilized formulations such as sucrose based formulations are known to be not very cost effective. Therefore, there is a need of better formulations of the pegylated interferon alpha conjugates than the existing ones.
The present invention provides a formulation of stable pegylated interferon with melezitose as a cryoprotectant, a buffer and a stabilizer. It is for the first time that pegylated interferon has been formulated using melezitose as a cryoprotectant to render stability to the lyophilized compound.
SUMMARY OF THE INVENTION
The present invention provides a novel composition of pegylated interferon comprising pegylated interferon and a cryoprotectant wherein the cryoprotectant is melezitose.
In one aspect, the composition further comprising, a buffer, a stabilizer, a solvent and a cryoprotectant wherein the cryoprotectant is melezitose either alone or in combination with other cryoprotectants like disaccharide, sugar alcohols.
In another aspect of the present invention, the buffer of the above said formulation is selected from the group consisting of phosphate, succinate, acetate, citrate, histidine or glycinate buffers either alone or in combination.
In yet another aspect, the above said composition comprises the surfactant which is a poloxamer or a polysorbate. The most preferred poloxamer is poloxamer 188 and the preferred polysorbate is polysorbate 20 or polysorbate 80.
The invention also encompasses a process for preparing a lyophilized composition of pegylated interferon comprising providing a solution which comprises a pegylated interferon, one or more cryoprotectants, a buffer, a stabilizer, wherein the cryoprotectant is Melezitose either alone or in combination with other cryoprotectants like disaccharide, sugar alcohols and the buffer maintains the pH of the formulation from 4.0 to 8.0 wherein the lyophilization process comprises the steps

of (a) formulating the pegylated interferon of the said formulation in a solution (b) aliquoting the solution in vials (c) placing the vials in a lyophilizer chamber at ambient pressure and at a temperature between -50°C and 24°C (d) subjecting the loaded vials for 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. Performing the primary drying step at a temperature between -40°C and -15°C for 15 to 45 hours and performing the secondary drying step at a temperature between 15°C and 35°C for 4 to 12 hours (f) unloading the vials containing the final lyophilized cake at ambient pressure and room temperature.
DESCRIPTION OF THE DRAWINGS
Figure 1 shows the stability of the final drug product during its shelf life
Figure 2 shows the physical appearance of the final drug product in the vial, physical appearance of the cake and the drug product after reconstitution with the reconstitution medium.
Figure 3 shows the SEC-HPLC Chromatogram during its initial storage (Blue line) and after one year storage (Red line). The percentage of Mono PEGylated protein is retained throughout the study period.
DESCRIPTION OF THE INVENTION
The present invention provides novel pharmaceutical composition of pegylated interferons that are stable during long-term storage at room temperature, as well as withstand deleterious effects that occur during lyophilization process.
The present invention is not limited to specific chemicals for the solution components in the novel pharmaceutical compositions. The present invention is also not limited by the concentrations of the components in the novel pharmaceutical compositions.
PEG-interferon alpha conjugates according to the present invention are Interferon alpha molecules or.their variants covalently linked to one or more PEG molecule/s. Suitable PEG molecules may be those well known and used in preparing PEGylated biological products for

therapeutic use. The PEG-interferon alpha conjugates of the present invention may comprise of Interferon alpha-2a, Interferon alpha-2b or Interferon alpha-2c and their suitable variants.
PEGylated Interferon alpha conjugates are interferon alpha molecules covalently linked to one or more Polyethylene glycol molecules. Commercially available PEGyalted interferon alpha conjugates include PEGASYS (PEGylated Interferon Alpha 2a) from Hoffmann La Roche and PEG-INTRON from Schering Plough (PEGylated Inteferon Alpha 2b).
40kDa branched Lysine mPEG-NHS PEG is used in PEGASYS. The PEG used in 40kD branched Lysine mPEG Interferon alpha 2a consists of two monomethoxy PEG chains, each with an average molecular weight of 20kD. The carboxyi group of the lysine linker is activated to an 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 epsilon group and another at the lysine alpha amino group.
PEG-INTRON is a covalent conjugate of Interferon alpha 2b with a linear 12kD PEG molecule. It is synthesized by the reaction of purified recombinant interferon alpha 2b with an electrophilic derivative of PEG, succinimidyl carbonate PEG (SC-PEG), in 100rnM sodium phosphate with pH 6.6. 12kD SC-PEG is a linear mono-methoxy polyethlylene glycol which 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 fractioned by ion-exchange chromatography to yield predominantly mono-pegylated interferon alpha 2b.
Lyophilization is a process of freeze-drying a composition wherein a frozen aqueous mixture is treated to remove water. Commonly, the process involves the sublimation of water from the frozen aqueous solution, usually under reduced pressure conditions. After lyophilization, the pegylated interferon alpha conjugate can be stored for extended periods of time.
PEGylated Interferon alpha conjugates, however, are subject to damage during and after lyophilization. Hence, there is a need for a novel lyophilized formulation of Pegylated Interferon alpha conjugates produced by lyophilization of an aqueous formulation that allow stabilization of Pegylated interferon alpha conjugates during and after lyophilization.

The present invention protects pegylated interferon alpha conjugates from damage by including them in formulations that prevent damage during and after lyophilization. While the present invention is not limited to a particular formulation, the formulations that are anticipated here utilize a suitable buffers), suitable stabilizer(s), suitable cryoprotectant(s) and/or lyoprotectant(s), a bulking agent(s) and solvents, in addition to the pegylated interferon alpha conjugate. Various possible combinations of the selected groups of buffers, stabilizer and cryoprotectants can be used to prepare the novel formulation of the present invention.
The buffer system, which may be used comprises of phosphate, succinate, histidine, glycinate and the like, either alone or in suitable combination, which provides the desired pH range. The preferred pH range is between 4.5 to 7.1, preferably 6.5 to 7.1 and most preferably 6.8. The preferred molar concentration of the buffer is in the range of 0.001 to 0.5 M. Preferred buffers may be selected from sodium phosphate, sodium succinate, potassium succinate, histidine chloride, sodium glycinate or their suitable combinations. Other buffer systems may also be used which maintain the desired pH range.
Stabilizer used in the present invention is either a poloxamer or a polysorbate. The stabilizers may also be selected from the group consisting of glycerol, dimethylsulfoxide, polyvinyl alcohol (PVA), polyethylene glycol (PEG) and 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 term "cryoprotectants" generally includes agents, which provide stability to the protein from freezing-induced stresses, however the term also includes agents that provide stability. Examples of cryoprotectants include different saccharides and polyols e.g. sucrose, trehalose, melizitose, mannitol, Sorbitol, etc., either alone or in suitable combinations.
The term cryoprotectant includes agents that provide stability to the protein during water removal from the system during the drying process, presumably by maintaining the proper conformation of the protein through hydrogen bonding.
Cryoprotectants can also have lyoprotectant effects, therefore, the terms "cryoprotectant" and "lyoprotectant" are used interchangeably, herein.

Suitable solvent for the present formulation is water; preferably the solvent may be water for injection.
Other suitable excipients may be optionally added to the formulation. Such excipients include glycine at suitable concentration so as to further stabilize the formulation.
The novel pharmaceutical compositions.of PEG-Interferon alpha conjugates are prepared using suitable combinations of a buffer, stabilizer, cryoprotectant(s) and/ or lyoprotectant(s) and a solvent, optionally with other excipients and suitably lyophilized and stored as a dry powder to be reconstituted before use.
Following example illustrate the described pharmaceutical compositions of the present invention and the means of carrying out the invention to obtain a stable pharmaceutical dosage form of pegylated interferon alpha conjugates.
Example 1
PEG-interferon alpha conjugate prepared as per techniques known in the art was formulated as described in Table 1.
Table 1

S.No. Ingredient Concentration (mg/ml)
1. PEG12000 Interferon a 2b 0.1
2. Melezitose 80
3. Poloxamer 188 0.5
4. Sodium Phosphate Dibasic Anhydrous 1.5
5. Sodium Phosphate Monobasic Dihydrate 1.5
6. Water for Injection q.s to 1.0 ml
The formulation given in Table 1 is prepared and subsequently lyophilized. After lyophilizatiqn the samples were stored at 2 0C to 8 °C and at different periods of time, samples were reconstituted with water for injection for analysis. The reconstituted samples were analyzed for visual clarity, in-vitro antiviral activity, and the level of free interferon.

Lyophilization was carried out by placing the above specified solution in glass containers followed by loading the glass containers into a lyophilizer at ambient pressure and temperature. Loading the filled vials into the lyophilizer may be performed at ambient pressure and at a temperature between -50 °C and 4°C. The loaded containers may be subjected to freezing at ambient pressure for 4 to 10 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 90 millibar (mbar), preferably between 250 and 100 mbar. The primary drying step may be performed for 15 to 25 hrs at a temperature between -38°C and -15°C while secondary drying step may be performed for 5 to 12 hour's at a temperature between 15 °C to 35 °C. After completion of lyophilization process, stopper the vials under vacuum. Unloading the glass containers containing the lyophilized powder may be performed at ambient pressure and room temperature or below. The detailed information regarding lyophilization cycle is shown in Table 2.
Table 2

S. No. Step Temperature (°C) Vacuum
(μ-bar) Time
(hh:mm)
1. Loading -50 — ...
2. Freezing -50 — 8:00
3. Evacuation — 300 —
4. Drying -35 260 16:00
5. Drying -22 100 12: 00
6. Drying 20 80 8:00
7. Drying 30 60 6:00
8. Drying 35 60 4:00
After lyophilization, vials were collected and stored at 5 (±3) °C, until it was used for further analysis. Samples were reconstituted with water for injection for analysis at different periods of time, as specified in Table 2. The reconstituted solutions were checked for visual clarity. Stability of PEG-interferon alpha conjugate was assessed for its in-vitro antiviral activity and level of free interferon (degree of depegylation) present in the reconstituted solution, as shown in Table 3.
Table 3

Time Storage Temperature % Mono PEGyated IFN % Free IFN Visual Clarity
Initial 5°C 94.8 2.0 Clear
6 months 5°C 94.2 2.2 Clear
9 months 5°C 94.0 2.4 Clear
12 months 5°C 91.6 3.6 Clear

The novel formulations of pegylated interferon alpha conjugates are prepared using suitable combinations of a buffer, stabilizer, cryoprotectant(s) and/or lyoprotectant(s) and a solvent, and lyophilized and stored as a dry powder to be reconstituted before use.
The formulations prepared such; contain an effective amount of biologically active pegylated interferon and are useful in treating several diseases such as Hepatitis B and C and various other cancer indications. They are preferably used as injectable aqueous solutions.
The novel lyophilized and stabilized formulations of pegylated interferon alpha conjugates described in the present invention have the following advantages.
1. Involve Operational simplicity.
2. Improves product stability in lyophilized condition in long-run
3. Involve use of novel cryoprotectants and/or iyoprotectants, which are less hygroscopic in nature.
4. Provide better physical strength to the lyophilized formulation.
5. Identifying and proving effectiveness of novel sugars with biopharmaceutical product explores newer possibilities of using the same as formulation components.
6. All the above factors contribute to the cost effectiveness of the process of this invention.

We Claim
1. A pharmaceutical composition comprising PEGylated interferon and a cryoprotectant wherein the cryoprotectant is melezitose.
2. A pharmaceutical composition of claim 1, further comprising buffer(s), stabilizer(s), and a solvent.
3. A pharmaceutical composition as claimed in claim 1, wherein the buffer is selected from phosphate, succinate, acetate, citrate, histidine or glycinate buffers either alone or in combination.
4. A pharmaceutical composition as claimed in claim 1, wherein the stabilizer is selected from poloxamer or polysorbate.
5. A pharmaceutical composition as claimed in any one of the preceding claims, wherein said interferon alpha molecules are selected from the group consisting of interferon alpha 2a, interferon alpha 2b, interferon alpha 2c or their suitable variants.
6. A pharmaceutical composition of claim ] wherein the pegylated interferon is interferon alpha covalently attached to linear or branched polyethylene glycol.
7. A pharmaceutical composition of any one of the preceding claims comprising PEG-interferon alpha conjugate, melizitose as a cryoprotectant, Polysorbate as a surfactant, sodium phosphate as a buffer and water as a solvent.
8. A process of lyophilizing the pharmaceutical composition in any of the preceding claims to obtain a lyophilized powder.
9. A process of preparing a lyophilized composition of PEG-Interferon alpha conjugates comprising, combining a suitable buffer, a suitable stabilizer, a suitable cryoprotectant and a solvent, as claimed in any of the preceding claims, wherein the said cryoprotectant is melezitose and subsequently, lyophilizing the mixture to obtain a lyophilized powder.

10. The lyophilized powder of any preceding claims, wherein said powder comprises 0.08% of said PEG-interferon alpha conjugates as measured by the mass of the interferon alpha, 3.6% of said sodium phosphate, 0.12% of said polysorbate and 96.2% of said melezitose, by weight.