A STABLE COMPOSITION OF PEG-INTERFERON ALPHA 2b CONJUGATE

FIELD OF THE INVENTION
The present invention relates to the field of biopharmaceutical sciences. More specifically, it relates to a stable composition of PEG-interferon conjugates and process for the preparation of the same.

BACKGROUND OF THE INVENTION
With increase in the number of applications of proteins, both natural and recombinant, the need for stable protein compositions is growing. As the protein is instable in liquid condition and also since the logistics of product shipping and use, precludes the storage as an aqueous solution, the protein is lyophilized (free-dried) and presented as solid forms. Developing the recombinant protein pharmaceuticals has proved to be very challenging because of both complexity of protein production, purification and the limited physical and chemical stability of proteins. Instability is one of the main reasons for the administration through injection rather than oral administration. To overcome the instability proteins have to be in solid forms to have the extended shelf life.

PEGylated Interferon alpha-2b is an antiviral drug and is said to have a dual mode of action both antiviral and on the immune system. This drug is approved around the world for the treatment of chronic hepatitis. Like other proteins PEGylated Interferon alpha-2b is also subject to degradation during the process of freeze drying and also during its storage in shelf life.

PEGylated Interferon alpha-2b, experiences a variety of instabilities during the process of freeze drying and later during storage. Such major instabilities include aggregation, deamidation, oxidation, the Maillard reaction, hydrolysis, and disulfide bond formation/ exchange. Many factors effect these instabilities, including storage temperature, glass transition temperature of the composition, residual moisture content, composition ‘pH’, crystallization of amorphous excipients, and presence of destabilizing excipients or contaminants. These instabilities may be minimized by proper selection of composition pH, residual moisture content, and more importantly, composition stabilizers, such as sugars/polyols, polymers, salts, and surfactants.

Even after successful lyophilizations, the long term stability may still be very limited, especially at higher temperatures. Pegylated interferon composition and freeze-drying in the presence of 8% sucrose and 0.1% ploysorbate-80 (tween-80) and phosphate buffer is stable at 250C, but it is not stable at 400C for longer periods as per the US patent, patent number 6180096.

A stability of 400C is especially critical for tropical countries, wherein the temperature is high. As none of the process and compositions available in the market are suitable for composition and logistics in tropical zone, the present invention attempts to solve and overcome the difficulties of prior art.

The present invention of the composition and lyophilization for the pegylated interferons are stable at 400C for longer time periods.

OBJECTIVES OF THE INVENTION:
The object of the present invention is to develop a storage stable composition comprising PEGylated Interferon alpha-2b.

Another object of the invention is to develop a stable composition of PEGylated Interferon alpha-2b that is stable at higher temperatures and for a longer period of time.

Another aspect of the present invention is to develop a stable composition of PEGylated Interferon alpha-2b of PEG Interferon alpha-2b that maintains not only the chemical stability but biological activity at higher temperatures.

Yet another object of this invention is to provide a process for the preparation of stable composition of PEGylated Interferon alpha-2b.

SUMMARY OF THE INVENTION
The present invention relates to a novel, stable composition of PEGylated Interferon alpha-2b. The novel composition comprising of amino acids in conjunction with sugars, buffers, salts and optionally other pharmaceutically acceptable excipients conferred stability to the active ingredient for extended periods over significant range of temperatures.

DETAILED DESCRIPTION OF THE INVENTION:
Accordingly present invention provides a storage stable composition comprising PEGylated Interferon alpha-2b.

The stable composition of the present invention comprises of PEGylated Interferon alpha-2b, amino acids, disaccharides, buffers, surfactants and other pharmaceutically acceptable excipients.

The stable composition of the present invention comprises PEGylated Interferon alpha-2b. PEG-Interferon alpha conjugates refer to Interferon alpha-2b molecule covalently attached to a PEG molecule through urethane bond predominantly either through lysine or histidine amino acids. Alternatively interferon conjugates can be obtained through covalent linkage at lysine residues of interferon with 40kDa branched PEG. In a preferred embodiment the present invention comprises interferon alpha-2b produced from E. coli.

The composition also includes a buffer. Buffers suitable for the maintenance of pH of the composition are sodium phosphate buffers (give a wide range of buffers) with pH in the range of 6-5-7.5, the most preferred pH being 6.8. The sodium phosphates used are dibasic sodium phosphate anhydrous and monobasic sodium phosphate dihydrate, the equal mass of concentrations 0.05 to 0.1 molar, are preferred for the present invention.

The composition of present invention includes a stabilizing agent. The stabilizing agent is selected from a group comprising surfactants. The surfactant used for the present invention is polysorbate-80. In general, the amount of stabilizing agent is most preferably 0.1mg/ml.

The composition of the present invention also includes amino acids. The amino acids are selected from the group comprising neutral charge. Preferably the amino acid is glycine or alanine. Most preferably the amino acid is glycine. The amino acid is generally present in low quantities, most preferably the amino acid is in the range of 0.05 to 2.0mg/ml.

The composition of the present invention also includes Cryoprotectants. The cryoprotectant is selected from a group comprising of sugars, polyols, polymers, amino acids, non-aqueous solvents, and surfactants. More preferably, the cryoprotectant is sugar, preferably a disaccharide, most preferably, the cryoprotectant is sucrose. The cryoprotectant is present in an amount of 50 to 100mg/ml, most preferably 80mg/ml.

The composition of the present invention also comprises of other pharmaceutically acceptable excipients.

The composition of the present has an extended storage life time without any detoriation of biological activity. The compositions as described herein maintain the chemical and biological stability for a period of at least 3 months at 400C.

The composition of the present invention is also synergistic in that the ingredients when constituted together as per the principles herein, yield a composition having stability for over 3 months at 40?C.

The invention is described in detail herein below with respect to the following examples, which are provided merely for illustration and are not intended to restrict scope of invention in any manner. Any embodiments that may be apparent to a person skilled in the art are deemed to fall within the scope of present invention.

EXAMPLE 1:
This example provides a description of a composition of the present invention and protection of PEG-Interferon conjugates during lyophilization and shelf life.

Description of the composition:
Component mg/vial*
PEG-Interferon alpha-2b conjugates 0.1
Sodium phosphate dibasic anhydrous 0.75
Sodium phosphate monobasic dihydrate 0.75
Sucrose 40
Polysorbate-80 0.05
Glycine 0.5
Water for injection q.s 0.5ml*
*Amount contained in a labeled volume of 0.5ml
After lyophilization, the resulting cake/powder is stored, over a period of six months. The reconstitution is done with water for injection for the analysis. The analysis is done for the visual clarity, purity by SEC-HPLC for the degree of conjugation, protein content, and biological activity etc. The results of the stability described here are few of the important tests and are as follows:
Stability data on 80mcg vial
Time
months Temp
0C Description After
Reconstitution Protein content Purity of PEGIFN on SEC-HPLC Activity assay
(IU/mg)
Spec NA White solid to powder CCS NLT 160mcg/ml 95.0-100% monoPEG NLT 0.4X108
T0 NA White solid CCS Conform 96.5 0.64 X108
T1 5 White solid CCS Conform 96.5 0.63X108
25 White solid CCS Conform 96.5 0.60 X108
40 White solid CCS Conform 96.4 0.64X108
T2 5 White solid CCS Conform 96.5 0.62X108
25 White solid CCS Conform 96.5 0.63 X108
40 White solid CCS Conform 96.4 0.61X108
T3 5 White solid CCS Conform 96.5 0.63 X108
25 White solid CCS Conform 96.5 0.63 X108
40 White solid CCS Conform 96.3 0.64 X108

NA: not applicable; CCS: clear colorless solution; NLT: not less than SEC: size exclusion chromatography

The results shows that the appearance after reconstitution and the protein content have no change when compared to initial results for a period of 3 months at 400C (Accelerated conditions). The change degree of conjugation (degradation to IFNs and polymers) is same as that of the initial results for a period of 3 months at 400C. Basing upon these results it is clear that the lyophilized product of PEG-Interferon alpha-2b conjugates is stable for 3 months at 400C.

EXAMPLE 2:
Description of the composition:
Component mg/vial*
PEG-Interferon alpha-2b conjugates 0.1
Sodium phosphate dibasic anhydrous 0.75
Sodium phosphate monobasic dihydrate 0.75
Sucrose 40
Polysorbate-80 0.05
L-Alanine 0.5
Water for injection q.s 0.5ml*
*Amount contained in a labeled volume of 0.5ml

The compositions containing Glycine, lyophilized cake is very rigid when compared with the compositions that contain Alanine. And also the pattern of stability is better with glycine composition rather than alanine.

EXAMPLE 3:
Description of the composition:
Component mg/vial*
PEG-Interferon alpha-2b conjugates 0.1
Sodium phosphate dibasic anhydrous 0.75
Sodium phosphate monobasic dihydrate 0.75
Mannitol 25.0
Polysorbate-80 0.05
NaCl 4.5
Water for injection q.s 0.5ml*
*Amount contained in a labeled volume of 0.5ml

EXAMPLE 4:
Description of the composition:
Component mg/vial*
PEG-Interferon alpha-2b conjugates 0.1
Sodium phosphate dibasic anhydrous 0.75
Sodium phosphate monobasic dihydrate 0.75
Trehalose 10.0
Polaxymer-188 0.05
Water for injection q.s 0.5ml*
*Amount contained in a labeled volume of 0.5ml
EXAMPLE 5:
Description of the composition:
Component mg/vial*
PEG-Interferon alpha-2b conjugates 0.1
Sodium phosphate dibasic anhydrous 0.75
Sodium phosphate monobasic dihydrate 0.75
Polyethylene glycol 20.0
Glycine 10.0
Water for injection q.s 0.5ml*
*Amount contained in a labeled volume of 0.5ml

EXAMPLE 6:
Description of the composition:
Component mg/vial*
PEG-Interferon alpha-2b conjugates 0.1
Sodium phosphate dibasic anhydrous 0.75
Sodium phosphate monobasic dihydrate 0.75
L-Ariginine 25.0
Glycine 25.0
Water for injection q.s 0.5ml*
*Amount contained in a labeled volume of 0.5ml

EXAMPLE 7:
Description of the composition:
Component mg/vial*
PEG-Interferon alpha-2b conjugates 0.1
Sodium phosphate dibasic anhydrous 0.75
Sodium phosphate monobasic dihydrate 0.75
Human serum albumin 0.5
Glycine 10.0
Water for injection q.s 0.5ml*
*Amount contained in a labeled volume of 0.5ml

Results from example 03 to 07:
Composition of pegylated interferon with PEG (Example 05) and composition with L-arginine (Example 06) failed during lyophilization by having collapsed cake. The compositions with HSA, Trehalose and mannitol are not effective in stabilizing the protein over a course of time at both real time and accelerated conditions.