FIELD OF THE INVENTION

The present invention relates to an improved lyophilized composition of PEG Interferon alpha conjugates that can be stored at elevated temperatures and a method for producing the same.

BACKGROUND OF THE INVENTION

Interferons (IFNs) are a family of structurally related cytokines with a hallmark function of antiviral activity. IFNs exhibit a diversity of biological functions, represented by antiviral activity, antitumor activity and immunomodulatory effects. Interferons can be further classified as IFN-a, IFN-p\ IFN-y. IFN- a could be further classified as IFN-a 2a and IFN a-2b. IFN a-2b is used to treat hepatitis (hepatitis-B and C) and genital warts. IFN is currently recommended for patients with compensated chronic Hepatitis-B with detectable HBsAg, HBeAg and HBV DNA.

Since interferon, like any other protein, is subjected to degradation and immunogenicity, an improved version, administered in a pegylated form was developed for administration to the patients. Pegylation is the process of covalent attachment of polyethylene glycol (PEG) polymer chains to another molecule, normally a drug or therapeutic protein. The covalent attachment of PEG to a drug or therapeutic protein can "protect" the active agent from aggressive reactions of the host's immune system (hence the reduced immunogenicity and/or antigenicity), increase the hydrodynamic size (size in solution) of the agent which prolongs its circulatory time by reducing its clearance.

In its native form some PEG Interferon conjugates in a liquid solution have got a limited stability. Hence there is a need to develop a lyophilized product to overcome the deterioration. However, even after successful lyophilization, the long term stability may still be very limited, especially at higher temperatures around 40°C. It may be noted that the lyophilized product is usually prepared in the form of a cake like structure. The integrity of the cake like structure and the anti collapsive property of the cake like structure is important for the effective stability of the product. Anti collapsive property of cake defines the integrity of the shape of cake initially prepared.

Prior art compositions of the pegylated interferon have seemingly paid less attention to the quality and the anticollapsive property of the lyophilized powder. In fact, most prior art documents do not even mention the quality of the cake and have sometimes erroneously referred to the product as being available as a powder. The retention of this "cake" like structure formed due to lyophilization process is integral to the stability of the product.

Pegylated interferon compositions of prior art have been shown to be stable at best at 25°C, but were found unstable at 40°C for extended periods of time.

In temperature conditions as seen to exist in the tropical Countries such as India, stability at 25°C and 40°C, even if it were for shorter periods of exposure is critical. It was noted that these products tends to expose to higher temperature around 40°C accidentally/ unavoidably by patients and pharmacies handling these products. At higher temperature such as 40°C, even intermittent exposure for short duration led to an unstable product. This has also been proved by accelerated studies conducted in the laboratory. In fact it was often noted that the lyophilized cake of the prior art composition collapsed and lost its "intactness" at such temperature (40 °C).

As none of the process and compositions available in the market are suitable for storage and logistics in the tropical zone, the present invention attempts to solve and overcome these difficulties of prior art.

OBJECT OF THE INVENTION

An object of the invention is to provide an improved lyophilized composition of PEG interferon alpha that has enhanced stability and a method for producing the same.

SUMMARY OF THE INVENTION

The present invention relates to an improved lyophilized composition of pegylated Interferon alpha-2b conjugate and a method for producing the same. The composition comprises a pegylated Interferon alpha-2b, a cryoprotectatant, a buffer, and optionally other pharmaceutically acceptable excipients. The composition of the present invention is stable for extended periods of two years at 25°C and six months at higher range of temperatures around 40 C, without any degradation.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

Prior art compositions comprising PEG interferon alpha 2b, comprising stabilizers such as trehalose/sucrose have been found to degrade due to higher temperature in India. Hence such prior art product required cold chain in order to maintain the stability of the drug.

Products that have to be transferred in cold chain requires

1. Transport by air wherein the field has to be informed about the date and time of arrival.

2. Transport in insulated packaging boxes with ice packs. These boxes usually maintain the correct temperature for at least 72 hours after leaving the supplier.

3. To prevent interruption in the cold chain, transport of the cold boxes to the place of destination is recommended and additionally there are usual instructions to place the drugs in a refrigerator upon arrival.

4. Shipments must be accompanied by a cold chain monitor card, data loggers etc. It is obvious for those in the field that in order to maintain the conditions as exampled above increases the transit costs of each shipment and eventually has bearing on the price and shelf life of the product.

To avoid such transit related inconveniences, the present invention provides a storage stable composition of pegylated Interferon alpha-2b that is stable even at 25°c for at least 24 months period and at 40°C for at least 6 months period. In addition, the storage stable composition of the present Invention exhibit anticollapsive property. Anticollapsive property defines as retaining of initial intact shape and structure as seen at the time of initial primary packaging without the loss of the original shape; color and nature of the lyophilized "cake".

Accordingly, the present Invention is drawn to a composition comprising a pegylated Interferon alpha-2b, a starch based cryoprotectant, a buffering agent, a stabilizing agent, and optionally other pharmaceutically acceptable excipients wherein the composition is stable at 25°C for two years and 40°C for period of six months.

Conjugated pegylated Interferon alpha-2b of the stable composition disclosed in the present invention may refer to Interferon alpha-2b molecule covalently attached to a PEG molecule of 12kDa through urethane bond. This urethane bond is either through lysine or histidine amino acids predominantly.

In a preferred embodiment the present invention may comprises interferon alpha-2b produced from E. coll More preferably, the product is prepared through specific processes and preparations as enumerated in patent application: 2722/CHE/2009.

The amount of pegylated interferon alpha-2b present in the composition may be in the range of 0.05mg to 2.0mg/ml. Most preferably, the amount of pegylated Interferon alpha-2b may be in the range of 0.07mg to 0.3mg/ml

The buffer suitable for the maintenance of pH of the composition may be selected from a sodium phosphate buffer or a Histidine buffer, preferably, the buffer may be sodium phosphate buffer. The sodium phosphate buffer may comprise of dibasic sodium phosphate anhydrous and monobasic sodium phosphate dihydrate or monohydrate. The buffer is selected such that the pH of the composition is maintained in the near neutral range.

It is preferred that the pH of the composition may be maintained in the range of 6-5-7.5. The amount of the buffer present in the equal mass of composition for monobasic and dibasic may be in the range of O.lmg/ml to lOmg/ml, more preferably, 1.5mg/ml.

The stabilizing agent present in the composition may be selected from the group comprising wetting agent and a surfactant. Preferably, the stabilizing agent may be selected from the group comprising Tween-20, Tween-80, Pluronic F-68, Briz-35. Most preferably, the stabilizing agent may be Pluronic F-68.

The amount of stabilizing agent may be present in a range of 0.05-2.0mg/ml, and most preferably, l.Omg/ml.

The cryoprotectant present in the composition of the present invention may be selected from the group comprising a sugar, a polyol, a polymer, an amino acid, a non-aqueous solvent, and a surfactant. Preferably, the cryoprotectant may be a starch, more preferably cryoprotectant may be a cyclic oligosaccharide. The cyclic oligosaccharide may be a cyclodextrin. The cyclodextrin may be selected from the group comprising a-Cyclodextrin (aCD), p-Cyclodextrin (pCD), 2-Hydroxypropyl-p-cyclodextrin (HPpCD; Kleptose® HPB), Sulfobutylether P-cyclodextrin sodium salt (SBEpCD; Captisol®), Randomly methylated P-cyclodextrin (RMpCD), y-Cyclodextrin (yCD), 2-Hydroxypropyl-y-cyclodextrin (HPyCD)., preferably the cyclodextrin may be 2-Hydroxy Propyl p-Cyclodextrin.

The cryoprotectant may be present in the range of 10-200mg/ml, most preferably, cryoprotectant may be lOOmg/ml.

In an aspect, the composition may optionally comprise other pharmaceutical excipients. The excipients may be selected from the group comprising aminoacid, preferably, the aminoacid may be glycine.

The amount of aminoacid present in the composition is 10-30mg/ml, preferably, 20 mg/ml

It is observed that the composition of the present invention is stable and that there is no collapse of the lyophilized cake for extended periods of time at 25°C for at least 24 months period and at 40°C for at least 6 months period. In particular, the composition of the present invention maintains the physical integrity, chemical and biological stability, not less than 95.0% at 25°C for at least 24months period and at 40°C for at least 6 months period.

In an aspect, the present invention also provides a method for preparing the improved composition of the PEGInterferon alpha-2b. The method comprising the steps of:

i) mixing of PEGInterferon alpha-2b, a buffering agent, a cryopreservant, a stabilizing agent and pharmaceutical excipients in a fixed ratio in water to obtain a clear solution;

ii) adjusting the pH of the solution obtained in step (i);

iii) making the volume and sterilization of the Solution obtained in step (ii);and

iv) lyophilization of the solution obtained in step (iii);

The Conjugated pegylated Interferon alpha-2b used in step (i) may be an Interferon alpha-2b molecule covalently attached to a PEG molecule of 12kDa through urethane bond. This urethane bond is either through lysine or histidine amino acids predominantly.

In a preferred embodiment the present invention may comprises interferon alpha-2b produced from E. coll The amount of pegylated interferon alpha-2b used in step( i) may be in the range of 0.05mg to 2.0mg/ml. Most preferably, the amount of pegylated Interferon alpha-2b may be in the range of 0.07mg to 0.3mg/ml

The buffering agent used in step (i) may be selected from a sodium phosphate buffer or a Histidine buffer, preferably, the buffer may be sodium phosphate buffer. The sodium phosphate buffer may comprise of dibasic sodium phosphate anhydrous and monobasic sodium phosphate dehydrate or monohydrate .
The amount of the buffer present in the equal mass of composition for monobasic and dibasic may be in the range of O.lmg/ml to lOmg/ml, more preferably, 1.5mg/ml.

The stabilizing agent used in step (i) may be selected from the group comprising wetting agent and a surfactant. Preferably, the stabilizing agent may be selected from the group comprising Tween-20, Tween-80, Pluronic F-68, Briz-35. Most preferably, the stabilizing agent may be Pluronic F-68.

The amount of stabilizing agent used in step (i) may be present in a range of 0.05-2.0mg/ml, and most preferably, 1 .Omg/ml.

The cryoprotectant used in step (i) may be selected from the group comprising a sugar, a polyol, a polymer, an amino acid, a non-aqueous solvent, and a surfactant. Preferably, the cryoprotectant may be a starch, more preferably cryoprotectant may be a cyclic oligosaccharide. The cyclic oligosaccharide may be a cyclodextrin. The cyclodextrin may be selected from the group comprising a-Cyclodextrin (aCD), P-Cyclodextrin (pCD), 2-Hydroxypropyl-p-cyclodextrin (HPpCD; Kleptose® HPB), Sulfobutylether p-cyclodextrin sodium salt (SBEpCD; Captisol®), Randomly methylated p-cyclodextrin (RMpCD), y-Cyclodextrin (yCD), 2-Hydroxypropyl-y-cyclodextrin (HPyCD)., preferably the cyclodextrin may be 2-Hydroxy Propyl P-Cyclodextrin.

The cryoprotectant used in step (i) may be present in the range of 10-200mg/ml, most preferably, cryoprotectant may be lOOmg/ml.

The pH adjusted in step (ii) may be maintained in the range of 6-5-7.5.

The sterilization of the solution obtained in step (iii) may be carried out by filter sterilization,

The composition of the present Invention exhibit biological activity/stability and chemical stability in hold time studies after the composition is prepared, but before the lyophilization steps.

Subsequent to the preparation of composition by addition of excipient, the composition may be subjected for lyophilization.

The lyophilization in step (iv) may be carried out by dispensing the solution obtained from step iii into the glass containers up to the desired volume and half stoppered so as to allow the vapors to go from the glass containers during the process of drying. The vials with half stoppers are then placed on the lyophilizer shelves. The lyophilization process is then started with the initial freezing step followed by primary drying and then the secondary drying. The lyophilization process is designed in such a way that the moisture after the drying cycle remains less than 2.5%. Preferably, the lyophilized powder has a moisture content of between 0.5% and 2.0%, and more preferably less than 1.5%.

Yet in another aspect of the invention, it is observed that the chemical and biological stability of the composition is also maintained for extended periods of about one month after the rehydration step.

Without being limited by the theory, it is proposed that the optimum use of the various ingredients at the said ratio is synergistic. The synergistic composition maintains the stability of the composition before and after the lyophilization, post reconstitution rendering a more stable product. The composition of the present invention permits the lyophilized products to achieve a rigid cake that maintains anti collapsing property, biological activity and chemical stability.

ADVANTAGES

1. The improved composition of the present invention is stable for 24 months at 25 C and at 40 C for a period of at least 6 months and hence minimizes the cost of the shipment as it can be transported under room temperature to the destination.

2. The composition of the present invention contains higher grade of pure PEGInterferon alpha-2b conjugate which is greater than 95.0%. This level of purity is retained throughout the shelf life period of 24 months at least.

3. In the composition of the present invention the purity levels of PEG interferon is maintained even without the lyophilization process.

4. The composition of the present invention takes less time for lyophilization as compared to the prior art composition and hence reduces the cost of production significantly.

5. The lyophilized cake prepared from the composition of the present invention exhibit good and improved anti collapsing property as compared to the prior art composition. The lyophilized cake prepared from the composition of the present invention exhibit improved dissolution profile. The composition of the present invention exhibit chemical stability even after rehydration process, for a period of one month, when rehydrated product is stored at 2-8°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.

EXAMPLES

Example-01: Various PEG Interferon conjugates compositions Fl, F2, F3, F4 and F5 is prepared by the method as disclosed in the specification. The detail of each composition is provided in TableOl:

Table 1:Various Composition of PEGInterferon alpha 2b conjugates.

* Amount of WFI used before lyophilization

Example-02: Hold time studies of the composition Fl and F2 before lyophilization.

The preparation of the composition before lyophilization was subjected to the hold time studies. This stability study was continued for a period of 3months at 5 C. The results are within the specified limit over a period of 3 months for the composition-1 but not for composition-2 as shown in table 02.

Table-02: Stability data of Compositions Fl and F2:

ND: Not Determined

As indicated from the experimental data provided in the Table 01 above, the composition Fl prepared according to the present invention exhibit better chemical stability and biological activity as compared to the F2. Thus, it can be concluded that the composition disclosed in the specification is synergistic and impart stability to the PEG conjugate composition

Example-03 Stability studies of Fl and F2 after lyophilization:

The composition of the present invention as described herein is tested for stability studies post reconstitution. The lyophilized cake in vials is stored, over a period of 24 months. The reconstitution is done with sterile water for injection. The stabilities parameter such as visual clarity, purity by SEC-HPLC for the degree of conjugation, protein content, and biological activity etc were analyzed for different time period, wherein the different time period are as follows: T0:Initially, T3:3 months, T6:6 months, T9:9 months, T12.12 months, T18 :18 months, T24:24 months. The protein content is determined by UV absorbance at 280nm and biological activity is determined by antiviral assay i.e, challenging the cells having interferon alpha-2b with Encephalo Myo Carditis Virus. The results of the stability of Fl and F2 are described in Table 03 and Table 04:

Table 03: Stability studies for composition-1 (2-Hydroxy Propyl-P-Cyclodextrin composition) after lyophilization in lOOmcg vial

Table 04: Stability studies for composition-2 (Sucrose composition) after lyophilization on 100 mcg vial

The results from Table 3 and 4 shows that the composition Fl of the present Invention exhibits better physio-chemical stability as compared to F2 after lyophilization. Also, Fl exhibit better biological stability post reconstitution over the time period as compared to F2. Also, Fl exhibits better anticollapsing property as compared to F2. Thus it can be concluded that the composition of the present is synergistic and impart better physio-chemical and biological stability to the PEG interferon alpha 2b conjugate.

Example-04 Stability studies for Composition-3 post reconstitution.

The composition of the present invention as described herein is tested for stability studies. The lyophilized cake in vials is stored, over a period of 12 months. The reconstitution is done with sterile 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 for different time period. The protein content is determined by UV absorbance at 280nm and biological activity is determined by antiviral assay i.e, challenging the cells having interferon alpha-2b with Encephalo Myo Carditis Virus. The different time period are as follows: TO: Initial, T3:3 months, T6:6 months, T9:9 months. The results of the stability is described in Table 05:

Table-05: Stability studies for Formulation-3 on lOOmcg vial

Also, the critical analysis is done for Composition F3, F4&F5 for Physical integrity of the lyophilized cake, visual clarity after reconstitution, purity by SEC-HPLC for the degree of conjugation, protein content, and biological activity etc. The different compositions F3, F4 and F5 prepared as per the present Invention exhibit enhanced physical, chemical and biological stability similar to Fl. Thus it can be concluded that compositions prepared with different concentration of the component according to the present Invention exhibit enhanced physical, chemical and biological activity as compared to prior art composition F2.

We Claim:

1. An improved stable composition comprising a PEG conjugate, a cryoprotectant, a buffering agent, a stabilizing agent and optionally other pharmaceutically acceptable excipient wherein the composition is stable at 40°C for period of six months.

2. The composition as claimed in claim 1, wherein the PEG conjugate is PEG Interferon alfa-2b conjugate.

3. The composition as claimed in claim 1, wherein the amount of PEG Interferon alfa-2b conjugate is in the range of 0.07mg to 0.3mg/ml.

4. The composition as claimed in claim 1, wherein the buffering agent is selected from the sodium phosphate buffer or a histidine buffer, preferably, the buffer is sodium phosphate buffer.

5. The composition as claimed in claim 4, wherein the amount of buffer present in equal masse of composition for monobasic and dibasic in the range of O.lmg/ml to lOmg/ml, more preferably, 1.5mg/ml.

6. The composition as claimed in claim 1, wherein the stabilizing agent is selected from a surfactant, and a wetting agent, preferably, stabilizing agent is selected from the group comprising Tween-20, Tween-80, Pluronic F-68, Briz-35, preferably, the surfactant is Pluronic F-68.

7. The composition as claimed in claim 1, wherein the stabilizing agent is present in the range of 0.05-2.Omg/ml preferably, l.Omg/ml.

8. The composition as claimed in claim 1, wherein the cryoprotectant is selected from the group comprising sugar, starch, polyol, polymer, aminoacid, non aqueous solvent, preferably, the cryoprotectant is starch more preferably, cryoprotectant is cyclic oligosaccharide most preferably, cryoprotectant is cyclodextrin.

9. The composition as claimed in claim 8, wherein the cyclodextrin is selected from the group comprising a-Cyclodextrin (aCD), p-Cyclodextrin (PCD), 2-Hydroxypropyl-P-cyclodextrin (HPpCD; Kleptose® HPB), Sulfobutylether p-cyclodextrin sodium salt (SBEpCD; Captisol®), randomly methylated P-cyclodextrin (RMpCD), y-Cyclodextrin (yCD), 2-Hydroxypropyl-y-cyclodextrin (HPyCD, preferably, the cyclodextrin is 2-Hydroxy Propyl P-Cyclodextrin.

10. The composition as claimed in claim 1, wherein the cryoprotectant is present in the range of 10-200mg/ml, preferably 100mg/ml.

11. The composition as claimed in claim 1, wherein the optional pharmaceutical excipient is aminoacid, preferably the aminoacid is glycine.

12. The composition as claimed in claim 11, wherein the glycine is present in the range of 10mg-30mg/ml, preferably glycine is 20mg/ml.