PEG-INTERFERON CONJUGATES

FIELD OF THE INVENTION

The present invention relates to the field of biopharmaceutical sciences. More specifically, it relates to novel PEG-interferon conjugates and process for the preparation of 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-α2a 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 the interferon, like any other protein, is subjected to degradation and immunogenicity, when administered to a patient, it is always, administered in the PEGylated form. 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 "mask" the agent from the host's immune system (reduced immunogenicity and antigenicity), increase the hydrodynamic size (size in solution) of the agent which prolongs its circulatory time by reducing renal clearance.

PEGylated interferon is known and available in the market. The marketed preparation comprises of IFN-a-2a/2b that are predominantly conjugated to the PEG by histidine residues. However, the marketed preparation is difficult to produce, as it involves several processes during protection such as pegylation and purification such as ion-exchange chromatography and gel permeation chromatography. Hence the resultant product is very costly. More over, the product has inherent disadvantages such as possible inconsistency in producing similarly modified form of peginterferon because this process involves more number of expensive and difficult steps. Hence, there is a need in the market to for an efficacious PEG-interferon conjugate that is easy to produce, cheap and at the same time having better efficacy than marketed products and lesser side effects.

OBJECTIVES OF THE INVENTION:

The object of the present invention is to develop a PEG-interferon a-2b conjugate.

Another object of the invention is to develop a PEG-interferon conjugate through a simple and cost-effective process

Yet another object of the invention is to develop a PEG-interferon conjugate that is efficacious and less toxic than the PEG-interferon of prior art.

Yet another object of this invention is to provide pharmaceutical compositions of PEG-interferon conjugates.

SUMMARY OF THE INVENTION

The present invention relates to a novel PEG-interferon-conjugate and a process for producing the conjugate. In the novel PEG-interferon conjugates of the present invention, the interferon is conjugated to the PEG through lysine. 3

The present invention relates to conjugate of Interferon covalently linked to a polymer such polyethylene glycol predominantly at the lysine residue to the protein, wherein preferentially, one polymer strand gets linked to one interferon molecule. The conjugate is attached at the lysine such as at the positions of Lys31, Lysl21/134, Lysl33, Lysl31, Lysl64, Lysl21, Lys83 and Lysll2 of the interferon molecule.!)The final pharmaceutical product is a combination of the various positional isomers.

The conjugation of the PEG at lysine is achieved by the novel process of production and purification of interferon. The conditions of this novel and inventive process allow the interferon to conjugate at the lysine residue the processes include production, purification of interferon and dialysis during the purification of interferon.

The said conjugates of the present invention are simple to produce and purify, cost effective, efficacious and less toxic from the products of prior art.

BRIEF DESCRIPTION OF THE DRAWINGS:

Fig1: Flow chart of the process for production interferon alpha-2b

Fig 2: Flow chart of the process for production PEGylated interferon -a.

Fig 3: Chromatographic profile of the positional isomers of PEGylated interferon-a. Positional isomers were isolated by using strong cation exchange chromatography.

Fig 4: Percentage of positional isomers of PEG-Interferon in comparison with published literature

DETAILED DESCRIPTION OF THE PRESENT INVENTION

Unless specific definitions are provided, the nomenclature utilized in connection with, and the laboratory procedures, techniques and methods described herein are those known in the art to which they pertain. Standard chemical symbols and abbreviations are used interchangeably with the full names represented by such symbols.

IFN-α as used herein, means interferon, as obtained by isolation from human leukocyte , obtained by recombinant DNA techniques in prokaryotic or eukaryotic host cells, including but not limited to bacterial, fungal, yeast, mammalian cell, transgenic animal, transgenic plant and insect cells, as well as salts, functional derivatives, precursors and active fractions thereof. The interferon could also be produced from synthetic sources.

Novel peg-interferon conjugates of the present invention may be produced by the steps comprising

(I) production and purification of the interferon

(II) pegylation of interferon predominantly at lysine residues

(III) purification of mono PEGylated interferon

The novel peg-interferon conjugates of the present invention are conjugated. The conjugate attached at the lysine such as at the positions of Lys31, Lysl21/134, Lysl33, Lysl31, Lysl64, Lysl21, Lys83 and Lysll2. Further the novel peg-interferon conjugates are less toxic and more efficacious that peg-interferon conjugates of prior art.

The novel peg interferon conjugates may be produced by the method comprising the following steps:

(I) Production and purification of interferon

Human IFN alpha-2b refers to proteins having the amino acid with the sequence ID 1. The Human Interferon alpha-2b may be produced by genetic engineering techniques and/ or recombinant techniques, wherein the sequence is introduced into a suitable microorganism and the microorganism can be propagated. The clone with the sequence ID 1 may be introduced into any prokaryote. The prokaryote is preferably E. coli. The sequence may be introduced by general techniques such as restriction digestion, ligation and transformation.

The propagation of the microorganism may be carried out by culturing methods. More preferably the propagation is carried out by fermentation techniques. The fermentation may be carried out with optimum conditions of pH, temperature and dissolved oxygen. In general, the pH of the bioreactor may be kept in the near neutral range and the temperature may be kept in the ambient range.

Subsequent to propagation, the microorganism or cells may be lysed to release the amplified sequence. The lysis may be carried out by chemical, mechanical or other means known in the art. Preferably the lysis is carried out by mechanical means.

The sequence is extracted by means of high concentration of denaturants. Preferably the denaturants are selected from a group consisting of urea and guanidine hydrochloride. The concentration of the denaturants may range form 6M to 8M. The sequence is further refolded and purified by column chromatography followed by dialysis.

The column chromatography may be carried out by using reverse phase matrix more preferably source-30 matrix. The elute in organic phase from the column chromatography is subjected to dialysis in to aqueous buffer which is a dialysis buffer. The dialysis buffer may be either tris buffer of sodium phosphate buffer more preferably sodium phosphate buffer. The pH of the sodium phosphate buffer may range from 6.5-8.0. The dialysis can be carried out using any material cellulose, more preferably regenerated cellulose. The process time of dialysis may range from 5hrs to 72hrs. The ratio of elute and dialysis buffer may be in 1:200. Process of dialysis may be carried out lower temperatures preferably at 2-8°C. The final product may be identified using SDS-PAGE. Dialysis was performed to separate the residual contaminants and improperly folded rhIFN a-2b. Correctly folded rhIFN a-2b remained in the solution during the process of dialysis, the improperly folded and the contaminant proteins were precipitated. Unstable protein intermediates precipitated during the transition of organic phase to aqueous phase. The process of dialysis not only helps in removing the traces of acetonitrile and TFA but also removes the improperly folded and contaminant proteins. This method of direct dialysis of RP pool has not been reported earlier till date. Purification of recombinant human

Interferon involves multiple chromatographic steps like Immunoaffinity chromatography, Reverse Phase chromatography, Cation exchange chromatography, and gel permeation chromatography as described in the patent 4765903. Multiple chromatographic purification steps consumes more time and also expensive. The method described here is a simple, rapid, cost effective and highly efficient which can be very valuable for industrial production of rhIFN a-2b and other recombinant proteins.

In accordance with the present invention, during the process of dialysis the lysines get exposed. As the process of dialysis is very slow, this can enable the lysine exposure.

(II) Pegylation of interferon

The activation of polymers (polyalkylene oxides) is done by treating the hydroxyl end group into reactive functional group so as to facilitate the conjugation with the protein. The process is referred as "activation" and the product is called as "activated polymer N activated PEG.

In accordance with the present invention, the activated polymers are reacted with the protein such as interferon, so that the polymer attachment occurs preferably at the €-amino group of the Lysine residues interferon molecule and to a lesser extent on the €-amino group of the histidine residues and N terminal cysteine residue.

Polymers that are suitable will vary by molecular weights from 200-35000 daltons as described in the US patent 5951974. The whole of US patent 5951974 is incorporated herein. Polymers having a molecular weight ranging from about 200 to 35000 are selected for the present invention. Molecular weights around 5000 to 12000 are particularly preferred. The polymer is selected in such a manner that it is highly soluble at room temperature. The polymer may be activated by coupling the succinimidyl carbonate functional group to the hydroxyl end. The interferon is PEGylated in solution with the activated polymer. The reaction is carried out such that the activated polymer is present in excess molar concentration with respect to the interferon.

More specifically, the conjugation reaction may be initiated by the addition and incubation with sodium lauryl sulfate. The conjugation process was carried out by the addition of excess molar of activated poly ethylene glycol at 23°C-27°C for 1-2 hrs. Further, the reaction may be quenched with the addition of 20mM-50mM glycine, to have the mixture of positional isomers of conjugated PEGylated interferon.

Preferably each isomer contains a single polymer attached to the interferon molecule. In alternate embodiment there can be more than one polymer attached to one molecule of interferon.

(Ill) Purification of Mono PEG ylated Interferon a-2b:

The product of step (ii) is subjected to a further purification step. The present invention although produces a substantial quantities of positional isomers, it can also contain predominantly the residual PEG, non-PEGylated interferon and higher forms of PEGylated IFN. The mixture can also contain buffer, SDS and glycine.

The solution containing the polymer protein conjugate may first undergo a process of diafiltration or dialysis against buffer across a low molecular weight cut-off tangential flow ultra-filtration, so as to remove the buffer salts, SDS and other small molecules like glycine to obtain a filtrate.

After filtration, the filtrate is further subjected to column chromatography. The material for chromatography may be selected from the group consisting of ion exchange media. The quaternary amine coated anion exchange resins are particularly preferred for this separation. The anion exchange matrix is packed and equilibrated with the buffer. The buffer having the same pH and strength as like the diafiltration buffer is used for the equilibration. The diafiltered mixture was then adsorbed on to column and washed with excess of buffer to remove the unbound residual PEG. After washing, a linear gradient was applied on to the column with the increase salt concentration.

The fractions containing monoPEGylated IFN are separated and are substantially pure. Other diPEGylated forms of IFN and non-PEGylated interferons are further separated as the remaining fractions at different strengths. The monoPEGylated interferon is at least 95.0% pure in a single step chromatography arid has diPEGylated interferon and non-PEGylated interferon as impurities.

PEGInterferon alpha-2b conjugate is >95.0% pure as per the published literature (Current Pharmaceutical Design, 2002), where as the present invention conjugate is at least 95.0% pure. The purified monoPEGIFN conjugate is 95.0% and may be achieved in a single chromatography step which is cost effective.

The novel pegylated interferon conjugates of the subject application may be analyzed for the position of conjugation. The analysis may be carried out by any method known to a person skilled in the art.

The positional isomers are separated from a mixture based on differences in the net surface charges and can be eluted at different molar strengths to obtain substantially purified monoPEGylated interferons. The preferred conjugation of interferon alpha-2b in solution occurs at the Lysine residue. Without being limited by theory, it is suggested that the lysine residue gets exposed during the dialysis process of interferons allows further to enable the lysine conjugation. The conjugate attached at the lysine such as at the positions of Lys31, Lysl21/134, Lysl33, Lysl31, Lysl64, Lysl21, Lys83 and Lysl 12. The lysine conjugates in the present invention are at least about 50%, up to 80% and are more preferably 70%. There are about 8 positional isomers that have the lysine conjugation.

Separation of such positional isomers can be achieved by means of ion-exchange chromatography, more particularly cation-exchange chromatography. As the conjugation process involves the attachment of polymer at different positions in pool of interferon molecules, each isomer will have its own charge in particular buffer. Depending upon the charge different positional isomers gets eluted from the column as the strength of starting buffer is increased. Cation exchange column with sulphonyl propyl functional groups is preferred for separation of the positional isomers.

The PEG-interferon conjugates as prepared above could be administered as a pharmaceutical composition. The pharmaceutical composition could be prepared by methods known in the art and may contain other pharmaceutical acceptable excipients.

The novel pegylated interferon conjugates of the present invention are less toxic and efficacious in comparison with that of peg interferon conjugates of prior art. An open-label, multi-centre, phase III clinical trial was conducted to evaluate the safety and efficacy of peginterferon alpha-2b for the treatment of chronic hepatitis B. Clinical trials were conducted both on HBeAg positive patients and HBeAg negative patients. The number of patients developed adverse events with product of the present invention and with published literature is given in Table 2. There were significant differences in the incidence of majority of adverse events between the two study products. The product of the present invention is also more efficacious than the product(s) of the prior art and is given in table 03. On comparing the efficacy of the product of the present invention with that of the published literature, it is observed that the product is almost 2 times more efficacious.

ADVANTAGES

1. The production of PEGylated Interferon alpha-2b is simple and cost effective which involves less number of chromatographic purification steps where the purity is achieved greater than 95.0% consistently.

2. PEGylated interferon of the subject application consists of significantly higher percentage of lysine modified PEGylated interferon molecules and this leads to more stable form of pegylation and in turn gives higher efficacy with lesser side effects.

3. The adverse effects of the product of the subject application are less than the other PEGylated interferons available in the market.

4. The product of the subject application also has a better safety profile. These results show that peginterferon alpha-2b developed by the process as disclosed herein has a better safety profile and also better virological and biochemical response in patients with chronic hepatitis B infection.

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:

Preparation of interferon alpha-2b:

Preparation of LB media was done and adjusted the pH of the media to 7.0±0.2 with sodium hydroxide. Media was sterilized by steam sterilization at 120-122 °C and 1.5KG pressure. Innoculation of glycerol stocks was carried out under laminar air flow unit (LAFU) and an orbital shaker. Orbital shaker is maintained at 36±1°C, 200±10 rpm for 7-8 hours. Sterilization of the media in the Fermentor, at 121°C for 15 minutes was done and the media is cooled down to 35°C. After the incubation period the seed of volume 5-6% was inoculated in to a 22L fermentor to have a batch volume of 15±1L. Preparation of dextrose, IPTG, ammonia solution was done for the feed requirements of the culture.

Fermentor was operated by maintaining the temperature of 35±1°C, pH 7.0±0.2, dissolved oxygen 100%, back pressure 0.5±0.02, and air of 0.5±0.02vvm. During the fermentation process agitation to 650 rpm and air to 0.9wm were gradually increased to maintain the DO in a range of 40-50%. pH is maintained in the range of 7.0±0.2 through out the process with 50% ammonia solution. Culture was grown for 4-5hrs and then was induced with 0.5mM-0.1mM IPTG to express the Interferon alpha-2b protein as inclusion bodies. After induction culture was grown for another 7-8hrs and harvested. The harvested culture was centrifuged to separate the cells.

Lysis of harvested cells was performed preferentially by mechanical means with Dynomill. Homogenous mixture at 17800rpm was prepared for lysis by suspending the cells and buffer (lOmM Tris, 5mM EDTA and 150mM NaCl at pH 8.0±0.2) in 1:1.5 ratio. Lysis of cells was achieved by disruption in a dynomill for 3 successive passes. The suspension of disintegrated cells was then extracted by centrifugation at 14900g for 15-30min. The extract may be suspended in a buffer containing 2-4M urea. The suspension was then incubated for 60-90min while being magnetically stirred. It was then centrifuged at 14900g for 15-30min as before and the supernatant was discarded. The extract was then suspended in buffer containing 0.5-1.0M NaCl and 0.25-0.75%Triton X-100. Then incubation of cells for 60-90min can be performed while being magnetically stirred. Then the mixture was centrifuged at 14900g for 15-30min to separate the extract. This extract was washed with buffer for several times and the inclusion bodies pellet was collected by centrifugation at 14900g for 15-30min.

The inclusion bodies pellet was suspended in 20 folds (20ml/gm of IB) of solubilization buffer (lOOmM Tris and 6M guanidine hydrochloride at pH 8.0). This solubilization was done for over night stirring with a magnetic bead at 2-8° C. The solubilized solution was then centrifuged at 14,900# at 4° C for 20-30 min. The protein concentration of the solubilized protein was determined by UV method and then the protein concentration was adjusted to 5.0mg/ml by adding the excess of same buffer. Refolding buffer (lOOmM Tris and 0.1M L-Arginine at pH 8.0) was pre chilled at 2-8°C. The solubilized sample was then renatured by adding to refolding buffer in 3 aliquots at 2-3 hrs intervals. The renatured solution was incubated without stirring for 60-65 hrs at 2-8°C. After the incubation period the refolded solution was concentrated to 10 times the original volume by using ultra filtration cassettes (Sartorius lOkDa).

Either silica based or polystyrene based matrix can be used as Reverse phase matrix for the purification. Elution gradient can be either Ethanol/Acetonitrile mixture or TFA/Acetonitirle based mixture. For the present invention TFA/Acetonitrie mixture is preferred. Reverse phase column (Source-30 RPC) was equilibrated with 4 column volumes of 0.1% trifluoroacetic acid (TFA) in water. The refolded concentrate was loaded on to the column through high flow rate pump. The column was then washed with 6 column volumes of 0.1% TFA. Then the column was connected to HPLC and ranned the gradient by using acetonitrile as elution solvent as shown in table 01. The gradient up to 38% of acetonitrile removes the aggregates and other low molecular weight components. As the hydrophobicity of folded form is less gets eluted in the initial % and later the improperly folded IFN. The fractions for each % were collected separately, and then the samples were analyzed on SDS-PAGE gel. The pooled correctly folded fractions from SDS-PAGE, was dialyzed against sodium phosphate buffer. Dialysis bag of 6-8kDa cutoff (Spectrapore) was used for the process. The dialysis was carried out in 1:200 folds buffer at 2-8° C for overnight. The dialyzed sample was then clarified by centrifugation at 14,900g and the supernatant was collected as purified Interferon alpha-2b. The purified IFN is thoroughly diafiltered using tangential flow system to remove the traces of solvent. Figure 1 shows the sequential steps for the purification of interferon alpha-2b.

Example 2: Pegylation of Interferon a-2b:

Purified IFN a-2b around 2.0mg/ml was taken, adjusted the conductivity to a molar strength of 100mM, to a pH of 6.5 with sodium phosphate buffer and incubated for 2-4hrs for the stabilization. Activated PEG12000 (SC-PEG12000) was added in the presence of 0.2% Sodium Dodesyl Sulphate, incubated at 25°C/2hrs for the conjugation and quenched the reaction by adding 20mM glycine.

Example 3: Purification of monoPEGylated interferon:

The above pegylation mixture is thoroughly diafiltered against 5mM tris buffer at pH 8.3 by using tangential flow system with molecular cutoff 10000 daltons. The diafiltered sample is then loaded to strong anion Ion-Exchange column which is pre equilibrated with the same buffer. The bound protein is eluted using 5mM tris buffer containing 100mM NaC1 at pH 8.3. MonoPEGylated interferon elutes at particular ionic strength and the diPEGylated interferon and non-PEGylated interferon gets eluted at different ionic strengths. The collected peaks are further analyzed on SDS-PAGE and pooled for the monoPEGylated interferons. Figure 2 shows the sequential steps of purification of PEGylated interferon.

Example 4: Analysis of positional isomers of monoPEGylated interferon conjugates:

Purified monoPEGylated interferon was diluted in 10mM sodium phosphate buffer at pH 5.8 to a concentration of 1.6mg/ml and loaded on to a cation exchange column with sulphonyl propyl functional groups. A linear shallow gradient was applied with increasing strength of the same buffer to elute the positional isomers at different concentration of the gradient. There are about 15 different peaks, in which there are about 13 isomers of monoPEGylated interferon. Figure 3 shows the chromatographic elution profile of the positional isomers. Figure 04 shows the chromatographic area in comparison with published literature [Yu-Sen Wang et al, Advanced Drug Delivery Reviews 54 (2002) 547-570].

Example 5: Evaluation of safety and efficacy of PEG-interferon.

An open-label, multi-centre, phase III clinical trial was conducted to evaluate the safety and efficacy of peginterferon alpha-2B, produced and analysed as per examples 1-3, for the treatment of chronic hepatitis B. Ninety-two patients with chronic hepatitis B were screened at 3 investigational centers. Among them, twenty-three patients did not meet the eligibility criteria. The remaining 69 patients consisting of 43 HBeAg-positive and 26 HBeAg-negative, who consented to participate in the trial, were enrolled into the study. PEG-IFN 100 ug was administered subcutaneously once a week for 28 weeks and 50 |j,g once a week for subsequent 25 weeks. Sixty-five (43 HBeAg-positive and 22 HBeAg-negative) patients, who completed the study period of 52 weeks, constituted efficacy evaluable patients. The investigational product - PEG-IFN was well tolerated and there were no serious adverse events. As a result, there was an excellent compliance in 99.2 ± 1.9% of patients. The study of various safety parameters like vitals, haematology and biochemical parameters showed that there were no changes in any of the parameters of clinical significance throughout the study period of 52 weeks except an improvement in liver function tests. None of the patients on the product of the subject application reported any serious adverse event. On the other hand, 12% HBeAg positive patients were reported to have developed serious adverse events.

The number of patients (%) who developed adverse events with product of the present invention and with published literature is given in Table 2. The product of the present invention is also more efficacious than the product(s) of the prior art. On comparing the efficacy of the product of the present invention with that of the published literature, it is observed that the product is almost 2 times more efficacious. The results are tabulated in Table 3.

Table 01: Program preferred for RP-HPLC

Table 2: % of patients with adverse events with PEG-IFN product of present invention and published results:

Table 3: Comparison of efficacy of PEG-IFN (Product of present invention Vs Published data PEG products)

We claim
1. A process for producing novel peg-interferon conjugates comprising the following steps

(I) production and purification of the interferon

(II) pegylation of interferon predominantly at lysine residues

(III) purification of monoPEGylated interferon

2. A process as claimed in claim 1, wherein the novel peg-interferon conjugates are conjugated at the lysine residues at the positions of Lys31, Lysl21/134, Lysl33, Lysl31, Lysl64, Lysl21, Lys83 and Lysll2.

3. A process as claimed in claim 1 step (I), wherein the interferon has the sequence ID 1.

4. A process as claimed in claim 1, step (I) wherein the interferon is produced by recombinant technique preferably in E. coli

5. A process as claimed in claim 1, step (I) wherein, the interferon containing E. coli is propagated by fermentation techniques, preferably at neutral pH and ambient temperature.

6. A process as claimed in claim 1, step (I) wherein the amplified sequence is lysed by mechanical means.

7. A process as claimed in claim 1, step (I) wherein the amplified sequence is extracted using denaturants selected from the group consisting of urea and guanidine hydrochloride at a concentration ranging from 6molar to 8molar.

8. A process as claimed in claim 1, step (I) wherein the sequence is purified by using reverse phase matrix, preferably source-30 matrix to obtain the eluate.

9. A process as claimed in claim 1, step (I) and claim 8 wherein the eluate is dialysed against dialysis buffer by material cellulose, more preferably regenerated cellulose.

10. The method as in claim 9, consisting of a step of dialysis wherein the organic phase of interferon is exchanged with the aqueous phase of the said dialysis buffer.

11. The method in claim 9, wherein the organic phase of comprises of organic solvent such as acetonitrile.

12. The method in claim 9, wherein the said buffer is a phosphate buffer.

13. The method in claim 9, wherein the said phosphate buffer is a sodium phosphate buffer.

14. The method in claim 9, wherein the pH of the said sodium phosphate buffer from about 6.5-8.0.

15. The method of claim 9, wherein the process time of dialysis is about 5hrs-72hrs.

16. The method of claim 9, wherein the ratio of elute and dialysis buffer from about 1:200.

17. The method of claim 9, wherein the process of dialysis is carried out at a temperature of2-8°C.

18. A method of preparing the composition of claim 9, comprising the formation of pure interferon protein folded and purified in a way to enable lysine conjugation.

19. A process as claimed in claim 1, step (II), wherein the pegylation of interferon is carried out with a polyethylene glycol having a molecular weight in the range of 200-35000 daltons, more preferably, in the range of about 5000 to 20000 and most preferably in the range of 5000 to 12000.

20. A process as claimed in claim 1, step (II) and claim 19, wherein the polymer is activated by coupling the succinimidyl carbonate functional group to the hydroxyl end.

21. A process as claimed in claim 1, step (II) and claim 11, wherein the polymer is present in excess molar concentration with respect to the interferon.

22. A process as claimed in claim 1, step (II) and claim 11, wherein, the conjugation is carried out by the steps comprising

(I) Initiation by the addition and incubation with sodium lauryl sulfate.

(II) Addition of excess molar of activated poly ethylene glycol at 23°C-27°C for 1-2 hrs.

(III) Quenching of reaction with the addition of 20mM-50mM glycine, to have the mixture of positional isomers of conjugated PEGylated interferon.

23. A process as claimed in claim 1, step (III), wherein the purification of MonoPEGylated Interferon a-2b is by diafiltration to obtain a filtrate.

24. A process as claimed in claim 1, step (III), and claim 14, wherein the filtrate is subjected to column chromatography using quaternary amine coated anion exchange resins

25. The pharmaceutical composition of claim 1, wherein the said predominant lysine derived PEG-Interferon alpha-2b is 50-80%.

26. Novel peg-interferon conjugates as claimed in claim 1 as and when administered as a pharmaceutical composition

27. Novel peg-interferon conjugates as claimed in claim 1 for use in treatment of chronic hepatitis B