EP3221450A1

EP3221450A1 - An improved process for the preparation of pharmacopoeial grade interferon alpha 2b

Info

Publication number: EP3221450A1
Application number: EP15823122.5A
Authority: EP
Inventors: Sanjeev Kumar Sharma; Surya Bhushan KUMAR; Rohan PARAB; Mohanish KANKONKAR; Conchita D'SOUZA; Sachin BACHATE; Ratnesh Kumar Sharma
Original assignee: Unichem Laboratories Ltd
Current assignee: Unichem Laboratories Ltd
Priority date: 2014-11-18
Filing date: 2015-02-11
Publication date: 2017-09-27
Also published as: WO2016079598A1

Abstract

The present invention relates to a simple and scalable process for the production of pharmacopoeial grade Interferon alpha 2b by fed-batch fermentation followed by purification using chromatography techniques. The invention also relates to the recombinant expression of interferon alpha 2b in soluble form by fermentation to obtain high yields. The process involves clarification and chromatographic steps for the purification of Interferon alpha 2b.

Description

"AN IMPROVED PROCESS FOR THE PREPARATION OF PHARMACOPOEIAL GRADE INTERFERON ALPHA 2B"

FIELD OF INVENTION:

The present invention relates to the production of Interferon alpha 2b ( IFN a2b ) by a process of fed-batch fermentation followed by purification using chromatography techniques. The process is simple, scalable and yields IFN a2b of pharmacopoeial grade.

BACKGROUND OF THE INVENTION

Interferons are cytokines that are naturally secreted by various cell types in the human body and various other species. Three major types of interferons have been identified. Type I includes interferon alpha, beta, epsilon, kappa and omega. Type II includes interferon gamma ( IFN-γ). Type III includes interferon lambda ( Outzen, M. et al ). Multiple subtypes of interferon alpha and betas exist, (Bekisz et al ) which in turn have allelic forms e.g. Interferon alpha subtype 2 has at least 3 allelic forms; IFN alpha 2a, IFN alpha 2b, IFN alpha 2c,

Interferons play a central role in the normal immunological response and exhibit a wide breadth of biological activities, some of which include antiviral, antiproliferative and immunomodulatory. These actions make interferon a most promising agent for the treatment of various diseases. (Khaitov, R.M. et al; Petska, S. et al ). To date, IFN alphas are approved for the treatment of cancers like hairy cell leukemia, malignant melanoma, follicular lymphoma, genital warts, AIDS- related Kaposi sarcoma and viral diseases like chronic hepatitis B and C.

With such widespread therapeutic uses of interferon alphas, abundant production of the same is necessary. Recent applications of recombinant DNA techniques have enabled the cloning of human genes in bacterial, yeast and mammalian cells, leading to the production of rare biologicals in abundant quantities (Baron E et al ). Industrial production of interferons by use of this technique is widespread using mammalian, yeast and bacterial expression system (EP 1097995 ).

At present, interferons expressed in E.coli are widely used (Baron, E. et al, 1990 ). Use of E. coli as an expression host is beneficial due to its rapid growth for achieving higher cell mass and also the ease of introducing recombinant vectors in the cells. A major drawback with the use of E. coli expression systems is the aggregation of the protein of interest into insoluble inactive forms. Here, the protein which is expressed at a high rate in the cell does not remain in the active soluble form but forms aggregates of denatured protein. Recovery of the insoluble aggregates and their refolding into soluble active forms is in most cases inefficient and leads to compromising yields.

Most prior arts teach processes which produce interferon in insoluble form. Chung, Jong-Sang et al ( WO2005 054287 ) teaches a process for the production of recombinant IFN a2b as inclusion bodies using E.coli. Isolation of inclusion bodies was carried out by cell lysis, followed by denaturation and ageing. The protein solution was concentrated and antibody affinity chromatography was performed. The elute was refolded in a urea containing buffer. The refolded solution was purified by cation exchange chromatography followed by gel filtration column chromatography. Obermeier (US4845032 ) describes a process for the purification of IFN alpha from crude IFN preparation by solubilization in a chaotropic agent followed by refolding. The renatured protein is then purified by hydrophobic interaction chromatography.

Eventually, the present inventors also carried out a process wherein IFN a2b was expressed as insoluble inclusion bodies by recombinant E. coli BL21 DE3 Gold strains. The inclusion bodies were subjected to refolding after solubilization in Guanidine containing buffer. 20-25% of active protein was obtained after refolding. The refolded protein was purified by various chromatographic steps. Final protein majorly contained met-IFN a2b.

Permeating the problem, another approach can be applied to avoid formation of inactive aggregates and obtain the soluble active form, maximally. Prior art teaches processes based on the said approach.

Gerhard Bodo et al (US5196323 A ) describes a process for the purification of soluble Interferon alpha by tandem chromatography, DE52 cellulose in tandem with affinity column comprising of monoclonal anti interferon IgG obtained from mouse ascites fluid coupled to BrCN-activated Sepharose 4B. The elute is then processed on cation exchanger followed by lyophilization.

Schilling, Ralf, Diederich, Bettina ( WO 2012 160027 A l ) discloses a process for the recombinant expression of an Interferon alpha in E. coli BL21 strains carrying a mutation in the genes encoding thioredoxin reductase and glutathione oxidoreductase whereby a more oxidative environment is created which facilitates the formation of disulphide bonds. This invention describes the construction of vector for soluble expression of human IFN a2b in E.coli. The invention also describes a chromatographic method for purification of the biologically active human IFN a2b.

Hauptmann et al (US5710027 A ) describes a process wherein Interferon alpha 2c is expressed as a soluble protein secreted in the periplasm of E. coli. The soluble protein is purified by adsorption chromatography followed by hydrophobic interaction chromatography, cation exchange chromatography and finally anion exchange chromatography.

Lohray et al ( US20060223 145 A l ) describes a process for the production of soluble IFN alpha 2b in Pischia pastoris. Fermentation is carried out with runtimes ranging between 48- 108 hrs. IFN alpha 2b is then purified using various chromatographic steps to obtain final product.

The disclosures made by prior arts teach the use of refolding techniques and use of antibodies for chromatographic purification. Production of IFN a2b by inclusion bodies yields met-IFN which does not satisfy pharmacopoeial requirement. Use of refolding is disadvantageous in that the efficiency of obtaining biologically active form from the inactive form is low in many cases. Use of antibodies in the purification process is not cost effective. Eventually, such a process may not be scalable. Similarly, the prior art teaches the soluble expression of IFN alpha, but they do not provide a process that may be scalable and industrially feasible. Longer fermentation runtimes are used in prior art for soluble expression of protein. Hence, the need arises for a suitable process that is simple, scalable while simultaneously giving high yield of IFN a2b of pharmacopoeial grade.

The present invention discloses a process which is simple, scalable and economical. Production of interferon a2b in soluble form by fermentation at low temperature in shorter period simplifies the process in comparison to prior art. Also, the purification process involves chromatographic steps which are simple and cost effective means of providing a product of pharmacopoeial grade. Thus the process avoids formation of insoluble forms of interferon a2b and use of antibody-linked resins as described by prior art.

SUMMARY OF THE INVENTION:

The present invention relates to a process which is scalable for industrial purpose to produce IFN a2b of pharmacopoeial grade. This aspect of the invention describes a process that involves fermentation of the recombinant E.coli BL21 Gold strains under controlled conditions to obtain IFN a2b in the soluble forms. Soluble IFN a2b isolated after fermentation is purified to pharmacopoeial grade using series of chromatographic techniques.

In another aspect, the present invention describes the preparation of IFN a2b by aerobic fed batch fermentation of recombinant E. coU BL21 Gold strains while providing conditions suitable for its soluble expression followed by the release of soluble IFN a2b in the supernatant by disruption of the cells and treatment of supernatant to remove nucleic acids.

In another aspect of the invention, a scheme for the purification of recombinant IFN a2b is described comprising the steps a ) microfiltration; b ) cation exchange chromatography; c) hydrophobic interaction chromatography; d) gel filtration chromatography; and e ) silica based chromatography.

DETAILED DESCRIPTION

By simple process, inventors mean the simplicity with respect to the ease of operation on the industrial scale. Scalable process means a process that is reproducible from laboratory scale to industrial scale with similar operations.

Interferon alpha 2b, IFN alpha 2b, Interferon a2b and IFN a2b mean the same and are used interchangeably.

The unit of time is considered as hour and is represented by h or hr, the plural being hrs.

The present invention describes a simple and scalable process to produce recombinant IFN a2b of pharmacopoeial grade. This aspect of the invention discloses the process involved in soluble expression of recombinant IFN a2b using fed batch fermentation, wherein the initial fermentation is carried out at 30°C to 40 ^" C and then at 18' C to 25 during induction phase followed by purification by a sequence of column chromatography.

One aspect, the invention describes a process for the culturing of recombinant E. coli BL21 DE3 Gold strains containing plasmid pET 20b harboring a gene coding for IFN a2b cloned in between the sites Nde I and Bam HI. in a seed medium comprising 2° o Luria HiVeg broth (w/v). 0.75° o Na_:HP0₄ (w/v). 0.5° o Dextrose (w/v). 0. 1° o MgS0₄.7H_:0 (w/v). Ampicillin to a final concentration of l OOng/ml and 0. l° o (v/v) trace metal solution of FeS0₄. ZnS0₄. CoCb. NaMo0₄. CaCb. MnCb. CuS0 or ¾B0₃ 111 Hydrochloric acid. The process is carried out for 12-15 hrs.

Another aspect of the invention discloses an aerobic fed batch fermentation of recombinant E. coli BL21 DE3 Gold strains in a production medium comprising 1% yeast extract ( w/v ), 1.2% Dextrose ( w/v ), 0.3% KH₂P0₄ ( w/v ), 1 .25% K HP0₄ ( w/v ), 0.5% i M I₄ S()₄ ( w/v ), 0.05% NaCl ( w/v ), 0. 1 % MgS0₄.7H₂0 ( w/v ) and 0. 1% ( v/v ) trace metal solution of FeS0₄. ZnS0₄. CoCb. NaMo0₄. CaCb. MnCk CuS0₄ or H3BO3 111 Hydrochloric acid. Ampicillin was added to a final concentration of 100 ng ml. Additionally, for increase in the cell mass, feeding may be initiated with a suitable carbon and nitrogen source. The suitable carbon source can be selected from Glucose or Glycerol, preferably 75% ( w/v ) of Glycerol. The suitable nitrogen source can be selected from tryptone, peptone or yeast extract, preferably 40% I w V ) of yeast extract. Feeding may be initiated after 2 log hrs, at predetermined feeding rates, maintaining C :N ratio in the range of 3 : 1 to 6: 1 , preferably with the C :N ratio of 4: 1. Those skilled in the art may vary the rates and the amounts as per their suitability, since they are specific for the parameters demonstrated for the particular batch sizes.

In another aspect of the present invention the best suitable feeding rates during fermentation were surprisingly observed to be 1.5-4 gmL 'h^"1 till 4^th hour, 4-8 gmL^" V¹ during 4^Ul to 22^nd hour and 1.5-4 gmL V¹ after 22^nd hr of fermentation. In yet another aspect of the invention, the initial growth of the culture was carried out with 1 -2 vvm aeration, dissolved oxygen maintained at 50%-60% and pH maintained at 6.6-7.0 with alkali such as Sodium Hydroxide. The temperature maintained during initial growth was 30-40°C preferably 37°C. During induction phase the temperature was reduced and maintained at 18-25°C preferably 22°C. Surprisingly it was observed that lower temperature supported the solubility of IFN a2b in the cytoplasm while increase in temperature above 28°C resulted in the formation of insoluble aggregates in the cells. This is non obvious as compared to prior art. Further it was surprisingly observed that fermentation run times can be lowered. Fermentation runtime is at least 22-28 hrs preferably 25 hrs.

In yet another aspect of the invention, expression of IFN a2b was carried out by appropriately inducing the production of IFN a2b with an inducer such as lactose or isopropyl thio-galacto-pyranoside (IPTG) preferably IPTG at a concentration of 200 μΜ to 1000 μΜ, preferably 1000 μΜ. Induction was optimally performed at mid log phase, when the cells were active, with the cell density measuring at least 40-60, preferably 50-55 at 600 nm. Expression of 10 to 15% of Interferon a2b was observed with at least 90% solubility in the cytoplasm at the end of 25 log hrs, as demonstrated by SDS-PAGE analysis of the total cell lysate and lyses supernatant. Fermentation by this mode yields at least 1400- 1600 mg soluble IFN alpha 2b per liter of broth as analyzed by densitometry method.

In yet another embodiment, isolation of soluble IFN a2b was carried out by harvesting cells by centrifugation to obtain the cell pellet, which was resuspended in a suitable buffer, pre-chilled to a temperature of 6°C- 10°C before lysis to avoid denaturation of protein during cell disruption. Lysis is carried out on a homogenizer (MiniDebee) under high pressure of 16000-20000 psi at pH 8.

In another aspect, the present invention describes a process for the isolation and purification of soluble interferon a2b to obtain pharmacopoeial grade IFN a2b. In this aspect of the invention, the cell lysate obtained after fermentation was chemically treated by using different concentrations of anionically charged neutralizers like polyethyleneimine or protamine sulplate preferably polyethyleneimine. The concentration of polyethyleneimine was varied from 0 1 % to 0.5%, preferred concentration being 0 25% of polyethyleneimine. Addition of these anionically charged neutralizers specifically polyethyleneimine, to the cell lysate under mild stirring for 1 5-30 minutes aided the flocculation of majority of the nucleic acid and nucleoprotein contaminants. The pH of the resulting solution was reduced to 4.8-5.0 and stirred for another 15-30 minutes. The contaminants were removed by centrifugation to obtain a clear solution.

The clear solution was incubated at 25-37°C for at least 48 hours under moderate stirring to further enhance precipitation of impurities and thus form a homogenous preparation of interferon a2b. At the end of 48 hours of incubation, precipitation of impurities rendered the solution turbid. 1 5% SDS PAGE analysis confirmed the above results depicting minimum loss of protein of interest in the precipitate.

In yet another aspect of the invention, the incubated turbid solution was subjected to clarification by microfiltration on a Ο. ΐ μ hollow fiber filtration system, pre- equilibrated with acidic buffers like sodium acetate, sodium citrate or ammonium acetate, preferably ammonium acetate buffer of pH 4.8-5.3. Filtration was carried out at a transmembrane pressure (TMP) of 2-4 psi. Approximately 90% recovery of the IFN a2b in the permeate was achieved by diafiltration of the retentate with equal volume of the buffer until absorbance of permeate at 280 nm is less than 1.5. As a variation, centrifugation and dead end filtration may be used to achieve the same. Recoveries were calculated based on analysis of the permeate by RP-HPLC.

Another aspect of the invention is to obtain a pharmacopoeial grade IFN a2b by a purification process comprising the steps: a) cation exchange chromatography;

b ) hydrophobic interaction chromatography; c) 5KDa crossflow filtration for concentration; d) gel filtration chromatography; and e) silica based chromatography.

In this aspect of the invention the permeate obtained after clarification was subjected to a cation exchanger such as SP Sepharose or CM Sepharose FF preferably CM Sepharose FF ( GE Healthcare). The cation exchanger was pre- equilibrated with the buffer selected from Sodium Acetate, Sodium Citrate, or Ammonium Acetate, preferably Ammonium Acetate. The concentration of the Ammonium Acetate was from 20 mM to 100 niM preferably 50 mM, with pH 4.8- 5.3. The cation exchanger adsorbed the protein of interest. After complete loading, the column was washed with 5 column volumes of the equilibration buffer. Elution was carried out using the buffers selected from Sodium Acetate, Ammonium Acetate and Sodium Citrate. The preferred buffer being Ammonium acetate containing at least 0. 15 M to 0.4 M Sodium Chloride or Ammonium Sulfate, preferably 0.2 M ammonium sulfate with pH of 4.8 to 5.3. Purity of IFN a2b at this stage is at least 60° o

Elute from cation exchanger was loaded onto a hydrophobic interaction chromatographic resin such as Phenyl Sepharose HP or Butyl Sepharose HP preferably Phenyl Sepharose HP equilibrated with the buffer selected from Sodium Acetate, Ammonium Acetate and Sodium Citrate, preferably with Ammonium Acetate buffer containing 1M Ammonium Sulphate. Binding of protein was achieved after a final concentration of 1M Ammonium Sulphate was made up in the protein load. Elution was carried out with 20 mM to 100 mM Ammonium Acetate preferably 50mM Ammonium Acetate buffer with pH 4.8 to 5.3 having conductivity of 4 to 6 mS cm. The elute contained 75-80% or pure IFN a2b.

In another aspect of the invention, the elute after hydrophobic interaction chromatography was concentrated at least 25-30 times on a 5 KDa crossflow filter to obtain a minimum volume of protein solution. At least 90% protein was recovered at this stage. The concentrated protein was loaded onto a gel filtration resin, like Superdex 75, Sephadex G25-G75, Sephacryl S-200 or Sephacryl S- 100, preferably Sephacryl S- 100 equilibrated with buffers ranging from Sodium Acetate, Sodium Citrate or Ammonium Acetate preferably Ammonium Acetate buffer having pH 4.0 - 4.5. Protein was eluted by passing equilibration buffer through column and the eluted fractions containing IFN a2b were pooled together. Purity of protein at this stage was at least 85-90%. The pooled fractions were polished on silica based C4 reverse phase column with a linear gradient from 35% Acetonitrile to 45% Acetonitrile in water with 0. 1% Trifluoroacetic Acid. 95-99% purity was achieved at this stage. The pure protein was lyophilized to obtain final powder.

As a variation to the above aspects, the process for the preparation of IFN a2b was carried out by aerobic fed-batch fermentation at higher temperature. In this the fermentation of recombinant E. coll BL21 DE3 Gold strains in the above mentioned production medium with same feeding rates and feeding ratios was carried out at higher temperature. Fermentation of the culture with 1 -2 vvm aeration, dissolved oxygen maintained at 50-60% and pH maintained at 6.6-7.0 with alkali such as NaOH was carried out while maintaining the temperature throughout the runtime at 37 . Exp ression of IFN a2b was effected by appropriately inducing the IFN a2b with an inducer, like isopropyl thio-galacto-pyranoside (IPTG ) at a concentration of 200 μΜ to 1000 μΜ, preferably 1000 μΜ. At the end of the fermentation run of at least 10- 14 hrs, IFN a2b was produced as inclusion bodies. Although higher cell densities obtained in fermentation and lowered fermentation run time due to operation at higher temperatures of 35-40°C as compared to the soluble expression of the cells, the product thus obtained was in an inactive form of inclusion bodies. The process of converting the inactive form to the active one then requires a refolding step. The final purified protein predominantly consists of methionine as the starting amino acid. Presence of methionine as the starting amino acid is undesirable and requires the use of specific enzymes to cleave off the methionine. Analytical separation techniques of the met-IFN a2b from IFN a2b are tedious. Chromatographic separation is also not possible for the two due to similar isoelectric point, very small difference in molecular weight, similar hydrophobicity and similar charge which results in a mixture of the two components in the final product.

The difficulties involved in the cleavage of methionine from met-IFN a2b by enzymatic methods and in the separation of met-IFN a2b from IFN a2b, in the final product, favors a process for the production of IFN a2b by a low temperature fermentation method which gives a methionine free IFN a2b as the first amino acid in the final product.

The present invention provides a process for the soluble expression of recombinant IFN a2b using low temperatures of 20-25°C with runtimes of 22-28 hrs, yielding 1400- 1 500 mg per litre of fermentation broth. The present invention also demonstrates the purification of IFN a2b using simple chromatographic steps which yields purified biologically active IFN a2b of pharmacopoeial grade.

The present invention thus provides a process for the soluble expression of recombinant IFN a2b using low temperatures of 20-25°C, with runtimes of 22-28 hrs, yielding 1400- 1 500 mg per litre of fermentation broth. The present invention also demonstrates the purification of IFN a2b using simple chromatographic steps. This process yields purified biologically active IFN a2b of pharmacopoeial grade. The present invention therefore provides a simpler process for the production of soluble IFN a2b and its purification. The process is economical and apt for industrial scaleup.

The above mentioned description is illustrative and does not limit those skilled in the art to cany out variations in the mentioned details.

Examples given below serve as references concerning the production and purification of IFN a2b

Inoculum preparation

Recombinant E.coli BL21 DE3 Gold strains were grown in a medium comprising of 2% I. una HiVeg broth ι w v i.0.75% Na₂HP0₄ (w/v), 0.5% Dextrose ι w v ).0.1% MgS0₄.7H₂0 I w V ). ampicillin to a final concentration of 100 ug ml and 01% (v/v) trace metal solution for 12-16 hrs. The medium provided optimal growth.

EXAMPLE 1: Soluble Expression of recombinant IFN alpha 2b at low temperature

Fed batch mode of fermentation was carried out for the production of soluble IFN alpha 2b wherein 10% inoculum was transferred to production medium comprising 1% yeast extract (w/v), 1.2% Dextrose (w/v), 0.3% KH P0 (w/v), 1.25% K HP0 (w/v), 0.5% (NH₄)₂S0₄ (w/v), 0.05% NaCl (w/v), 0.1% MgS0₄.7H₂0 (w/v), 0.1% (v/v) trace metal solution, Ampicillin ( ΙΟΟμ/ml). Fermentation is carried out with 1- 2 vvm aeration, dissolved oxygen at 50-60% and pH maintained at 6.6-7.0 with alkali. Initial growth is carried out at a temperature of 37°C. Temperature is then decreased and maintained at 22°C during the induction phase. Fermentation runtime is 22-28 hrs, preferably, 25 hrs. Induction to trigger the production of IFN alpha 2b is carried out by addition of IPTG at a final concentration of 1000 μΜ. Fermentation by this mode yields at least 1400- 1500 mg of soluble IFN alpha 2b per litre of broth.

EXAMPLE 2: Recombinant IFN alpha 2b inclusion bodies at higher temperature

Fed batch mode of fermentation was carried out for the production of IFN alpha 2b wherein 10% inoculum was transferred to production medium comprising 1% yeast extract ι w v i. 1 .2% Dextrose ι w v i. 0.3% KH₂P0 ( v ). 1 .25% Κ ΗΡ0 ι w v i. 0.5% i M I₄ hS()₄ i \v 0.05% NaCl i \v v i. 0. 1% MgS0₄.7H₂0 i \v v i. 0. 1% ( v/v ) trace metal solution, Ampicillin ( Ι ΟΟμ/ml ). Fermentation is carried out with l -2vvm aeration, dissolved oxygen at 50-60%, pH maintained at 6.6-7.0 with alkali. Temperature throughout the fermentation run was 37°C. Induction of IFN alpha 2b gene is carried out by addition of IPTG at a final concentration of Ι ΟΟΟμΜ. With increase in temperature during the fermentation run, inclusion bodies containing aggregated IFN alpha 2b were obtained.

EXAMPLE 3: Isolation of recombinant IFN alpha 2b from cells

Cell pellet is obtained by centrifugation of the fermentation broth. Pellet is resuspended in a suitable buffer at pH 8.0 and lysed on a homogenizer (MiniDebee ) under high pressure of 18000 psi, to release soluble IFN alpha 2b in the supernatant. Analysis of the supernatant and pellet by SDS-PAGE reveals the presence of at least 90% IFN alpha 2b in the supernatant.

EXAMPLE 4: Removal of Nucleic acid impurities

Supernatant is treated with at least 0.25% polyethyleimine followed by stirring for 15-30 mins. The pH was then reduced to 4.8-5.0 and the solution left on stirring for 15-30 mins. The solution was then centrifuged to obtain a clear supernatant, which was further incubated at 37°C for at least 48 hours, on stirring, to precipitate impurities and to allow the formation of a homogenous preparation of interferon alpha 2b. At the end of incubation period, a sticky precipitate is formed which majorly contains protein impurities. The solution is turbid and minimum loss of protein of interest is observed after SDS-PAGE analysis of the precipitate.

EXAMPLE 5: Clarification of the protein solution by niicrofiltration

The protein solution is filtered on a Ο. ΐ μ hollow fiber unit pre-equilibrated with ammonium acetate buffer at a TMP of 2-4psi to obtain a clear permeate. The remaining retentate is diafiltered in continuos mode with ammonium acetate buffer to recover the interferon alpha 2b in the permeate more than 90% interferon alpha 2b is recovered in the permeate.

EXAMPLE 6: Purification of I FN a2b

Clarified protein solution is loaded onto a cation exchange resin CM Sepharose FF preequilibrated with ammonium acetate buffer, with pH 5.0-5.3 which adsorbs the protein of interest. After loading, column is washed with 5 column volumes of ammonium acetate buffer pH 5.0-5.3.Elution is carried out using ammonium acetate buffer containing 0.2 M ammonium sulfate at pH 5.0-5.3. To the elute obtained from cation exchanger ammonium sulphate is added to a final concentration of 1 M. This solution is loaded onto Hydrophobic interaction chromatography column Phenyl Sepharose HP equilibrated with ammonium acetate buffer containing 1 .0 molar ammonium sulphate salt, pH 5.0-5.3. After complete loading the column is washed with ammonium acetate buffer containing 1.0 molar ammonium sulphate salt, pH 4.0. Elution is carried out using ammonium acetate buffer having conductivity of 4 to 6 mS cm. The elute contains 75-80% pure IFN alpha 2b. Elute is concentrated 25-30 times using on a 5 KDa crossflow filter maintaining a TMP of 2-4 psi. More than 95% recovery is obtained. The concentrated solution is loaded on a gel filtration column, Sephacryl S- 100 pre-equilibrated with ammonium acetate buffer with pH 4.0-4.5. Fractions containing IFN a2b are pooled and elute shows a purity of at least 90%. The pooled fractions are polished on silica based C4 reverse phase column with a linear gradient of 35% acetonitrile to 45 % acetonitrile in water with 0 1 % trifloroacitic acid; 98-99% purity is achieved at this stage. The pure protein is lyophilized to obtain final powder.

EXAMPLE 7: Preparation of IFN alpha 2b as inclusion bodies and its purification

Recombinant E.coli BL21 DE3 Gold strains were grown in a seed medium for 12- 15 hrs. Fermentation for the production of insoluble IFN alpha 2b was carried out in fed batch mode, where 10% inoculum was transferred to the production medium containing ampicillin to a final concentration of 100 ng/ml. Controlled conditions are provided with 1 -2 vvm aeration, 50-60% DO, pH maintained as 6.6-7.0 with alkali and temperature maintained at 37°C. Fermentation runtime is 12- 14 hrs. Induction of IFN alpha 2b gene is carried out at mid log phase by the addition of IPTG at a final concentration of Ι ΟΟΟμΜ. Cell pellet harvested by centrifugation of the fermentation broth, is resuspended in a suitable buffer and lysed on a homogenizer ( iniDebee) under high pressure to release IFN a2b inclusion bodies (IBs ). IBs are recovered as a pellet by centrifugation. 3 buffer washes are given to the IBs to remove cell based impurities. The IBs are then solubilized in a Guanidine containing buffer and refolded in a suitable buffer containing arginine. Refolded solution is loaded onto a HIC column in presence of at least 1.2 M NaCl at pH 8.5, The elute obtained is diafiltered to reduce the conductivity of the protein solution before binding it on an anion exchange resin like Q Sepharose HP. pH based elution is then performed. Fraction containing IFN a2b is then polished on silica based reverse phase C4 column with 35% acetonitrile to 45% acetonitrile in water with 0. 1% trifloroacitic acid; 95-99% purity is achieved at this stage. The pure protein is lyophilized to obtain final powder.

EXAMPLE 8: Preparation and purification of soluble IFN alpha 2b

Recombinant E.coli BL21 DE3 Gold strains were grown in a seed medium for 12- 15hrs. Fermentation for the production of soluble IFN alpha 2b was carried out in fed batch mode, where 10% inoculum was transferred to the production medium containing ampicillin to a final concentration of l OO.ug ml Controlled conditions are provided with l -2vvm aeration, 50-60% DO and pH maintained as 6.6-7.0 with alkali. Initial growth is carried out at 37°C but reduced to 22°C during the induction phase. Fermentation runtime is 25hrs. Induction of IFN alpha 2b gene is carried out at mid log phase by the addition of IPTG at a final concentration of Ι ΟΟΟμΜ. Cell pellet harvested by centrifugation, of the fermentation broth is resuspended in a suitable buffer and lysed on a homogenizer (MiniDebee) under high pressure to release soluble IFN alpha 2b in the supernatant. Supernatant is treated with 0.25% polyethyleimine on stirring followed by reduction of pH to 4.8-5.0. The solution was centrifuged to obtain a clear supernatant, which was further incubated at 25- 35°C for at least 48 hours, on stirring. The resultant turbid solution is clarified on a 0. 1 μ hollow fiber unit at a TMP of 2-4 psi to obtain clear permeate.

Clarified protein solution is loaded onto a cation exchange resin, CM Sepharose FF which captures the protein of interest. Elution is carried out in an ammonium acetate buffer containing 0.2M ammonium sulfate at pH 5.0-5.3. To the elute obtained ammonium sulphate is added to a final concentration of 1 M and this solution is loaded onto Phenyl Sepharose HP resin. Elution is carried out with ammonium acetate buffer having conductivity of 4 to 6mS cm. The elute contains 75-80% pure IFN alpha 2b. Elute is run on a 5KDa crossflow filter at 2 to 4 psi transmembrane pressure to concentrate it 25-30 times to obtain minimum volume of retentate. More than 95% recovery is obtained. The concentrated solution is loaded on a gel filtration column, Sephacryl S- 100 pre-equilibrated with an ammonium acetate buffer with pH 4.0-4.5. Fractions containing IFN a2b are pooled and have a purity of at least 85-90%. The pooled fractions are polished on silica based C4 reverse phase column with a linear gradient of 35% acetonitrile to 45% acetonitrile in water with 0. 1% trifloroacitic acid; 98-99% purity is achieved at this stage. The pure protein is lyophilized to obtain final powder of pharmacopoeial grade. Yields obtained by processing 3L fermented batch are tabulated in Table 1

Table 1 :

Interferon a2b product specification compliance with Indian/European Pharmacopoeia

Lyophilized powder obtained as per process of the present invention was analyzed as per European Pharmacopoeia specifications. The tests included related substance, SDS-PAGE, Isoelectric focusing, Endotoxin, host cell protein, host cell DNA. Findings are presented in Table No. 2

Table no 2:

Sr. no. Test Specification as per European Pharmacopoeia Compliance

In the chromatogram obtained with the test

Related solution, the area of any peak, apart from

01 Complies Protein principal peak, is not greater than 3% of total

area of the entire peak. The sum of area of any peak other than principal peak is not

grater than 5% of the total area of all of the peaks.

The profile of chromatogram obtained with

Peptide the test solution should also correspond to

Complies mapping that of the chromatogram obtained with the

reference solution.

The principal bands of the electropherogram obtained with the test solution ( a)

Isoelectric

corresponds in position to the principal band Complies focusing

in the electropherogram obtained with reference solution( a)

Reducing Condition:

The electropherogram obtained with test solution ( a ) under reducing condition may show additional bands but no such band is more intense than the band obtained with reference solution d. Not more than 3 such bands should be more intense than the principal band obtained with reference solution

Non Reducing Condition:

SDS- The electropherogram obtained with test Complies

PAGE

solution under non reducing condition may show in addition to principal band, less intense bands with molecular masses higher than the principal band. No such band is more intense than the principal band in elecopherogram obtained with reference solution d. Not more than 3 such bands are more intense than the principal band in electropherogram obtained with reference solution.

Host cell

Limit as per approved by WHO guideline Complies Protein

Host cell

Limit as per approved by WHO guideline Complies DNA

Bacterial

Not more than 100 EU/mg of Protein Complies Endotoxin

Claims

CLAIMS We Claim:

1. A process for the preparation of phaniiacopoeial grade Interferon alpha 2b ( IFN a2b) comprising the steps:

a. fed batch feniientation of recombinant E. coli in a suitable medium, wherein the initial fermentation is earned out at 3()"C to 4()"C and then at 18"C to 25"C during induction phase; and wherein the feeding rate during feniientation is 1.5-4 gmL 'h^"1 till 4^th hour. 4-8 gniL^"1!!^"1 during 4^th to 22"^d hour and 1.5-4 ginl V after 22"^d hrs of feniientation to produce feniientation product;

b. isolation and clarification of the feniientation product obtained in step a. to obtain cinde IFN a2b; and

c. purification of the cinde IFN a2b obtained in step b comprising the steps:

i. initial purification of cinde IFN a2b by cation exchange chromatography;

ii. partial purification of elute obtained from step i by Hydrophobic interaction chromatography;

iii. concentrating the elute obtained from step ii by crossflow filtration;

iv. further purification of the concentrated elute obtained in step iii by gel filtration chromatography to obtain purified elute; and

v. polishing the purified elute obtained from step iv by reverse phase chromatography.

2. The process for the preparation of phaniiacopoeial grade IFN a2b as claimed in claim la. wherein the recombinant E coli used are E. coli BL21 Gold strains, preferably E. coli BL21 Gold DE3 strains.

3. The process for the preparation of phaniiacopoeial grade IFN a2b as claimed in claim la. wherein the fed batch feniientation is earned out for 22 - 28 hrs preferably for 25 hrs.

4. The process for the preparation of phaniiacopoeial grade IFN a2b as claimed in claim la. wherein the suitable medium for feniientation comprises: l° o yeast extract (w/v ). 1.2% Dextrose (w/v ). 0.3% KH_:P0₄ (w/v ). 1.25% K_:HP0₄ (w/v). 0.5% (NH₄ )_:S0₄ (w/v ). 0.05% NaCl (w/v ). 0. 1% MgS0₄.7H_:0 (w/v ). 0. 1% (v/v ) trace metal solution, and Ampicillin ( Ι ΟΟμ/ml ).

5. The process for the preparation of phaniiacopoeial grade IFN a2b as claimed in claim l a. wherein the preferred initial feniientation temperature is 30 to 40 °C. preferably 37°C.

6. The process for the preparation of phaniiacopoeial grade IFN a2b as claimed in claim la. wherein the preferred temperature during induction phase is I S to

25°C. preferable 22°C.

7. The process for the preparation of phaniiacopoeial grade IFN a2b as claimed in claim l a. wherein the production of IFN a2b is induced during induction phase using an inducer selected from IPTG. Lactose, preferably IPTG.

8. The process of claim 7. wherein the inducer is used at a concentration range of 200- 1000 μΜ. preferably 1000 μΜ.

9. The process for the preparation of phaniiacopoeial grade IFN a2b as claimed in claim la. wherein the feed is a Carbon ( C ) source and a Nitrogen (N ) source; and wherein both the sources are used independently or in combination.

10. The process for the preparation of phaniiacopoeial grade IFN a2b as claimed in claim 9. wherein the C :N ratio is in the range of 3 : 1 to 6: 1. preferred C:N ratio is 4: 1.

1 1. The process of claim 9. wherein the Carbon source is selected from glucose or glycerol, preferably glycerol and the Nitrogen source is selected from tryptone. peptone or yeast extract, preferably yeast extract.

12. The process for the preparation of phaniiacopoeial grade IFN a2b as claimed in claim lb. wherein the process of isolation of IFN a2b comprises the steps: a. lysis of the bacterial cells to isolate soluble IFN a2b;

b. chemical treatment of the soluble IFN a2b obtained in step a. , using anionicalK charged neutralizer; and

c. incubation of the chemically treated soluble IFN a2b obtained in step b.

13. The process as claimed in claim 1 1a. wherein lysis is earned out on a homogenizer under pressure of 16000-20000 psi at pH 8.

14. The process as claimed in claim l ib. an anionicalK charged neutralizer is selected from poK ethy leneimine. protamine sulphate more preferably poK ethyleneimine.

15. The process as claimed in claim 1 1c. wherein the chemically treated soluble IFN a2b is incubated at 25-33"C for 48 hi s at pH 4.8 to 5.3

16. The process for the preparation of phaniiacopoeial grade IFN a2b as claimed in claim lb. wherein clarification is earned out using 0. 1 μ cross flow filter.

17. The process for the preparation of phaniiacopoeial grade IFN a2b as claimed in claim lc ( i ). wherein the cation exchange resin is selected from SP Sepharose and CM Sepharose FF resin, preferabh CM Sepharose resin and the product is eluted using a buffer selected from Sodium Acetate. Sodium Citrate and Ammonium Acetate preferabh 20 niM to 100 niM Ammonium Acetate more preferabh 50 niM Ammonium Acetate with pH 4.8 to 5.3.

18. The process for the preparation of phaniiacopoeial grade IFN a2b as claimed in claim lc ( ii ). wherein hydrophobic interaction chromatographic resin is selected from Butyl Sepharose HP or Phenyl Sepharose HP resin preferab Phenyl Sepharose HP and the product is eluted using a buffer selected from Sodium Acetate. Ammonium Acetate or Sodium Citrate, preferabh 20 niM to 100 niM Ammonium Acetate more preferabh 50 niM Ammonium Acetate with pH 4.8 to 5.3.

19. The process for the preparation of phaniiacopoeial grade IFN a2b as claimed in claim lc ( iii ). wherein the elute is concentrated using 5 KDa crossflow filtration.

20. The process for the preparation of phaniiacopoeial grade IFN a2b as claimed in claim lc ( iv). wherein gel filtration resin is selected from Superdex 75. Sephadex G25-G75. Sephacryl S-200 or Sephacryl S- 100 preferabh Sephacryl S- 100 resin and the product is eluted using a buffer selected Sodium Acetate. Ammonium Acetate or Sodium Citrate, preferabh 20 niM to 100 niM Ammonium Acetate more preferabh 50 niM Ammonium Acetate with pH 4.8 to 5.3.

21. The process for the preparation of phaniiacopoeial grade IFN a2b as claimed in claim lc (v ). wherein reverse phase chromatography is earned out using silica based C4 reverse phase column with a linear gradient from 35° o Acetonitrile to 45° o Acetonitrile.