FORM 2
THE PATENTS ACT 1970
(39 of 1970)
&
The Patents Rules, 2003
PROVISIONAL/COMPLETE SPECIFICATION
(See section 10 and rule 13)
TITLE OF THE INVENTION: PROCESS FOR THE PREPARATION OF INTERLEUKIN-2

1. APPLICANT
(a) Name: Intas Biopharmaceuticals Limited
(b) Nationality: Indian
(c) Address: Gautam Bakshi (IN/PA-1069)
Intas Biopharmaceuticals Limited Intellectual Property Management (IPM) Plot No: 423/P/A/GIDC Sarkhej-Bavla Highway Moraiya, Tal.: Sanand Ahmedabad-382 210 Gujarat, India.

2. PREAMBLE TO THE DESCRIPTION
The present invention relates to the process for the preparation of lnterleukin-2 using exponential feeding strategy. The process of the present invention was found to be economically viable and feasible.

COMPLETE
The following specification particularly describes the invention and the manner in which it is to be performed.

4. DESCRIPTION (Description shall start from next page.) Enclosed in separate sheet
5. CLAIMS (not-applicable for provisional specification. Claims should start with the preamble "I/We claim" on separate page)
6. DATE AND SIGNATURE (to be given at the end of last page of specification)


7. ABSTRACT OF THE INVEN
separate page)
iven along with complete specification on

Note:-
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*Name of the applicant should be given in full, family mane in the beginning.
"Complete address of the applicant should be given stating the postal index no./code,
state and country.
"Strike out the column which is/are not applicable.

FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003
PROVISIONAL/COMPLETE SPECIFICATION (See section 10 and rule 13)
Title: PROCESS FOR THE PREPARATION OF INTERLEUKIN-2
Intas Biopharmaceuticals Limited
An Indian company having its registered office at:
Plot No: 423/P/A/GIDC
Sarkhej-Bavla Highway
Moraiya, Tal.: Sanand
Ahmedabad-382 210
Gujarat, India
The following specification describes the invention.

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PROCESS FOR THE PREPARATION OF INTERLEUKIN-2
INTRODUCTION TO THE INVENTION
The present invention relates to the process(s) for the preparation of lnterleukin-2.
lnterleukin-2 (IL-2) belongs to a family of cytokines. IL-2 is normally produced by the body during an immune response. When environmental substances (molecules or microbes) gain access to the body, these substances (termed antigens) are recognized as foreign by antigen receptors that are expressed on the surface of lymphocytes. Antigen binding to the T cell receptor (TCR) stimulates the secretion of IL-2, and the expression of IL-2 receptors IL-2R. The IL-2/IL-2R interaction then stimulates the growth, differentiation and survival of antigen-selected cytotoxic T cells via the activation of the expression of specific genes. IL-2 is necessary for the development of T cell immunologic memory, one of the unique characteristics of the immune system, which depends upon the expansion of the number and function of antigen-selected T cell clones.
A recombinant form of IL-2 is manufactured by Chiron Corporation with the brand name Proleukin. It is used for the treatment of cancers (malignant melanoma, renal cell cancer).
US Patent 5,849,883 describes processes for isolating and purifying granulocyte colony stimulating factor (G-CSF) from a G-CSF producing microorganism. The simplified processes include steps of lysing the microorganism and separating insoluble material; extracting the material with deoxycholate (optionally); solubilizing and oxidizing the G-CSF in the presence of

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a denaturant solubilizing agent and an oxidizing agent; removing the denaturant solubilizing agent; subjecting to ion exchange chromatography; and recovering the purified protein. The process as described in this patent document is for the isolation and purification of G-CSF describing only about the downstream process(s). Further the protein G-CSF as described in the said patent document is different from the protein of the present invention IL-2.
The process(s) for the preparation of conjugates of IL-2 are addressed by the present invention.
SUMMARY OF THE INVENTION
The present invention relates to the process(s) for the preparation of lnterleukin-2.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to the process for the preparation of lnterleukin-2. The process of the present invention was found to be economically viable and feasible.
In context of the present invention, the process for the preparation of lnterleukin-2 comprises using genetically modified E.Coli in a fermenter followed by harvesting and isolation of inclusion bodies (IB), refolding, extraction and purification of interleukin-2 protein. The purification is done using the following steps: purification, concentration and polishing to obtain pure IL-2.
In an embodiment, interleukin-2 was manufactured using genetically modified E.Coli in a fermenter by exponential feeding strategy under controlled conditions and induction with animal source free media (ASF). Fermentation was followed by harvesting and extraction of the inclusion bodies by cell disruption,

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centrifugation and buffer washes respectively. Inclusion bodies were refolded and purified by ion exchange, hydrophobic interaction chromatography and gel filtration chromatography to obtain pure IL-2 in the formulation buffer. Final bulk was formulated and stored under specified conditions.
In one embodiment, the process may include the following steps and procedures for the growth of E.coli, isolation of inclusion bodies (IB), refolding, extraction and purification of interleukin-2 protein. First E.coli was grown in seed media and then the developed seed (inoculum) was transferred to the fermenter for further growth. The initial production fermenter's Optical Density (OD) was maintained at desired value by transferring seed inoculum. Then initial parameters may be maintained to the specified set points. Feeding may be given by exponential growth strategy, followed by induction by elevating the temperature, decreasing of rpm to a specified seed and finally post induction feeding. Isolation of Inclusion bodies may comprise of harvesting, then the biomass may be separated by centrifugation from the fermentation broth. After centrifugation, biomass may be homogenized with dithiothreitol (DTT) and inclusion bodies may be isolated from E.coli by cell disrupter using desired pressure. The lysate may be centrifuged to remove cell membrane and debris. After that, lysate may be homogenized with resuspending the buffer to chelating agent for divalent metal ions and stir for specified conditions. Then, the buffer may be separated by centrifugation. Finally inclusion bodies may be washed with water for injection (WFI) to remove impurities. The over expression of protein in bacteria may result into improper folded protein, which are there in the cells in the form of insoluble inclusion body. Oxidizing agent acts as a catalyst for oxidation of insoluble inclusion body (IL-2 molecule) which has two sufhydril groups that can form one disulfide bond. This converts biologically inactive IL-2 to active from. During the refolding step the sulfhydril groups of IL-2 molecules get oxidized and form disulfide bonds.

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Further, the unfolded protein may be refolded to regain its native structure for biological activity. The refolding may be carried out in the presence of suitable denaturant which acts as a solubilizing agent. The denatured protein may be oxidized and refolding may be carried out for 36hours at 25°C. The resultant mixture contains detergent, which is removed using anion exchanger. The detergent may bind to the anion exchanger and protein comes out as flow through. The resultant protein mixture, which contains IL-2 may be turbid due to removal of detergent. The IL-2 may be extracted from this mixture by pH precipitation. Most of the host cell related impurities are precipitated at about pH 6 and IL-2 may be in solubilized form, which is recovered by filtration. The obtained extract may be subsequently purified using adsorptive chromatography to get the required purity. Finally gel filtration chromatography may be carried out to obtain pure IL-2 in the formulation buffer.
In yet another embodiment, lauryl sarcosine sulfate (LSS) was found to be useful as a surfactant in context of the present invention.
In an embodiment, the concentration of LSS used in the process for the preparation of lnterleukin-2 ranges from about 1% to about 5%.
In another embodiment, copper sulfate was found to be useful as an oxidizing agent in context of the present invention.
In yet another embodiment, the concentration of copper sulfate used in the process for the preparation of lnterleukin-2 ranges from about 0.1 mM to about 0.3 mM.
In context of the present invention, the process for the purification consists of purification step, concentration and polishing step. The purification technique used can be either cation or anion exchange chromatography or a combination of both. The concentration step can be a cross flow filtration using appropriate

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membrane or any adsorptive chromatography. The buffer exchange step can be dialysis using membrane filtration or chromatography on a suitable resin.
In context of the present invention, the purification techniques known to the one skilled in the art for the purification of therapeutic proteins are but not limited to, ion exchange chromatography such as cation and/or anion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography and the like.
Further, ion exchange resins found useful are but not limited to anion exchange resins such as Q - Sepharose, DEAE sepharose, Unosphere - Q, Amberlite with its various grades and the like; cation exchange resins like SP Sepharose, CM-Sepharose, Unospere S, Amberlite MB-50 with its various grades and the like.
In context of the present invention, one or more pharmaceutically acceptable excipients may be used optionally for the preparation of IL-2 conjugates or mixture(s) of conjugates of IL-2. These pharmaceutically acceptable excipients may include but are not limited to stabilizing agents, co-solvents, solubilizing agents, buffers and pH adjusters, chelating agents, oxidizing agents and the like.
Solubilizing or suspending agents that is surfactants may include anionic surfactants such as chenodeoxycholic acid, 1-octanesulfonic acid sodium salt, sodium deoxycholate, glycodeoxycholic acid sodium salt, N-lauroylsarcosine sodium salt or lauryl sarcosine sulfate (LSS), lithium dodecyl sulfate, sodium cholate hydrate, sodium lauryl sulfate (SLS) and sodium dodecyl sulfate (SDS); cationic surfactants such as cetylpyridinium chloride monohydrate and hexadecyltrimethylammonium bromide; nonionic surfactants such as N-decanoyl-N-methylglucamine, octyl a-D-glucopyranoside, n-Dodecyl b-D-maltoside (DDM),

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polyoxyethylene sorbitan esters like polysorbates such as Tween 80, Tween 20 and the like.
Various buffers and pH adjusters include but not limited to acetate, citrate, phosphate or combinations thereof; acetic acid, hydrochloric acid, sodium hydroxide, sodium acetate trihydrate and the like.
Further chelating agents include but not limited to ethylenedinitrilotetra acetic acid and its salts and the like; oxidizing agents include but not limited to oxygen sparging, oxidized glutathione, cystine, zinc sulfate and the like.
The following examples will further illustrate certain aspects and embodiments of the invention in greater detail and are not intended to limit the scope of the invention.
EXAMPLE
Example 1
Process for the preparation of IL-2
Manufacturing process:
Seed media:

I S.No. Ingredients Quantity(g/L)
I1 Papaic Soya peptone 10
2 Yeast extract 5
3 Ampicillin 0.1
Seed media was prepared in duplicates. Fermentation media:

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S.No. Ingredients Quantity(g/L)
1 Papaic Soya peptone 12
2 Yeast extract 24
3 Di-potassium hydrogen phosphate) 5.71
4 Potassium dihydrogen phosphate 2.85
5 Manganese chloride 0.004
6 Strucktol 673 0.44 ml 7
Glucose
Ampicillin 0.1
Cells were inoculated at pH 7 of fermentation media
Feed media composition:

S.No. Ingredients Quantity(g/L)
1 Papaic Soya peptone 23.5
2 Yeast extract 11.77
3 Ampicillin 0.1
4 Strucktol 673 0.44 ml
5 Di-potassium Hydrogen phosphate 4.42
6 Potassium dihydrogen phosphate 2.21
7 Glucose 19.6

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Production of rHu IL-2 from E.coli
(Upstream processing & Primary down stream processing)

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*Abbreviations:
Ø WFI= Water For Injection
Ø WCB= Working Cell Bank
Ø wm= volume of air per volume of medium per minute
Ø DTT=Dithiothreitol
Ø KPSI=kilo pounds per square inch
Ø RCF= Relative Centrifugal Force
Ø IB=lnclusion Bodies
Ø RT= Room Temperature
Ø DEAE= Diethylaminoethyl
Ø CM= Carboxymethyl
Ø HIC= Hydrophobic interaction Chromatography
Ø LSS= Lauryl sarcosine sulfate

Dated: 31st day of May 2007