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 GRANULOCYTE COLONY-STIMULATING FACTOR (G-CSF)
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 GRANULOCYTE COLONY-STIMULATING FACTOR (G-CSF)
INTRODUCTION TO THE INVENTION
The present invention relates to the process(s) for the preparation of Granulocyte Colony-Stimulating Factor or mixture(s) of Granulocyte Colony-Stimulating Factor.
Granulocyte Colony-Stimulating Factor (G-CSF) is a glycoprotein, growth factor or cytokine produced by a number of different tissues to stimulate the bone marrow to produce granulocytes. It also stimulates the survival, proliferation, differentiation and function of neutrophil granulocyte progenitor cells and mature neutrophils. G-CSF is produced by endothelium, macrophages, and a number of other immune cells. The natural human glycoprotein exists in two forms of a 174-and 180-amino-acid-long protein of molecular weight 19,600 grams per mole. The more-abundant and more-active 174-amino acid form has been used in the development of pharmaceutical products by recombinant DNA (rDNA) technology.
The recombinant human G-CSF synthesised in an E. coli expression system is called filgrastim. The structure of filgrastim differs slightly from the structure of the natural glycoprotein. Filgrastim is a water-soluble 175 amino acid protein with a molecular weight of approximately 19 kilodaltons (kD). Filgrastim (Neupogen®) and PEG-filgrastim (Neulasta®) are two commercially-available forms of rhG-CSF (recombinant human G-CSF).
Chinese patent application no.CN 1718740 describes a fermenting method for preparing recombinant human granulocyte colony stimulating factor which include steps such as screening bacterial strain, culturing in the class-one seed liquid, culturing in class-two seed liquid, inoculating basic fermenting and induced

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fermenting while supplementing material every one hour when the OD600 is 2 and terminating the fermentation. Its advantages disclosed are high output and high activity.
The process(s) for the preparation of G-CSF are addressed by the present invention.
SUMMARY OF THE INVENTION
The present invention relates to process(s) for the preparation of Granulocyte Colony-Stimulating Factor (G-CSF) or mixture(s) of G-CSF by high cell density fermentation process using for E.coli so as to get high yield of inclusion bodies and henceforth G-CSF.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to process(s) for the preparation of G-CSF or mixture(s) of G-CSF. Also the present invention was found to be economically viable and feasible by virtue of reduction in the number of batches to be taken.
In an embodiment of the present invention, high cell density fermentation of E.coli with animal source free media was done using two-stage open loop fed batch technique.
In one embodiment, step gradient mode of feeding was utilized for the preparation of G-CSF or mixture(s) of G-CSF. In first stage of fed batch, carbon source was fed with a predetermined gradient and at the second stage nitrogen was given in a single lot. DO was kept constant with stirrer and oxygen enrichment in cascade mode.

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In another embodiment, the first stage of fed batch in which carbon source was fed with a predetermined gradient may begin at about 7 to about 8 hours of fermentation to about 12 to about 13 hours and may finish at about 12 hours to about 13 hours of fermentation.
In yet another embodiment, in the first stage of fed batch, the rpm of the stirrer may be from 400 rpm to about 700 rpm.
In an embodiment, about six to seven carbon feeds were given in the first stage of fed batch, which may continue with one to two hour intervals in between the carbon feeding.
In an embodiment, the second stage of fed batch in which nitrogen source was fed with a predetermined gradient may begin at about 13 hours to about 14 hours of fermentation and may finish at about 25 hours to about 26 hours of fermentation.
In another embodiment, in the second stage of fed batch, the rpm of the stirrer may be from 350 rpm to about500 rpm.
In yet another embodiment, harvest may begin at about 25 hours to about 28 hours.
In yet another embodiment, the yield of Inclusion bodies ranges from about 10 gm/lit to about 15 gm/lit.
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.

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EXAMPLE
Example 1
Process for the preparation of rHu G-CSF from E. coli by High Cell Density
fermentation
Upstream processing & Primary down stream processing:

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"Abbreviations:
> WFI= Water For Injection
> OD= Optical Density
> DOC= Dissolved Oxygen Concentration
> WCB= Working Cell Bank
> wm= Volume of air per volume of medium per minute
> KPSI= Kilo pounds per square inch
> RCF= Relative Centrifugal Force
> IB= Inclusion Bodies
> RT= Room Temperature