FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003
PROVISIONAL/COMPLETE SPECIFICATION (See section 10 and rule 13)
Title: PROCESS(S) FOR THE PRODUCTION OF ISOFORMS OF RECOMBINANT HUMAN ERYTHROPOIETIN
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(S) FOR THE PRODUCTION OF ISOFORMS OF RECOMBINANT
HUMAN ERYTHROPOIETIN
INTRODUCTION TO THE INVENTION
The present invention relates to the process(s) for the production of isoforms of recombinant human erythropoietin (rHu EPO) from stirred tank bioreactor using fed-batch process.
Erythropoietin (EPO) is a glycoprotein hormone, which stimulates red blood cells (RBC) production by a process known as erythropoiesis. EPO is produced in the kidney and stimulates the division and differentiation of committed erythroid progenitors in the bone marrow. In patients with renal insufficiency, serum EPO levels remain low despite the anemia. Inappropriately low serum EPO levels may also be seen in anemic patients with cancer, HIV infection, ulcerative colitis and sickle cell anemia. For all these indications and to decrease the rate of blood transfusion, EPO is established as an effective treatment.
Cloning of EPO gene in 1985 was a break-through to produce EPO using recombinant DNA technology. Since 1989 recombinant Human Erythropoietin (rHu EPO) is available as a drug and listed as a Pharmacopeial product. The rHu EPO is a 165 amino acid containing glycoprotein produced through recombinant DNA technology in animal cell lines such as Chinese Hamster Ovary (CHO) and Baby Hamster Kidney (BHK) cell lines. The rHu EPO has the same biological properties as endogenous erythropoietin secreted in humans. It has a molecular weight (MW) of about 30,600 daltons with carbohydrate moiety composing about 30% of MW. The presence of carbohydrate moiety known as glycosylation determines the extent of biological activity in vivo and the potency of rHu EPO.

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More the extent of glycosylation, more the isforms, which gives rise to high in vivo biological activity of rHu EPO. The rHu EPO is available as erythropoietin alpha and erythropoietin beta. Biochemically and clinically erythropoietin Alpha and erythropoietin Beta are indistinguishable.
United States Patent Application 20050069979 describes a method for producing a recombinant polypeptide of interest which method comprises: (a) providing a transformed eukaryotic host cell which comprises a nucleotide sequence which encodes the recombinant polypeptide of interest and which directs expression of the recombinant polypeptide of interest in the host cell; (b) providing a serum-free culture medium which comprises (i) water, a plant-derived peptone, an osmolality regulator, a buffer, an energy source, amino acids, a lipid source or precursor, a source of iron, non-ferrous metal ions and one or more vitamins and cofactors; and (ii) does not contain any full-length polypeptides; and (c) culturing the transformed eukaryotic host cell in the culture medium under conditions that allow for expression of the recombinant polypeptide of interest.
EP1428878 describes the production of recombinant glycoproteins, in particular rHu EPO, in mammalian cells, as well as methods of purifying the produced glycoproteins from the cell culture. The patent document discloses the production of recombinant human erythropoietin containing a maximum degree of sialylation at a higher pH range.
None of the above mentioned patent references describe cell culture production process parameters so as to control the production of isoforms of rHu EPO as addressed by the present invention.

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SUMMARY OF THE INVENTION
The process(s) for the production of isoforms of rHu EPO are described in context of the present invention. Stirred tank bioreactor based fed-batch production process(s) or cell culture production process(s) have been developed to control isoforms production of EPO.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to the production of isoforms of rHu EPO. The process(s) of the present invention was found to be economically viable and feasible.
In context of the present, recombinant Chinese hamster ovary (CHO) cells were used to produce rHu EPO alpha. These recombinant CHO cells were grown as suspension cells in serum free and animal component free cell culture medium in tissue culture flasks and spinner flasks. For the production of rHu EPO, recombinant CHO cells were cultured in stirred tank bioreactors in fed-batch mode for the desired period. During this period the rHu EPO is secreted by recombinant CHO cells into the culture medium. It was found that the different process parameters independently played a role in secreting isoforms of rHu EPO, which are desirable from the point of high in vivo biological activity.
In context of the present invention, cell culture production process parameters such as bioreactor mixing rate, mode of supply of gases, feed components and frequency of feeding have been found to control the production of isoforms of rHu EPO.
In an embodiment, CHO cells were cultured in stirred tank bioreactors in fed-batch mode for about 10 days to about 16 days.

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ln another embodiment, the mode of supply of gases (air, oxygen, carbon dioxide and nitrogen) into the bioreactor was done through headspace. It was observed that the direct supply of gases into the culture medium through a sparger decreased the production of isoforms. Supply of these gases through headspace by spraying over the liquid layer without directly introducing into the culture medium increased the production isoforms of rHu EPO with high in vivo biological activity.
In yet another embodiment, increase or decrease of dissolved oxygen (p02) concentration in the bioreactor from about 0% to about 5% or from about 60% to about 100% affected the secretion of isoforms of rHu EPO.
In one embodiment, increase or decrease of culture pH in the bioreactor affected the secretion of isoforms of rHu EPO. The pH found suitable in context of the present invention ranges from about 6.7 to about 7.5.
In an embodiment, production of isoforms was high when the mixing of culture contents in the bioreactor (agitation / stirring) rate was maintained at a defined speed rate. Decrease or increase in mixing rate beyond this range resulted in low yield or absence of isoforms. The rpm found suitable in context of the present invention ranges from about 125 rpm to about 600 rpm.
In another embodiment, feed component's addition into the bioreactor every day produced high concentration of isoforms whereas alternate day addition of feed components decreased the secretion of isoforms with high in vivo biological activity.

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In context of the present invention, addition or deletion of feed components such as but not limited to glucose, glutamine, amino acids, vitamins, non-animal derived hydrolysates, recombinant growth factors and the like played a role in the production of forms of rHu EPO. Change in the concentration of any one of these or all of these feed components resulted in increase or decrease in isoforms secretion with high in vivo biological activity.
In one embodiment, the method used for the quantification of rHu EPO secreted by the recombinant CHO cells was reverse phase-high performance liquid chromatography (RP-HPLC). Further, to determine the different isoforms, isoelectric focusing (IEF) was used, which utilizes isoelectric point (pi) of rHu EPO.
In context of the present invention, one or more desirable amino acids, recombinant growth factors may be used optionally for the production of isoforms of rHu EPO.