FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION (See Section 10 and Rule 13)
ERYTHROPOIETIN CONJUGATES
Intas Biopharmaceuticals Limited
An Indian company having its registered office at:
PlotNo:423/P/A/GIDC
Sarkhej-Bavla Highway
Moraiya, Tal.: Sanand
Ahmedabad-382 213
Gujarat, India
The following specification describes the invention.

FIELD OF THE INVENTION
The present invention relates to a conjugate of erythropoietin and a method of producing the same.
BACKGROUND OF THE INVENTION
Erythropoietin (EPO) is a glycoprotein hormone which stimulates red blood cells 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, inappropriately low serum EPO levels may also be seen in anemic patients with cancer, Human Immunodeficiency Virus (HTV) 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.
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 recombinant human erythropoietin (rHu EPO) has the same biological properties as endogenous erythropoietin secreted in humans. It has a molecular weight of about 36,000 daltons with carbohydrate moiety composing about 30% of molecular weight.
Pegylation technology has emerged as a means to improve the pharmacokinetic and pharmcodynamic properties of biopharmaceuticals. Some of the benefits of pegylation include improved clinical properties, enhanced solubility, sustained absorption and release, reduced immunogenicity and proteolysis, reduced clearance from circulation by the kidneys, increased dosing intervals due to higher in-vivo half-lives owing to increased circulation time and the like. The longer circulation of Erythropoietin results in beneficial therapeutic effects such as prolongation of it's presence in the human body, effective therapeutic treatment of disease and conditions thereof
Pegylated Erythropoietin (MIRCERA® from Roche) is a PEGylated recombinant form of human EPO. The erythropoietin used to generate MIRCERA® is the active substance of Neorecormon® (epoetin beta; Roche's recombinant EPO first approved for general medical use in the EU in 1996). The PEG moiety used is methoxypolyethylene glycol-succinimidyl butanoic acid (PEG-SBA); a 30 kDa linear chemically activated PEG. The PEG-SBA spontaneously forms amide linkages with either EPO's N-terminal amino group or with the E-amino group of an accessible

surface lysine residue (Lys 45 or Lys 52). The final product generated is a 60 kDa monopegylated product.
US5286637 describes about biologically active drug polymer derivatives, namely peptides or protein derivatives, are useful medicaments and are represented by the generic formula: RO-(CH2CH2O)n-(CO)-NH-X-(CO)-NH-Z wherein R represents a lower alkyl group, n is an integer comprised between 25 and 250, X when combined with adjacent NH and CO groups represents an amino acid or a dipeptide or tripeptide residue, and Z when combined with the adjacent NH group represents a biologically active peptide or protein or NH or NH2 containing drug residue.
Indian Patent 212429 describes about a conjugates of erythropoietin with poly(ethylene glycol) comprising an erythropoietin glycoprotein having at least one free amino group and having the in vivo biological activity of causing bone marrow cells to increase production of reticulocytes and red blood cells and selected from the group consisting of human erythropoietin and analogs thereof which have sequence of human erythropoietin modified by the addition of from 1 to 6 glycosylation sites or a rearrangement of at least one glycosylation site; said glycoprotein being covalently linked to "n" polyethylene glycol) groups of the formula-CO-(CH2)x(OCH2CH2)mOR with the carbonyl of each poly(ethylene glycol) group forming an amide bond with one of said amino groups; wherein R is lower alkyl; x is 2 or 3; m is about 450 to about 900; n is from 1 to 3; and n and m are chosen so that the molecular weight of the conjugate minus the erythropoietin glycoprotein is from 20 kilodaltons to 100 kilodaltons.
291/MUM/2005 describes about a conjugate of erythropoietin having higher bioactivity comprises an Erythropoietin glycoprotein having N-terminal a - amino group and aldehyde derivatives of polyethylene glycol, said erythropoietin glycoprotein being selected from the group consisting of human erythropoietin and analogs thereof, said aldehyde derivative of methoxy polyethylene glycol of the formula: PEG-R-CHO wherein R= alkyl group of formula -(CH2)n. wherein n=2 to 4. The formula of the conjugate of the present invention is: PEG-R-CONH-EPO wherein R=alkyl group of formula -(CH2)n. wherein n=2 to 4 Said erythropoietin according to the present invention is a recombinant erythropoietin (rhEPO) produced in animal cells; said erythropoietin conjugate is prepared by expressing, fermenting, and purifying rhEPO protein in recombinant animal cells and reacting EPO with PEG-AIdehyde to obtain N-terminaily pegylated EPO by reductive amination reaction and then further purified to obtain pure N-terminally pegylated EPO.

978/MUM/2004 describes about a non-glycosylated EPO with improved stability and half lives. The present invention also provides a process for the preparation of a non-glycosylated EPO with improved stability and half life. The invention provides an erythropoietin compound comprising a non-glycosylated human erythropoietin and Y-PEG_NHS.
US6340742 describes about erythropoietin glycoprotein products which have the in vivo biological activity of causing bone marrow cells to increase production of reticulocytes and red blood cells. These conjugates have an increased circulating half-life and plasma residence time, decreased clearance, and increased clinical activity in vivo. In addition, compared with conventional PEG-EPO conjugates, the conjugates of this invention have superior clinical properties. This invention also describes a method for the treatment of anemia in a human employing the novel erythropoietin glycoprotein products as well as a method for preparing the erythropoietin glycoprotein products.
US7128913 describes about conjugates of erythropoietin with poly(ethylene glycol) comprising an erythropoietin glycoprotein having an N-terminal alpha.-amino group and having the in vivo biological activity of causing bone marrow cells to increase production of reticulocytes and red blood ceils and selected from the group consisting of human erythropoietin and analogs thereof which have the sequence of human erythropoietin modified by the addition of from 1 to 6 glycosylation sites or a rearrangement of at least one glycosylation site; said glycoprotein being covalently linked to one poly(ethylene glycol) group of the formula-CO-(CH2)x(OCH2CH2)mOR wherein the -CO of the poly(ethylene glycol) group forms an amide bond with said N-terminal alpha.-amino group; and wherein R is lower alkyl; x is 2 or 3; and m is from about 450 to about 1350.
However, the bioavailability of commercially available protein therapeutics such as EPO is limited by their short plasma half-life and susceptibility to protease degradation. These shortcomings prevent them from attaining maximum clinical potency.
Compared to unmodified EPO (i.e. EPO without a PEG attached) and conventional PEG-EPO conjugates, the present conjugates have an increased circulating half-life and plasma residence time, decreased clearance, and increased clinical activity in-vivo. The conjugates of this invention have the same uses as EPO. In particular, the conjugates of this invention are useful to treat patients by simulating the division and differentiation of committed erythroid progenitors in the bone marrow in the same way EPO is used to treat patients.

SUMMARY OF THE INVENTION
An objective of the present invention is to provide conjugate wherein the said conjugate comprising an erythropoietin glycoprotein having at least one free amino group selected from the group consisting of human erythropoietin and analogs thereof wherein said glycoprotein being covalently linked to "n" poly (ethylene glycol) groups of the
CH3O(CH2CH2O)nOCO Formula I
with -CO- group of each poly (ethylene glycol) group forming an amide bond with one of said amino groups of glycoprotein wherein n is from 300 to 900, and "n" is chosen so that the molecular weight of the polyethylene glycol units is from 15,000 to 45,000 Daltons.
A conjugate according to the said invention having the formula
CH3O(CH2CH2O)nOCONH-P Formula-II
The present invention to provide a process for the preparation of conjugate as described above which comprising condensing the compound of Formula I.
The pharmaceutical composition comprising a conjugate as described in the present invention may be formulation in a suitable combination of pharmaceutically acceptable excipients.
The pharmaceutical composition comprising the conjugate may be formulated at a particular strength effective for administration by various means to a human patient experiencing blood disorders characterized by low or defective red blood cell production.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the SDS-PAGE with silver staining analysis for purity
Lane 1 is PEG-EPO
Lane 2 is 1% of PEG-EPO loaded in lane 1,
Lane 3 is 1% EPO protein of PEG-EPO loaded in Lane 1
Lane 4 is molecular weight marker.
Figure 2 shows SDS-PAGE Western blot for identity Lane 1 is PEG-EPO Lane 2 is blank

Lane 3 pre stained molecular weight marker
Figure 3 shows SE-HPLC for purity, principle peak is of Mono-Peg-EPO bulk protein, pre peaks species are poly Pegylated EPO and Post peak species is non-Pegylated EPO species.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a conjugate, said conjugate comprising an erythropoietin glycoprotein having at least one free amino group selected from the group consisting of human erythropoietin and analogs thereof wherein said glycoprotein being covalently linked to "n" poly (ethylene glycol) groups of the Formula I with CO group forming an amide bond with one of said amino groups of glycoprotein wherein n is from 300 to 900

wherein the molecular weight of the polyethylene glycol units is from 15,000 to 45,000 Daltons.
The term "Erythropoietin" or "EPO" refers to a glycoprotein, having the amino acid sequence encoding the human protein erythropoietin which is described in the US patent no.4,703,008. These terms also include analogs having 1 to 6 additional glycosylation sites for glycosylation. These terms also include both natural and recombinantly produced human erythropoietin.
The conjugates of this invention can be represented by Formula II
CH3O(CH2CH2O)nOCONH-P Formula II
In an embodiment, P may be selected from the group consisting of residues of human erythropoietin and analogs thereof having from 1 to 6 additional sites for glycosylation.
Preferably, the glycoprotein of the conjugates as defined above is a human erythropoietin.
The recombinant EPO was produced by growing recombinant CHO cells in a serum free medium. Recombinant human EPO was synthesized by recombinant CHO cells is secreted to the culture medium which is then purified by using chromatographic steps. The purified EPO thus obtained is used for further pegylation.

With the purified EPO monomethoxy-PEG-Nitro Phenyl carbamate/carbonate was added and mixed to obtain a Pegylated Erythropoietin. The obtained conjugated erythropoietin is purified further by using various chromatographic steps which is then used for further formulation.
In context of the present invention, one or more pharmaceutically acceptable excipients may be used optionally to prepare the formulations comprising PEG-EPO.
The invention will be better understood by reference to the following examples which illustrate but do not limit the invention described herein.
Example 1 Preparation of EPO
The rHu EPO is secreted in the harvest while the cells are adherent to the surface of roller bottles. Any cell debris or detached cells were removed by 0.45μ filtration. The harvest was loaded on the affinity column where rHu EPO binds and the rHu EPO is eluted using high salt. Elute of affinity column was desalted using the gel filtration column. The output after desalting is Ion exchange input. The Ion Exchange input is loaded on anion and cation Exchange columns; the bound protein was eluted using the salt gradient. Cation exchange output is pure protein buffer exchanged into the formulation buffer. The rHu EPO bulk was filtered by passing through 0.22-μ. filters in a laminar hood. This is rHu EPO bulk solution ready for manufacturing of Pegylated EPO bulk protein.
Example 2 Pegylation Reaction
PEG-EPO molecule is formed by the attachment of Erythropoietin with a functionalized Poly Ethylene Glycol through the amino group of the Lysine residue and N-terminals of protein. The said molecule is one where only one PEG reagent binds per protein molecule called the monopegylated conjugate.
The pH of EPO bulk protein used in pegylation was adjusted to 9.0 with 6% boric acid and 3% NaOH. Pegylation reaction was done by adding the solid PEG reagent to the protein solution after preconditioning the protein. The PEG reagent was added in a molar excess of 1 to 3 on activity basis. Peg reagent was allowed to dissolve and react by mild rocking in a falcon tube using platform rocker. Reaction was carried out for 18-20 hours at 4°C temperature in cold cabinate.

Analysis of the pegylation reaction was carried out through SEC-HPLC and SDS- PAGE to check the pegylation yield.
Pegylation Reaction:
The reaction mechanism is shown in below

Reaction mechanism of Peg- Erythropoietin conjugates formation.
Purification of mono Pegylated protein
Pegylation mixture was diluted to 5 fold with 20mM Sodium Acetate, pH 3.0 to. The purification of the protein was done through cation exchange chromatography using SP MacroCap Resin (GE Healthcare) with 20mM Sodium Acetate, pH 3.0. Diluted protein was loaded on equilibrated column, non conjugated PEG reagents was removed as column unbound, Di-PEG-EPO, Mono-PEG-EPO and Non Pegylated EPO was eluted by varying sodium chloride salt concentrations in 20mM Sodium Acetate, pH 3.O.. Fraction containing mono-Pegylated EPO protein was collected and concentrated through 30 kDa TFF membrane. Finally, Concentrated mono-PEG-EPO was buffer exchanged to formulation buffer by diafiltration and tested by various analytical techniques.
Example 3
SDS-PAGE with silver staining for purity
SDS-PAGE of purified PEGylated EPO was performed to visualize impurities differing in molecular weight. 4 % - 20 % SDS-PAGE gradient gel was prepared using former system. Test

samples were run at the constant voltage: 130 V and maximum current: 200 mA. Electrophoresis run until dye front completely elutes. Gel stained with silver stain. (Figure 1)
Western Blot
Western blot method is a combination of SDS-PAGE followed by blotting of SDS-PAGE. 8 % Acrylamide gel (90*30*1 mm) was used. Test sample was treated with loading buffer and loaded for separation. Electrophoresis was carried out at 130 volts till dye reached the bottom of gel. Poly-Vinylidene Di-Fluoride (PVDF) membrane was used for transfer. PVDF membrane was placed in blocking buffer (5 % skimmed milk powder (non-fat) in lx TBS) for 1 h with gentle shaking at RT. Membranes were treated with 1:1000 diluted primary antibody (Anti IFN raised in rabbit), prepared in blocking buffer for 1 h with gentle agitation at RT. 3 washes of (lx) TBS were given to membrane after 5 min interval time. Membranes were treated with 1:2000 diluted secondary antibody enzyme labeled (Goat anti rabbit ALP conjugate), prepared in buffer for 1 h with gentle agitation at RT. Three washes were given to membrane, after 5 min interval time. Color development was observed by adding the substrate solution (BCTP/NBT) followed by incubation in dark. (Figure 2)
Size Exclusion -High Performance Liquid Chromatography:
SE-HPLC was performed on the Agilent HPLC system. HPLC system was prepared with TSK G3000SWxl column with Diameter: 7.8 mm, Length: 300 mm Porosity: 250-A Particle size: 5 μ. The guard column used Porous silica gel TSK SWXL, catalogue No: 08543, diameter: 6 mm, Length: 400 mm Particle size: 7 um. The mobile phase used was phosphate buffer, pH 5.0 containing sodium chloride. The method was run at 0.5 ml/min flow-rate at 30 °C temperature for 40 min. Output was detected at 214 nm wavelength. (Figure 3)

Claims
1. A conjugate comprising an erythropoietin glycoprotein having at least one free amino group selected from the group consisting of human erythropoietin and analogs thereof wherein said glycoprotein being covalently linked to V poly (ethylene glycol) groups of the formula
CH3O(CH2CH2O)nOCO Formula I
with -CO- group of each poly (ethylene glycol) group forming an amide bond with one of said amino groups of glycoprotein wherein "n" is from 300 to 900, and "n" is chosen so that the molecular weight of the polyethylene glycol units is from 15,000 to 45,000 Dal tons.
2. The conjugate according to claim 1, of the formula CH3O(CH2CH2O)nOCONH-P wherein n is defined as claim I, and P is the residue of the glycoprotein without n amino group(s) which form amide linkage(s) with the poly (ethylene glycol) group(s).
3. The conjugate according to any preceding claim, wherein the glycoprotein is a human erythropoietin.
4. The conjugate according to any of the claims 1 to 3, wherein the glycoprotein has the sequence of human erythropoietin modified by the addition of from 3 to 6 glycosylation sites.
5. A composition comprising conjugates, each of said conjugates comprising an erythropoietin glycoprotein having at least one free amino group selected from the group consisting of human erythropoietin modified by the addition of from 1 to 6 glycosylation sites or a rearrangement of at least one glycosylation site; the glycoprotein in each said conjugate being covalently linked to "n" poly(ethylene glycol) groups of the formula CH3O(CH2CH2O)nOCO- with the -CO group of each poly (ethylene glycol) group forming an amide bond with one of said amino groups of glycoprotein wherein "n" is from 300 to 900, and "n" is chosen so that the molecular weight of the polyethylene glycol units is from 15,000 to 45,000 Daltons.
6. A pharmaceutical composition comprising a conjugate or a composition according to any of the claims 1 to 4 and a suitable combination of pharmaceutically acceptable excipients.

7. A process for the preparation of compounds according to any of the claims 1 to 4 which
process comprises condensing the compound of formula

with a erythropoietin glycoprotein as defined in any of the claims 1 to 4.
8. Compounds according to any of claims 1 to 3, whenever prepared by the process of claim
7.
9. A conjugate substantially as described herein before.