FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See Section 10 and Rule 13)
PROCESS FOR THE PURICATION OF HYPERGLYCOSYLATED ANALOGUE OF ERYTHROPOIESIS STIMULATING PROTEIN
Intas Biopharmaceuticab 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.

FIELD OF THE INVENTION
The present invention relates to a .process for the purification of hyperglocysylated analogue of erythropoietin.
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 (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.
Novel Erythropoiesis stimulating protein (NESP) [s an analogue of Erythropoietin with five N-linked carbohydrate chains and one O-linked carbohydrate chain. Due to its increased sialic acid containing carbohydrate content, NESP is biochemically distinct from recombinant human Erythropoietin (rHu EPO), having an increased molecular weight and greater negative charge. Compared with rHu EPO, it has an approximate three fold longer serum half-life, greater in-vivo potency.
US7217689 discloses about an analog of human erythropoietin having the in vivo biological activity of increasing hematocrit comprising- an amino acid sequence which differs from the amino acid sequence of human erythropoietin from residues 1-165 as shown in SEQ ID NO:26 at least by having one or more additional glycosylation site(s) and wherein a carbohydrate chain is attached to the additional site(s).
EP0228452 describes a method of purifying a biologically active erythropoietin from a liquid comprising the following chromatographic steps: reverse phase liquid chromatography and anion exchange chromatography.
EP0267678 describes about a method of producing at least 99% pure EPO, involving (a) culturing recombinant EPO-containing rodent epithelioid cells in nutrient medium free of serum to produce EPO-containing medium, (b) clarifying the medium of cell debris to yield clarified EPO-containing medium, (c) subjecting the EPO-containing medium to ion exchange

chromatography to yield partially purified EPO, (d) subjecting the partially purified EPO to reverse phase HPLC in an organic solvent to yield pure EPO in the organic solvent, and (e) removing the organic solvent.
EP083Q376 describe a method for purifying erythropoietin, wherein EPO from the culture supernatant is purified by dye affinity chromatography then by hydrophobic chromatography on an alkylated or arylated carrier, followed by hydroxy apatite, hydrophobic chromatography and finally by anion exchange chromatography.
EP1127063 describe a method for purifying erythropoietin comprising the following steps: differential precipitation, hydrophobic interaction chromatography, diafiltration, anion exchange chromatography, cation exchange chromatography, and molecular exclusion chromatography.
627/CHE/2005 disclose a method for purification of the erythropoiesis stimulating protein which comprise of the following downstream steps: a. Initial clarification and concentration using normal and tangential flow filtration procedures b. Ultra filtration / Dialysis filtration (based on tangential flow filtration) c. Chromo step - I: Affinity chromatography using lectin / m-amino phenyl based matrices. M-amino phenyl ligand based affinity medium would be more preferred. d. Chromo step - II: Ion-exchange chromatography (IEX) using Q-Sepharose anion exchanger e. Chromo step - III: Hydrophobic interaction chromatography (HIC) using butyl - Sepharose. Virus removal and sterile filtration g. Endotoxin removal h. Formulation.
It is necessary to have a method for the purification of hyperglocysylated analogue of erythropoietin, and preferably without cost-intensive chromatographic steps as well as extensive steps, which does not require the use of undesirable reagents. The hyperglocysylated analogue of erythropoietin obtained by the purification method according to the present invention is supposed to meet the criteria for purity, which are set forth by the admission authorities.
Similarly, the process should be preferably managed without a reversed phase chromatography, for example an RP-HPLC, In this type of chromatography, reagents like methyl cyanide are conventionally employed, which are difficult to remove from the protein and which can be harmful to human beings. Another disadvantage of the RP-HPLC is that often cost-intensive organic solvents are used, which increases the expenses for purification. Furthermore, organic solvents are questionable with respect to environmental damages and are difficult and dangerous to handle. Altogether, employing reversed-phase chromatography is often undesirable.

It is evident that, with respect to purity and glycosylation pattern, high demands are made on the
hyperglocysylated analogue of erythropoietin product obtained from the purification. These high
' standards can only be met by means of a specific purification method, which is specialized for
hyperglocysylated analogue of erythropoietin and which is the result of extensive studies and
analyses.
The present invention discloses a method for purifying biologically active form of hyperglocysylated analogue of erythropoietin, by means of which it is possible to obtain hyperglocysylated analogue of erythropoietin with satisfactory purity and yield. The said purification method can be performed with fewer chromatographic steps as possible in order to keep technical complexity and costs on a low level and to avoid high losses of protein.
SUMMARY OF THE INVENTION
The present invention relates to a method for the purification of hyperglocysylated analogue of erythropoietin from a solution, in particular from a culture supernatant, wherein the following steps a) to c) are performed in the given order: in step a) a first anion exchange chromatography, b) a mixed mode chromatography preferably hydroxy apatite chromatography, c) a second anion exchange chromatography d) gel permeation chromatography.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the SDS-PAGE profile for first anion exchange chromatography
Lane 1: Q sepharose eluate fraction 1 Lane 2: Q sepharose eluate fraction 2 Lane 3: Q sepharose eluate fraction 3 Lane 4: Q sepharose eluate fraction 4 Lane 5: Standard
Lane 6: Q sepharose eluate fraction 5 Lane 7: Q sepharose eluate fraction 6 Lane 8: Q sepharose eluate fraction 7
Figure 2 shows the SDS-PAGE profile for Hydroxyapatitie chromatography
Lane 1: Blank
Lane 2: HA input
Lane 3: UFDF II Permeate
Lane 4: HA Output
Lane 5: Standard
Lane 6: HA high salt fraction
Lane 7: Standard
Lane 8: Blank

Figure 3 shows the SDS-PAGE profile for Second anion exchange chromatography
Lane 1: DEAE Sephacel Input
Lane 2: DEAE Sephacel FT and wash
Lane 3: DEAE Sephacel Eluate fraction 2
Lane 4: DEAE Sephacel Eluate fraction 5
Lane 5: Standard
Lane 6: DEAE Sephacel Eluate fraction 6
Lane 7: DEAE Sephacel Eluate fraction 7
Lane 8: DEAE Sephacel Eluate high salt fraction
Figure 4 shows the SDS-PAGE profile for Gel permeation chromatography
Lane 1:GFC fraction 2 Lane 2: GFC fraction 3 Lane 3: GFC fraction 4 Lane 4: GFC fraction 5 Lane 5: Standard Lane 6: GFC fraction 6 Lane 7: GFC fraction 7 Lane 8: Blank
Figure 5 shows RP-HPLC Chromatogram
Figure 6 shows the IEF profile from the pi range between 2.5 to 5.0
Lane 1: Cell culture harvest
Lane 2: Blank
Lane 3: Hydroxyapatite chromatography elute
Lane 4: Blank
Lane 5: GFC Fraction 5
Lane 6: -GFC Fraction 6
Lane 7: Standard
Lane 8: Hydroxyapatite chromatography high salt Fraction
DESCRIPTION OF THE INVENTION
The present invention discloses a process for the purification of hyperglocysylated analogue of erythropoietin. .
In one embodiment of the present invention the purification is done by one or more anion exchange chromatography, a mixed mode chromatography preferably hydroxyapatide chromatography and a gel permeation chromatography.

The hydroxyapatite chromatography interacts with biomolecules by multiple modes. An electrostatic interaction occurs between positively charged calcium ions and negatively charged phosphate groups on support and charged moieties on biomolcule's surface.
In a preferred embodiment, the hyperglycosylated analogue of erythropoietin purification method comprises, in the given order, a first anion exchange chromatography, hydroxyapatite chromatography, second anion exchange chromatography, and a gel permeation chromatography.
The embodiments described herein may optionally encompass any of tangential flow filtration, concentration, diafiltration or ultrafiltration steps between the chromatographic steps.
The embodiments described herein may further comprise one or more viral inactivation steps, sterile filtration and nanofiltration steps.
The purification according to the present invention thus utilizes at least four chromatographic steps, i.e. a) first anion exchange chromatography, b) a hydroxyapatide chromatography c) second anion exchange chromatography d) a gel permeation chromatography.
The cultivation of the hyperglocysylated analogue of erythropoietin producing host cells is done in a cell culture medium which is free of proteins and animal components.
In a preferred embodiment, no reversed-phase chromatography takes place at any stage of the hyperglocysylated analogue of erythropoietin purification.
It has been found that the hyperglocysylated analogue of erythropoietin obtained by the method according to the present invention has purity at least >85% and more specifically >95% wherein the purity is determined by analytical HPLC.
The present invention also relates to pharmaceutical preparations containing the hyperglocysylated analogue of erythropoietin purified according to the present invention. Usually " hyperglocysylated analogue of erythropoietin is formulated in liquid form and as such is injected intravenously or subcutaneously. Suitable adjuvant in liquid formulations of hyperglocysylated analogue of erythropoietin are, for example, buffers, like for example phosphate buffers, salts, like for example sodium chloride, stabilizers for example amino acids, sugars and sugar alcohols, as well as surfactants, like for example polysorbate 20/80.

Preferably, the hyperglocysylated analogue of erythropoietin purified according to the present invention is recombinant human hyperglocysylated analogue of erythropoietin, produced in eukaryotic cells. Preferably it is produced in mammalian cells, particularly in CHO cells. According to conventional protocols cell cultivation is done with commercially available culture media.
The person skilled in the art must be familiar with the principles of chromatographic steps employed in the purification process according to the present invention; in any case, they are described in detail in the manuals or protocols of the chromatographic matrices suppliers.
The anion exchange chromatographic steps can be conducted by conventional, commercially available anion exchanger resins or membranes. Thus, in a preferred embodiment of the method according to the present invention, for example each of Q Sepharose or DEAE sephacel is employed in the first and second anion exchange chromatography. Particularly, Q Sepharose is used in first anion exchange chromatography and DEAE sephacel is used in the second anion exchange chromatography.
The mixed mode and gel permeation chromatographic steps can be conducted by conventional, commercially available mixed mode and gel permeation resins. Particularly, hydroxyapatite Type I is used in mixed mode chromatographic step and Superdex 75 is used in gel permeation chromatographic step.
The following example illustrates the present invention and the means of carrying out the invention to obtain the purified hyperglocysylated analogue of erythropoietin.
Examples
The examples described in detail below use starting material samples comprising hyperglycosylated analogue of erythropoietin obtained from culture supernatant medium from a bioreactor. Typically, the starting material is clarified first and then optionally concentrated and/or buffer exchanged prior to being captured on the first chromatographic step.
The semi-purified harvest is then subjected to one or more ion exchange chromatography and to hydroxyapatite chromatography followed by gel permeation chromatography.

Anion exchange chromatography removes highly basic Isoforms, other host cell proteins and media components and hydroxyapatite chromatography removes the host cell proteins and other product related impurities.
Anion exchange chromatography is effective to remove other undesired isoforms and gel permeation chromatography is used to remove the product related impurities i.e. aggregates as well as buffer exchange in formulation buffer.
Example 1
First Anion exchange chromatography
In this step all the proteins are captured and eluted according to their binding properties. This step is used to separate out highly basic isoforms from desired isoforms and host cell impurities.
Hydroxyapatite Chromatography
In this step host cell as well as product related impurities are removed.
Second Anion exchange chromatography
In this Step residual undesired isoforms and host cell impurities are removed.
The elute obtained in this step in then concentrated by ultrafiltration followed by diafiltration to a low pH buffer ( pH 3.0 to 3.7) and continuous circulation in the same buffer for 45 minutes to 60 minutes to inactivate the virus contaminants.
After low pH viral inactivation, it was further diafiltered against a suitable buffer of pH in the slightly acidic to neutral range and concentrated.
Gel permeation chromatography
In this step, the separation is based on molecular size.
Elute obtained from this step, is then preferably passed through a nano filter to remove viral contaminants, the filterate is then stored in sterile bottles at -20 aC.
The hyperglycosylated analogue of erythropoietin obtained can be formulated in the form of a liquid formulation with conventional pharmaceutically acceptable excipients as known in the art. The details of individual chromatographic steps are given below.

First Anion exchange chromatography
The clarified cell culture harvest is concentrated and diafiltered by ultrafiltration against lOmM Tris buffer, pH 7.0 and conductivity < 3.0 mS/cm followed by loading on an anion exchange column (Q Sepharose FF) pre-equilibrated with the same buffer. After washing with the equilibration buffer the bound protein is eluted with a gradient between 10 mM Tris buffer, pH 7.0 and IM sodium chloride in same buffer.
Hydroxyapatite Chromatography
The elute of Q Sepharose FF is concentrated and diafiltered against 2mM sodium phosphate buffer, pH 6.0 and conductivity <1.0 mS/cm and loaded to hydroxyl apatite column (Ceramic Hydroxy apatite, Typel 40 micron) pre-equilibrated with 2mM sodium phosphate buffer, pH 6.0. The flow through was collected. The column was washed with same buffer and pooled with the flow through which contains the desired isoforms.
Second Anion exchange chromatography
The hydroxyapatite out put is concentrated and diafiltered against 40 mM acetate, 2,5 mM CaCl2, pH 4.5, cond.1.5-2.0 mS/cm and loaded on anion exchange column (DEAE sephacel). After washing with the equilibration buffer, the bound protein is eluted with a gradient between 40 mM acetate, 2.5 mM CaClz, pH 4.5 and IM sodium chloride in same buffer.
Gel permeation chromatography
The high salt elute which contains the desired acidic isoforms is diafiltered against sodium citrate buffer ( 40 mM), ph 3.0 and circulated for an additional 1 hr in the same buffer for low pH viral inactivation followed by further Diafiltration with sodium phosphate buffer ( 20mM), pH 7.0 contains sodium chloride (75 mM) and concentration. The Ultrafiltration-diafiltration out put may further be loaded on gel permeation chromatography column (Superdex 75 or sephacryl) as an additional polishing step followed by nano filtration.
Chromatographic resins
The following chromatographic resins are currently employed in the purification process. Equivalent resins can also be employed in the purification process described in the present invention.

First anion exchange chromatography - Q Sepharose FF (GE Healthcare) Hydroxyapatite chromatography - HA Type I (Bio-rad) Second anion exchange chromatography - DEAE Sephacel (GE Healthcare) Gel permeation chromatography - Superdex 75 (GE Healthcare)
A novel process for the purification of hyperglocysylated analogue of erythropoietin described in the present invention has the following advantages;
1. Involves operational simplicity and robustness.
2. Avoids the usage of reverse phase and/or dye-binding affinity chromatography
3. With reduced cost.
4. Results in purity >85% with desire isoform profile and activity.

We Claim
1. A process for the purification of hyperglocysylated analogue of erythropoietin from a
culture supernatant produced by culturing eukaryotic host cells in culture medium,
comprising the following steps:
a) first anion exchange chromatography
b) hydroxyapatite chromatography
c) second anion exchange chromatography
d) gel permeation chromatography

2. A process of claim 1, further comprising at least one nanofiltration step or virus inactivation step.
3. A process of claim 1, wherein Q Sepharose is used for first anion exchange chromatographic step.
4. A process of claim 1, wherein DEAE Sephacel is used for second anion exchange chromatographic step.
5. A process of claim 1, wherein HA Type I is used for hydroxyapatite chromatographic step.
6. A process of-claim 1, wherein Superdex 75 used for Gel permeation chromatographic
step.
7. A process of claim 1, wherein the protein mixture is a host cell-free filtrate of the culture medium, which is subjected to diafiltration prior to the first anion exchange chromatography.
8. A process of claim 1, wherein the culture medium is and animal component free.
9. A process of claim 1, wherein the method does not comprise reverse phase and/or affinity (dye) chromatographic step.

10. A pharmaceutical composition comprising the purified hyperglycosylated analogue of erythropoietin according to any one of the preceding claims and one or more pharmaceutically acceptable excipients.