FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION (See Section 10 and Rule 13)
PROCESS FOR THE PURIFICATION OF Fc CONTAINING PROTEINS
Intas Biopharmaceuticals Limited
An Indian company having its registered office at:
Plot No.: 423/P/A/GIDC
Sarkhej-Bavla Highway
Moraiya, Tal.: Sanand
Ahmedabad-382 213
Gujarat, India
The following specification describes the invention.

FIELD OF THE INVENTION
The present invention relates to a process for the purification of proteins. More specifically, it relates to the purification of Fc fusion proteins.
BACKGROUND OF THE INVENTION
Proteins are important as these are used to cure a number of diseases including diabetes (e.g. Insulin), cancers (e.g. Interferon, monoclonal antibodies), heart attacks, strokes, cystic fibrosis (e.g. Enzymes, Blood factors), inflammation diseases (e.g. Tumor Necrosis Factors), anemia (e.g. Erythropoietin), hemophilia (e.g. Blood clotting factors) etc. One of the important challenges is the development of efficient and competent process for the large scale purification of these proteins. Numerous processes are available for the large scale purification of desired protein from the cell culture supernatants, but still it is difficult to separate the desired protein from a mixture. Once a cell culture supernatant comprising desired protein is .obtained, its purification from other proteins produced by the host cell is usually done by carrying a combination of chromatographic techniques.
Tumour necrosis factor is a polypeptide cytokine involved in inflammation and the acute phase response. TNF-alpha is present in larger quantities in persons with rheumatoid arthritis or Crohn's disease. Direct inhibition of TNF-alpha by the biological agents has produced significant advances in rheumatoid arthritis treatment and has validated the extra-cellular inhibition of this pro-infiammatory cytokine as an effective therapy. One such biological agent is Etanercept.
Etanercept (TNFR:Fc) is a dimeric fusion protein consisting of the extra-cellular ligand-binding portion of the human 75 kilo Dalton (p75) tumor necrosis factor receptor (TNFR) linked to the Fc portion of human IgGl. The Fc component of Etanercept consists of the CH2 domain, the CH3 domain and hinge region, whereas the CH1 domain is absent. It is produced through recombinant DNA technology in Chinese hamster ovary mammalian cells. It consists of 934 amino acids, and has an apparent molecular weight of approximately 150 kilo Dalton.
US564I870 disclose a process for purifying an antibody. In this process, a mixture containing the antibody and contaminants is subjected to low pH hydrophobic interaction chromatography (LPHIC) optionally at low salt concentration. The antibody is eluted from the column in the fraction which does not bind thereto. This process can be preceded and followed by other purification steps.

US7181395 disclose a process for purifying a protein by mixing a protein preparation with a solution having a first salt and a second salt, wherein each salt has a different lyotropic value, and loading the mixture onto a hydrophobic interaction chromatography column. The dynamic capacity of the column for a protein using the two salt combinations will be increased compared with the dynamic capacity of the column for either single salt alone.
WO2003102132 disclose a method for protein purification that involves the combination of non-affinity chromatography with High performance tangential flow filtration.
WO2008025747 disclose a process for the purification of an Fc-fusion protein having a pi between 6.9 and 9.5 comprising protein A or G affinity chromatography, cation exchange chromatography, anion exchange chromatography and hydroxyapatite chromatography.
WO2009053358 disclose a method for the purification of Fc-fusion proteins via blue dye affinity chromatography, in particular for the reduction of the amount of free Fc-moieties in an Fc- fusion proteins preparation.
In spite of these advanced chromatographic techniques, affinity chromatography is frequently used to purify Fc fusion proteins. The high cost and instability of affinity media however increase the ultimate cost of Fc fusion proteins, particularly those requiring high doses and/ or chronic administration. In addition, adequate purity often is not achieved unless several purification steps are combined, thereby further increasing cost and reducing product yield. Consequently, there is a need for processes that purify protein therapeutics or other polypeptide compounds fused to Fc component using fewer steps and without the need for a costly affinity step.
SUMMARY OF THE INVENTION
It is an objective of the present invention to provide a new process for the purification of Fc fusion protein which does not employ affinity chromatographic step.
In a first aspect, the present invention provides a process for the purification of an Fc fusion protein comprising the following steps:
• Anion Exchange Chromatography
• Cation exchange Chromatography
• Hydrophobic interaction chromatography

It is another objective of the invention to provide a pharmaceutical composition comprising the Fc fusion proteins purified according to the said purification process with one or more pharmaceutically acceptable excipients.
BRIEf DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates the present invention (i.e) a process of purification comprising the steps of
• Anion exchange chromatography
• Cation exchange chromatography
• Hydrophobic Interaction Chromatography
Figure 2 shows a flow chart of a specific embodiment of the present invention (i.e.) a process of purification comprising the steps of:
• Ultrafiltration/ Diafiltration
• Anion exchange chromatography
• Cation exchange chromatography
• Hydrophobic Interaction Chromatography
• Ultrafiltration/ Diafiltration
Fig 3 shows the SDS-PAGE profile of anion exchange chromatography
Lane 1; Cell harvest
Lane 2; Q Sepharose flow through and wash
Lane 3: Q Sepharose Eiuate Fraction I
Lane 4: Q Sepharose Eiuate Fraction 2
Lane 5: Q Sepharose Eiuate Fraction 3
Lane 6; Standard
Lane 7: Q Sepharose Eiuate Fraction 4
Lane 8: Q Sepharose Eiuate Fraction 5
Fig 4 shows the SDS-PAGE profile of Cation exchange chromatography
Lane 1: SP sepharose Load
Lane 2; SP sepharose flow through and wash
Lane 3: SP sepharose Eiuate Fraction 2
Lane 4. SP septotross. Elvate FtasSioYi 3
Lane 5: SP sepharose Eiuate Fraction 4
Lane 6: Standard
Lane 7: SP sepharose Eiuate Fraction 5
Lane 8: SP sepharose Eiuate Fraction 6
Fig 5 shows the SDS-PAGE profile of hydrophobic interaction chromatography
Lane 1: Low molecular weight marker
Lane 2: PS Load
Lane 3: PS flow through and wash

Lane 4: PS Eluate Fraction 1 Lane 5: Standard Lane 6: PS Eluate Main Fraction Lane 7: PS high salt Fraction Lane 8: Standard
Figure 6 shows the comparative SE-HPLC chromatograms of in-house Fc containing protein with Standard.
P
DETAILED DESCRITION OF THE INVENTION

The invention provides a process for the purification of Fc fusion proteins.
The purification process of the invention enables Fc containing proteins of high purity, which may be then formulated to the final medicament. It has the advantage of affording a high degree of purity without using affinity chromatography.
The present invention describes a process for the purification of Fc fusion protein comprising the following steps:
• anion exchange chromatography,
• cation exchange chromatography
• and hydrophobic interaction chromatography
The invention described herein may optionally encompass any of tangential flow filtration, precipitation, concentration, diafiltration or ultrafiltration steps between the chromatographic steps.
The invention described herein may further comprise one or more viral inactivation steps, sterile filtration and nano filtration steps.
The purification according to the present invention utilizes at least three major chromatographic steps i.e.
• anion exchange chromatography,
• cation exchange chromatography and
• hydrophobic interaction chromatography.
The cultivation of the Fc fusion protein producing host cells is done in a cell culture medium which is free of protein and animal components.

It has been found that the Fc containing proteins obtained by the purification process according to the present invention has purity at least >85% and more specifically >90% wherein the purity is determined by analytical HPLC technique.
The advantage of the present invention is that the purification process is devoid of a cost intensive reverse phase chromatography and an affinity chromatographic step and provides highly purified Fc containing proteins with the additional advantage of rendering the purified protein free of protein A leach.
The present invention also relates to a pharmaceutical preparation comprising the Fc fusion protein purified according to the present invention along with one or more pharmaceutically acceptable excipients.
Preferably, the Fc fusion protein purified according to the present invention is recombinant Fc fusion protein produced in eukaryotic cells. Preferably it is produced in mammalian cells, specifically in Chinese hamster ovary cells. According to conventional protocols cell cultivation is done with commercially available culture medium.
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 step can be conducted by conventional, commercially available anion exchange resins or membranes. Thus, in a preferred embodiment of the method according to the present invention, for example Q Sepharose or DEAE Sephacel is employed in the anion exchange chromatographic step. Particularly, Q Sepharose is used in anion exchange chromatographic step. Other suitable anionic exchange resin or membrane can also be employed.
The cation exchange chromatographic steps can be conducted by conventional, commercially available cation exchange resins or membranes. Thus, in a preferred embodiment of the purification process according to the present invention, for example SP Sepharose or source 30S is employed in cation exchange chromatography. Particularly, SP Sepharose is used in the cation exchange chromatographic step. Other suitable cationic exchange resin or membrane can also be employed.

The hydrophobic interaction chromatographic steps can be conducted by conventional, commercially available hydrophobic interaction resins or membranes. Thus, in a preferred embodiment of the purification process according to the present invention, for example Phenyl sepharose FF or Phenyl sepharose HP is employed in hydrophobic interaction chromatography. Particularly, Phenyl Sepharose is used in the hydrophobic interaction chromatographic step.
The following example illustrates the present invention and the means of carrying out the invention to obtain the purified Fc fusion protein.
Examples
The examples described in detail below use starting material samples comprising Fc fusion protein obtained from culture supernatant medium from the bioreactor. Typically, the starting material is clarified first and then optionally concentrated and/or bufer exchanged prior to being captured on (he first chromatographic step.
The semi-purified harvest is then subjected to anion exchange chromatography, cation exchange chromatography and hydrophobic interaction chromatographic steps.
Anion exchange chromatography removes highly basic isoforms, other host cell proteins, host cell DNA, endotoxins and media components and cation exchange chromatography removes host cell proteins and uncouple Fc chain and hydrophobic interaction chromatography removes dissociated molecule.
Example 1
Anion exchange chromatography
In this step all the proteins captured and eluted according to their binding properties. This step is used to separate highly basic isoforms from desired isoforms and host cell protein impurities. This step is carried out at about 25°C.
Cation exchange chromatography
In this step host cell proteins, uncoupled Fc chain and product-related impurities are removed. This step is carried out at about 25°C.
Hydrophobic Interaction chromatography
In this step host cell proteins as well as dissociated Fc fusion protein are removed. This step is carried out at about 25°C.

The Fc fusion protein can be formulated in the form of a liquid with pharmaceutically acceptable excipients as known in the art for human therapeutic use.
The details of individual chromatographic steps are given below.
Step 1 - Clarification, concentration, dialysis and filtration of harvest
The clarified cell culture harvest is concentrated and diafiltered by Ultrafiltration against 10mM Tris buffer, pH 8.5 and conductivity < 1.0 mS/cm followed by loading on an anion exchange column (Q Sepharose FF) pre-equilibrated with the same buffer.
Step 2 - Anion exchange chromatography
Ultrafiltration and diafiltration output is loaded on an anion exchange column (Q Sepharose FF) pre-equilibrated with the 10mM Tris buffer, pH 8.5 and conductivity < 1.0 mS/cm buffer. After washing with the equilibration buffer the bound protein is eluted with a gradient between 10 mM Tris buffer, pH 8.5 and 1M sodium chloride in the same buffer.
Step 3 - Cation exchange chromatography
Q out put is diluted with 10 mM sodium citrate buffer to reduce the conductivity to < 1.0 mS/cm and is loaded on cation exchange column (SP Sepharose FF) pre-equilibrated with the IOmM citrate buffer, pH 5.0 and conductivity < 1.0 mS/cm buffer. After washing with the equilibration buffer the bound protein is eluted with a gradient between IOmM citrate buffer, pH 5.0 and 1M sodium chloride in the same buffer.
Step 4 - Hydrophobic Interaction chromatography
Elute of SP Sepharose FF is processed for next chromatographic step by adding 1.0 M ammonium sulphate and pH is adjusted to 7.4 with NaOH. After filtration, sample is loaded on phenyl sepharose resin pre-equilibrated with 20mM Phosphate buffer containing 1.0 M ammonium sulphate, pH 7.4. After washing with the equilibration buffer the bound protein is eluted with a gradient between 20mM Phosphate buffer containing 1.0M ammonium sulphate and 20mM Phosphate buffer, pH 7.4.
A novel process for the purification of Fc fusion protein described in the present invention has the following advantages;

1. Involves operational simplicity and robustness.
2. Avoids the usage of reverse phase and/ or affinity chromatography which reduces the purification cost.
3. Results in purify >85% with desire isoform profile and activity.
4. Avoids contaminants leached from Protein A or Protein G resins.

We claim
1. A process for the purification of Fc fusion protein from a culture supernatant produced by
culturing eukaryotic host cells in culture medium, comprising the following steps:
a. Anion exchange chromatography
b. Cation exchange chromatography
c. Hydrophobic interaction chromatography
2. A process of claim 1, wherein Q Sepharose is used for1 anion exchange chromatographic step,
3. A process of claim 1, wherein SP Sepharose is used for cation exchange chromatographic step.
4. A process of claim 1, wherein Phenyl Sepharose is used for hydrophobic interaction chromatographic step.
5. A process of claim 1, further comprising at least one nanofiltration step or virus inactivation step.
6. A process of claim 1, wherein the method does not comprise reverse phase and/ or affinity chromatographic steps.
7. A method for purifying Fc fusion protein subjecting a liquid comprising Fc fusion protein to;
a. subjecting to anion exchange chromatography on a resin comprising quaternary
ammonium groups and eluting a first eluate with a buffer comprising NaCl and at a
pH between 6.5 to 9.0.
b. subjecting the first eluate to a step of cation exchange chromatography on a resin
comprising sulpho propyl group and eluting a second eluate with a buffer comprising
phosphate, NaCl and at a pH between 4.0 to 6.0.
c. subjecting the second eluate to hydrophobic interaction chromatography on a resin
comprising phenyl groups and eluting a third eluate with a buffer comprising
phosphate, ammonium sulfate at a pH between 5.0 to 8.0.
d. ultrafiltration of third eluate to concentrate.
e. subjecting the ultrafiltration output to nanofiltration to form a permeate.

8. The method according to any of the preceding claims, wherein the Fc-fusion protein comprises a ligand binding portion of a member of the tumor necrosis factor receptor (TNFR) superfamily.
9. The method according to any of the preceding claims, wherein the Fc-fusion protein comprises a heavy chain constant region wherein the constant region comprises the hinge, CH2 and a CH3 domain of an immunoglobulin IgGl.
10. A pharmaceutical composition comprising the purified Fc fusion protein according to any one of the preceding claims and one or more pharmaceutically acceptable excipients.