FORM 2
THE PATENTS ACT, 1970
(39 OF 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10; rule 13)
1. Title of the invention - Process for the purification of Fc fusion proteins
2. Applicant(s)
(a) NAME: INTAS BIOPHARMACEUTICALS LIMITED
(b) NATIONALITY: An Indian Company.
(c) ADDRESS: Plot No.: 423 P/A/GIDC
Sarkhej - Bavla Highway, Moraiya, Ta.: Sanand Ahmedabad-382213 Gujarat, India
3. PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention and the manner in which it is to be performed.

RELATED APPLICATIONS
This application is related to Indian Provisional Application 3743/MUM/2012 filed 31st Dec, 2012 and is incorporated herein in its entirety.
FIELD OF THE INVENTION
The present invention relates to a process for the purification proteins. More specifically, the invention provides a robust downstream purification process suitable for use in manufacturing of Fc fusion protein for human administration.
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 the mixture. Once a cell culture supernatant comprising desired protein is obtained, its purification from other proteins produced by the cell is usually done by carrying a different 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-inflammatory 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 Daiton (p75) tumor necrosis factor receptor (TNFR) linked to the Fc portion of human IgG1. The Fc component of Etanercept consists of the CH2 domain, the CH3 domain and hinge region, whereas the CH1 domain is absent. !t 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 !50 kilo Daiton,
US5641 870 discloses a process for purifying an antibody is provided. In this process, a mixture containing the antibody and contaminant 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.
Solutions for Purification of Fc-fusion Proteins - When platform processes are applied to fusion molecules, innovation and flexibility are needed, Mar 2, 2008 By; Douglas W. Rea, Michiel E. Ultee, PhD, Sharon X. Chen. Thomas P. Loisel BioPharm International Supplements.
US6008036 discloses a method for purifying viruses from a cell line culture by chromatography, comprising an anion exchange chromatography step followed by a cation exchange chromatography step and optionally a metal-binding affinity chromatography step. The method is particularly suitable for producing viruses for use in vaccines.
US6127526 disclosed a method for purifying proteins by Protein A chromatography is described which comprises the steps of: (a) adsorbing the protein to Protein A immobilized on a solid phase comprising silica or glass; (b) removing contaminants bound to the solid phase by washing the solid phase with a hydrophobic electrolyte solvent; and (c) recovering the protein from the solid phase.
US6333398 discloses a method for purifying proteins by Protein A chromatography is described which comprises the steps of: (a) adsorbing the protein to Protein A immobilized on a solid phase comprising silica or glass; (b) removing contaminants bound to the solid

phase by washing the solid phase with a hydrophobic electrolyte solvent; and (c) recovering the protein from the solid phase.
US6870034 discloses a method for purifying proteins by Protein A chromatography is described which comprises removing contaminants by washing the solid phase with various intermediate wash buffers,
US7122641 discloses a method is provided for separating a protein from one or more other proteins using hydroxyapatite chromatography in which the protein does not bind to hydroxyapatite but the other protein(s) does. In some embodiments, a second protein affixed to a solid support has been used previously to purify the protein by affinity chromatography, and small amounts of the second protein are introduced in the sample during this process. The protein being purified can comprise at least one constant antibody immunoglobulin domain. The second protein can bind to proteins comprising such a domain.
US71 81 395 discloses 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.
US7223848 discloses a process for dissociating Fc-containing molecules from complexes of Protein A/Fc-containing molecules or mixtures containing Fc-containing molecules and Protein A. The association, e.g., by hydrophobic interactions, between the Fc-containing molecules and Protein A can be reduced or inhibited by raising the p.H of dissociation. The pH of dissociation can be raised by addition of agents capable of inhibiting hydrophobic interactions, including buffers containing arginine and/or ethylene glycol, to the mixture, either prior to adding the mixture to the column chromatography substrate, after adding the mixture to the column chromatography substrate, or both prior to and after adding the mixture to the column chromatography substrate. Separation of Fc-containing molecules from Protein A can be performed on a number of different column chromatographic

substrates, including column chromatographic substrates contained in Q columns (Q column chromatography substrate), HIC columns (hydrophobic interaction column chromatography substrate), and IMAC columns (meta! chelate column chromatography substrate).
US7847071 discloses a method of purifying an antibody comprising the steps of: firstly, purifying an antibody by means of protein A affinity chromatography wherein the protein A is a native protein A or a functional derivative thereof, secondly, loading the purified antibody comprising a protein A-contaminant, wherein said protein A-contaminant is obtained upon eluting bound antibody from said protein A affinity chromatography, on a first ion exchanger under conditions which allow for binding of the protein A or its derivative, thirdly, collecting the antibody loaded onto the first ion exchanger in the flow-through of the first ion exchanger whilst a contaminant protein A is bound to the first ion exchanger, wherein the first ion exchanger is an anion exchanger, further purifying the antibody by loading on. binding to and eluting it from a second ion exchanger, and discarding a tail fraction of the eluate of the second ion exchanger such that a monomeric antibody fraction is enriched as a purified antibody pool.
US7807799 discloses a method for reducing leaching of protein A during protein A affinity chromatography is described which involves reducing temperature or pH of, or by adding one or more protease inhibitors to, a composition that is subjected to protein A affinity chromatography.
US20100022757 discloses a method for separating and purifying an Fc-containing protein from a fluid comprising at least a cation exchange chromatography purification step comprising: (a) binding an Fc protein containing fluid comprising an Fc-containing protein to a cation exchange resin; (b) washing the cation exchange resin with a buffer at a pH about 1 unit below the isoelectric point of the Fc-containing protein, the buffer having a conductivity of about 2 to 6 mS/cm; and (c) eluting the Fc-containing protein with a buffer at a pH about 1 unit below the isoelectric point of the Fc-containing protein with an increasing salt gradient.

US20100190961 discloses a method for reducing the concentration of free Fc-moieties in a fluid comprising an Fc-containing protein, the method comprising subjecting said fluid to cation exchange chromatography.
US20120202974 discloses a method for separating and purifying an Fc-containing protein from a fluid comprising at least a cation exchange chromatography purification step comprising: (a) binding an Fc protein containing fluid comprising an Fc-containing protein to a cation exchange chromatography resing; (b) washing the cation chromatography resin with a buffer at a pH about 1 unit below the isoelectric point of the Fc-containing protein, the buffer having a conductivity of about 2 to 6 mS/cm; (c) eluting the Fc-containing protein with a buffer at a pH about 1 unit below the isoelectric point of the Fc-containing protein with an increasing salt gradient; and (d) applying the eluent of step (c) to an anionic exchange chromatography matrix or a hydrophobic interaction chromatography matrix.
WO2003102132 discloses a method for protein purification that involves the combination of non-affinity chromatography with High performance tangential flow filtration.
WO2008025747 discloses a process for the purification of an Fc-fusion protein having a pH 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.
WO2011089212 discloses a method for depleting impurities, in particular host cell proteins (HCP) and DNA from cell culture supernatants by means of protein A chromatography using a novel washing buffer.
WO2013009526 discloses a method of purifying a Fc fusion protein produced in eukaryotic expression comprising an optimized protein A affinity chromatography step and two ion exchange chromatography steps both of which are operated in the bind and elute method.

Liu et al., discussed in mAbs 2, 2010. 480-499 about the basic unit operations such as harvest Protein A affinity chromatography and additional polishing steps along with alternative processes such as flocculation, precipitation and membrane chromatography and also covered platform approaches to purification methods development, use of high throughput screening methods, and offered a view on future developments in purification methodology as applied to monoclonal antibodies.
Fusion proteins get truncated while purification which affects the stability of the molecule. 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 fusion proteins using fewer steps without affecting the stability of the protein.
It is necessary to have a method for the purification of Fc fusion proteins and preferably without cost-intensive chromatographic steps as well as extensive steps. The Fc fusion proteins obtained by the purification method according to the present invention is supposed to meet the criteria for purity, yield which are set forth by the admission authorities.
OBJECT OF THE INVENTION
The main objective of the present invention is to provide a simple purification process for the p75 TNFR:Fc protein,
In a further object, the invention relates to a process for the purification of p75 TNFR:Fc protein comprising the following steps:
1) Providing a sample comprising a p75 TNFR:Fc protein
2) Binding the p75 TNFR:Fc protein present in the sample to a Protein A affinity chromatography resin
3) Editing the p75 TMFR:Fc protein from the Protein A resin, wherein the eluted product provides a second sample
4) Binding the second sample to a anion exchange (AEX) chromatography resin

5) Eluling the second sample from the A EX resin, wherein the eluted product provides a third sample
6) Binding the third sample to an hydrophobic interaction chromatography (H1C) resin
7) Eluting the third sample from the hydrophobic interaction chromatography (HIC) resin, wherein the eluted product provides purified p75 TNFR:Fc protein.
Another object of the present invention is to provide an operationally simle and robust process which avoid the use of reverse phase chromatography and costly resins.
Yet another object of the present invention is to provide a process of purification which results in >85% pure protein of interest with desire isoform profile and activity.
SUMMARY OF THE INVENTION
The present invention is based on the development of a purification process for p75 TNFR:Fc protein.
In a first aspect, the invention relates to a process for the purification of p75 TNFR:Fc protein comprising the following steps:
i) Providing a sample comprising a p75 TNFR:Fc protein
2) Binding the p75 TNFR:Fc protein present in the sample to a Protein A affinity chromatography resin
3) Eluting the p75 TNFR:Fc protein from the Protein A resin, wherein the eluted product provides a second sample
4) Binding the second sample to a anion exchange (AEX) chromatography resin
5) Eluting the second sample from the AEX resin, wherein the eluted product provides a third sample
6) Binding the third sample to an hydrophobic interaction chromatography (HIC) resin

7) Eluting the third sample from the hydrophobic interaction chromatography (HIC) resin, wherein the eluted product provides purified p75 TNFR:Fc protein.
In a second aspect, the invention relates to a process for the purification of p75 TNFR:Fc protein comprising affinity chromatographic step followed by anion exchange chromatographic step followed by hydrophobic interaction chromatic step.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a flow chart of a specific embodiment of the present invention (i.e.) a process of purification comprising the steps of:
fl) Protein A affinity chromatography
(2) Anion Exhange chromatography
(3) Hydrophobic Interaction chromatography
(4) Ultrafiltration/Diafiltration
(5) Nanofiltration
(6) Sterile Filtration (0.22 μm filtration)
Figure 2 shows the non reducing SDS-PAGE profile of batch-1 and batch-2.
Figure 3 shows the comparative SE-HPLC chromatograms of in-house Fc containing proteins with standards.
Figure 4 shows the comparative HIC-HPLC chromatograms of in-house Fc containing proteins with standards.
DETAILED DESCRIPTION OF THE INVENTION
The present invention discloses a process for the purification of Fc fusion proteins.
In one embodiment of the present invention the purification is done by affinity chromatography, anion exchange chromatography and hydrophobic interaction 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 nano filtration steps.
The purification according to the present invention utilizes at least three major chromatographic steps i.e. affinity chromatography, anion exchange chromatography and a 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.
In a preferred embodiment, no reverse-phase chromatography takes place at any stage of the Fc fusion protein purification.
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.
Affinity chromatographic step can be conducted by conventional, commercially available Protein A resins or membranes. Thus, in a preferred embodiment of the method according to the present invention, for example MabSelect or Mab Select Sure Particularly, MabSelect SuRe is used in Affinity chromatographic step.
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 Sepharose is employed in the anion exchange chromatographic step. Particularly. Q Sepharose is used in anion exchange chromatographic step.
The Hydrophobic interaction chromatographic step can be conducted by conventional, commercially available hydrophobic interaction resins or membranes. Thus, in a preferred embodiment of the method according to the present invention, for example Phenyl Sepharose or Butyl Sepharose is employed in the hydrophobic interaction chromatographic step. Particularly, Phenyl Sepharose HP is used in 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.
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 costly resins
3. With reduced cost.
4. Results in purify >85% with desire isoform profile and activity.
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 materia! 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 affinity chromatography, anion exchange chromatography and hydrophobic interaction chromatographic steps.
Affinity chromatography step to remove the media components and to capture the protein of interest in small volume for further purification. The output of affinity chromatography is

subjected to the low pH treatment for 60 minutes at pH < 4.0 for virus inactivation. The output of the low pH treatment is further purified by Anion exchange chromatography to remove the host cell protein, host cell DNA, other undesired basic isoforms and dissociated impurities. The partial purified Q output is now purified by HIC to reduce truncated forms, aggregates, misfolded and the remaining other impurities.
Example 1
Affinity chromatography
In this step protein of interest is captured and eluted in small volume for further purification. This step is carried out at about 25°C.
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.
Hydrophobic Interaction chromatography
This step is used to reduce truncated forms, aggregates, misfolded and the remaining other impurities. This step is carried out at about 25°C.
Ultrafiltration/ Diafiltration
The Fc fusion protein can be formulated in the form of a liquid formulation with pharmaceutically acceptable excipients as known in the art for human therapeutic use.
The details of individual chromatographic steps are given below.
Affinity chromatography
Clarified harvest is loaded on an affinity column (Mab select sure) pre-equilibrated with the 20mM Tris buffer, pH 7.4 containing 150mM sodium chloride. After washing with the equilibration buffer a low pH wash of pH 5.1 is given with 20mM acetate buffer. After this

the bound protein is eluted with 20mM acetate containing 100mM sodium chloride buffer. pH 3.6. The output of affinity chromatography is subjected to the low pH treatment for 60 minutes at pH < 4.0 for virus inactivation. After 60 minutes pH is adjusted to 7.4 with 3.0M Tris buffer.
Anion exchange chromatography
Affinity chromatography output is loaded on an anion exchange column (Q Sepharose FF) pre-equilibrated with 20mM Tris buffer, pH 7.4. After washing with the equilibration buffer a low p|-| wash of pH 4.8 is given with 20mM acetate buffer. After this the bound protein is eluted with a 20mM Tris buffer containing 300mM sodium chloride buffer, pH 7.4.
Hydrophobic interaction chromatography
Elute of Q Sepharose FF is processed for next chromatographic step by adding 0.8 M of ammonium sulphate salt. After filtration, sample is loaded on phenyl sepharose resin pre-equilibrated with 20mM Phosphate buffer containing 0.8 M ammonium sulphate, After washing with the equilibration buffer the bound protein is eluted either with 20mM Phosphate buffer containing 0.3 M ammonium sulphate buffer in step gradient mode or with 20mM Phosphate buffer in linear gradient mode.
Ultrafiltration/ Diafiltration & Nanofiltration
Hydrophobic Interaction chromatography output is then diafiltered and concentrated against a suitable buffer followed by Nano filtration and concentrated to prepare either the drug substance or drug product.
Example 2 - Purification of p75 TNFR:Fc proteins
The clarified cell culture harvest is loaded on to pre-equilibrated affinity chromatography column (Mab select sure). After washing with the equilibration buffer the bound protein is eluted with acetate buffer and then elute obtained from the above affinity chromatographic step is kept for low pH viral inactivation for 45-60 miti. After one hour pH is readjusted and loaded to the pre -equilibrated anion exchange column (Q Sepharose FF) with Tris buffer. After washing with the equilibration buffer a low pH wash is given to remove basic

(undesired) isoforms and the bound protein is eluted with Tris buffer containing sodium chloride. Output obtained from anion exchange chromatographic step is further purified by hydrophobic interaction chromatography (Phenyl Sepharose) after adding ammonium sulphate to the sample. The bound protein is eluted with ammonium sulphate in sodium phosphate. Out put obtained from hydrophobic interaction chromatographic step is then diafiltered and concentrated against a suitable buffer to prepare either the drug substance or drug product of p75 TNFR:Fc protein.

WE CLAIM,
1. A process of purifying a p75 TNFR:Fc protein from one or more impurities in a sample, comprising
the steps of:
1) Providing a sample comprising a p75 TNFR'.Fc protein
2) binding the p75 TNFR:Fc protein present in the sample to a Protein A affinity chromatography resin
3) eluting the p75 TNFR:Fc protein from the Protein A resin, wherein the eluted product provides a second sample
4) binding the second sample to a anion exchange (AEX) chromatography resin
5) eluting the second sample from the AEX resin, wherein the eluted product provides a third sample
6) binding the third sample to an hydrophobic interaction chromatography (HIC) resin
7) eluting the third sample from the hydrophobic interaction chromatography (HIC) resin, wherein the eluted product provides purified p75 TNFR:Fc protein.

2. A process according to claim 1. wherein the p75 TNFR:Fc protein is etanercept.
3. A process as claimed in claim 1, wherein MabSelect SuRe is used for Protein A affinity chromatographic step.
4. A process as claimed in claim 1, wherein the Q Sepharose FF is used for anion exchange chromatographic step.
5. A process as claimed in claim 1. wherein the phenyl sepharose SP is used for hydrophobic interaction chromatographic step.
6. A process of claim 1. optionally comprising at least one viral inactivation. ultrafiltration/diafiltration, nanofiltartion and/or sterile filtration (0.22 urn filtration).
7. A process according to any of the preceeding claim, comprising the steps of subjecting p75 TNFR:Fc protein to;

a. Protein A affinity chromatography on affinity column with pH based elution using 20 mM
Sodium acetate buffer with different pH- Equilibration at pH 7.4, pH 5.1 wash and elution at pH
3.6.
b. Subjecting the eluate of step (a) of affinity chromatography to a step of anion exchange
chromatography on Q Sepharose FF with pH based elution using 20mM Tris buffer containing
300mM sodium chloride buffer - Equilibration at pH 7,4. pH 4.8 wash and elution at, pH 7.4,
c. Subjecting the eluate of step (b) of anion exchange chromatography to a step of hydrophobic
interaction on phenyl sepharose resin wherein the elusion is carried out with 20mM Phosphate
buffer containing 0.3 M ammonium sulphate buffer in step gradient mode or with 20mM
Phosphate buffer in linear gradient mode.
d. Diafiltration of the eluate from step (c) to concentrate against suitable buffer
e. Subjecting the eluate from step (d) to nanofiltralion to form a permeate.
8. A purified p75 TNFR:Fc obtained by the process according to claim 1.
9. A pharmaceutical compsition comprising p75 TNI:R:Fc, purified according to any preceding claims with one or more pharniaceutically acceptable excipients.