Process for the purification of TNFR:Fc fusion protein

1. A process for purifying the TNFR:Fc fusion protein from the protein mixture comprising TNFR:Fc fusion protein and at least one HCP impurity containing protein degrading enzyme, the said process comprising:

a) obtaining protein mixture from the suitable mammalian expression system comprising TNFR:Fc fusion protein and host cell protein (HCP) impurities containing at least one protein degrading enzyme;

b) applying the protein mixture to Protein A affinity chromatography column;

c) eluting the TNFR:Fc fusion protein from Protein A affinity chromatography column wherein the eluted TNFR:Fc fusion protein is present in second protein mixture containing reduced amount of HCP impurity;

d) applying the second protein mixture to Hydrophobic interaction chromatography column;

e) eluting the TNFR:Fc fusion protein from a Hydrophobic interaction chromatography column wherein the eluted TNFR:Fc fusion protein is present in third protein mixture contains reduced amount of HCP impurity;

f) applying the third protein mixture to anion exchange chromatography column;

g) eluting the TNFR:Fc fusion protein from a anion exchange chromatography column wherein the eluted protein of interest is present in fourth protein mixture containing reduced amount of HCP impurity;

h) applying the fourth protein mixture to mixed-mode chromatography column;

i) eluting the TNFR:Fc fusion protein from a mixed-mode chromatography column wherein the eluted TNFR:Fc fusion protein is substantially free of HCP impurity containing at least one of the protein degrading enzyme.

2. The process as claimed in claim 1 , wherein the mixed-mode chromatography comprises anion exchange and hydrophobic interaction chromatography.

3. The process as claimed in claim 2 , wherein the mixed-mode chromatography is performed in flow-through mode.

4. The process as claimed in claim 3 , wherein the mixed-mode chromatography column is selected from Capto adhere (N-Benzyl-N-methyl ethanol amine), Capto MMC (MMC ligand), MEP Hypercel (4-marcaptomethyl pyridine), BEA Hypercel (hexylamine) and PPA Hypercel (phenylpropylamine).

5. The process as claimed in claim 4 , wherein the mixed-mode chromatography column is Capto adhere (N-Benzyl-N-methyl ethanol amine).

6. The process as claimed in claim 1 , wherein the protein mixture is applied on to the mixed-mode chromatography column in step (h) at a suitable pH in the range of about pH 6 to about pH 6.8.

7. The process as claimed in step (h) of claim 1 , wherein the protein mixture is applied on to the mixed-mode chromatography column at a suitable conductivity in the range of about 30 mS/cm to about 42 mS/cm.

8. The process as claimed in step (h) of claim 1 , wherein the mixed-mode column is equilibrated with a buffer comprising 20 mM histidine hydrochloride, 240 mM sodium acetate and 220 mM NaCl.

9. The process as claimed in claim 1 , wherein the eluted TNFR:Fc fusion protein in step (i) is ˜≥80% pure.

10. The process as claimed in claim 1 , wherein the TNFR:Fc fusion protein is Etanercept.

11. The process as claimed in step (b) of claim 1 , wherein the Protein A column is pre-equilibrated with an equilibration buffer comprising about 50 mM Tris-Cl at pH 8.5 and 18 mS/cm conductivity.

12. The process as claimed in claim 1 , wherein the step (b) further comprises:

a′) washing the bound TNFR:Fc fusion protein with a buffer having a pH in the range of about pH 8 to about pH 9 and/or

b′) washing the bound TNFR:Fc fusion protein with a buffer having a pH in the range of about pH 4 to about pH 5.

13. The process as claimed in claim 12 wherein the step (a′) comprises a buffer selected from Tris chloride, HEPES, Triethanolamine, Borate and Glycine-NaOH.

14. The process as claimed in step (c) of claim 1 , wherein the TNFR:Fc fusion protein is eluted with a linear gradient or a step gradient or a combination thereof.

15. The process as claimed in claim 14 , wherein the TNFR:Fc fusion protein is eluted with a combination of linear gradient and step gradient.

16. The process as claimed in claim 15 , wherein the linear gradient is achieved by mixing an elution buffer having a pH in the range of about 2 to about 3.5 and a wash buffer having a pH in the range of about 4 to 5 in a suitable ratio.

17. The process as claimed in claim 1 , wherein the protein degrading enzyme is removed by at least 90%.

18. The process as claimed in claim 1 , wherein the HCP impurities are selected from aggregates, misfolded protein, fragments, endotoxins, nucleic acids, viruses and proteases.

19. The process as claimed in claim 1 , wherein the hydrophobic interaction chromatography column is selected from Butyl Toyopearl 650 M resin, Toyopearl Phenyl-650, Butyl Sepharose 6 Fast Flow and Phenyl Sepharose 6 Fast Flow.

20. The process as claimed in claim 1 , wherein the anion exchange chromatography column is selected from Quaternary Ammonium, Sulfonic Acid, Diethylaminoethyl and Carboxymethyl.