Process for the purification of Fc-containing proteins
The invention relates to a process for reducing the concentration of free Fc-moieties in a fluid comprising an Fc-containing protein comprising a cation exchange chromatography step.
1. A method for reducing the content of free Fc-moieties in a fluid comprising an Fc-containing protein and free Fc-moieties and having a pH of at least one unit below the isoelectric point (pI) of the Fc-containing protein, the method comprising cation exchange chromatography comprising the steps of loading said fluid onto a cation exchange resin to absorb the Fc-containing protein, washing the cation exchange resin with a first buffer having a conductivity of 8.2 to 9.2 mS/cm and a pH of 6.0 to 7.0 to remove free Fc-moieties, and eluting the Fc-containing protein with a second buffer to produce an eluate comprising the Fe-containing protein, whereby the level of free Fe-moieties in said eluate relative to the level of Fe-containing protein has been reduced from said fluid by a factor of 5 to 15.
2. The method according to claim 1 , wherein the first buffer has a conductivity of 8.2 to 8.6 mS/cm.
3. The method according to claim 1 or 2 , wherein the first buffer comprises 75 to 125 mM sodium phosphate.
4. The method according to claim 1 , wherein the second buffer has a pH ranging from 7.0 to 8.5.
5. The method according to claim 4 , wherein the second buffer has a conductivity of 15 to 22 mS/cm.
6. The method according to claim 1 or 4 , wherein the cation exchange resin is a strong cation exchange resin.
7. The method according to claim 6 , wherein the cation exchange resin comprises SO 3 − groups.
8. The method according to claim 6 , wherein the resin comprises a cross-linked methacrylate matrix.
9. The method according to claim 1 or 4 , further comprising a purification step selected from affinity chromatography, anion exchange chromatography and hydroxyapatite chromatography.
10. The method according to claim 1 or 4 , wherein the Fc-containing protein comprises an Immunoglobulin (Ig) constant region.
11. The method according to claim 10 , wherein the constant region is a human constant region.
12. The method according to claim 10 , wherein the immunoglobulin is an IgG 1 .
13. The method according to claim 10 , wherein the constant region comprises a CH2 and a CH3 domain.
14. The method according to claim 1 or 4 , wherein the Fc-containing protein comprises an immunoglobulin variable region.
15. The method according to claim 14 , wherein the Fe-containing protein is an antibody.
16. The method according to claim 1 or 4 , wherein the Fc-containing protein is an Fc-fusion protein.
17. The method according to claim 16 , wherein the Fe-fusion protein comprises an extracellular domain of a member of the tumor necrosis factor receptor (TNFR) superfamily selected from the group consisting of TNFR1 and TNFR2.
18. The method according to claim 16 , wherein the Fe-fusion protein comprises an extracellular domain of BAFF-R, BCMA, or TACI.
19. The method according to claim 16 , wherein the Fe-fusion protein comprises a polypeptide selected from
a. amino acids 30 to 110 of SEQ ID NO: 2;
b. SEQ ID NO: 3; and
c SEQ ID NO: 4.
20. The method according to claim 1 , wherein free Fc-moieties constitute less than 5% of the total protein concentration of said eluate.
21. The method according to claim 1 , wherein free Fc-moieties constitute less than 2% of the total protein concentration of said eluate.
22. The method according to claim 1 , wherein free Fc-moieties constitute less than 1% of the total protein concentration of said eluate.
23. The method according to claim 1 , wherein free Fe-moieties constitute less than 0.5% of the total protein concentration of said eluate.
24. The method according to claim 1 , wherein free Fe-moieties constitute less than 0.2% of the total protein concentration of said eluate.
25. The method according to claim 1 , wherein free Fc-moieties constitute less than 0.1% of the total protein concentration of said eluate.
26. The method according to claim 9 , wherein said fluid comprising an Fc-containing protein is an eluate from an affinity chromatography purification step.
27. The method according to claim 26 , wherein said eluate having a reduced level of free Fc-moieties is subjected to anion exchange chromatography to produce an anion exchange chromatography flow-through comprising the Fc-containing protein.
28. The method according to claim 27 , wherein the anion exchange chromatography flow-through comprising the Fc-containing protein is subjected to hydroxyapatite chromatography to produce a hydroxyapatite chromatrography eluate comprising the Fc-containing protein.
29. The method according to claim 1 , wherein the first buffer has a conductivity of 8.4 to 9.2 mS/cm.