Removal of high molecular weight aggregates using hydroxyapatite chromatography

1. A method for purifying at least one antibody monomer from an antibody preparation containing high molecular weight aggregates (HMWA) comprising:

contacting a hydroxyapatite resin with the antibody preparation in load buffer comprising from 1 to 20 mM sodium phosphate and from 0.2 to 2.5 M NaCl, allowing the purified antibody to flow through the column, while impurity HMWA binds to the column.

2. The method of claim 1 , wherein the purified antibody contains less than 5% high molecular weight aggregates.

3. The method of claim 1 , wherein the purified antibody contains less than 1% high molecular weight aggregates.

4. The method of claim 1 , wherein the load buffer has a pH from 6.4 to 7.6.

5. The method of claim 4 , wherein the load buffer contains 3 mM or 5 mM sodium phosphate.

6. The method of claim 4 , wherein the load buffer contains 1 M or 0.35 M NaCl.

7. The method of claim 4 , wherein the load buffer has a pH of 6.8 or 7.2.

8. The method of claim 1 , wherein the antibody is an IgG, IgA, IgD, IgE, or IgM antibody.

9. The method of claim 1 , wherein the antibody is monoclonal, polyclonal, chimeric, humanized, or a fragment thereof.

10. The method of claim 1 , wherein the antibody is an anti-IL-21 receptor, anti-GDF-8, anti-Abeta, anti-CD22, anti-Lewis Y, anti-IL-13, or anti-IL-22 antibody.

11. The method of claim 1 , wherein the antibody has a basic pI.

12. The method of claim 1 , wherein the resin is ceramic hydroxyapatite type I or type II.

13. The method of claim 12 , wherein the resin is ceramic hydroxyapatite type I.

14. The method of claim 1 , wherein the purified antibody contains less than 300 ppm Protein A.

15. A method for purifying at least one antibody monomer from an antibody preparation containing high molecular weight aggregates (HMWA) comprising subjecting the antibody preparation to

(a) Protein A affinity chromatography,

(b) ion exchange chromatography,

(c) hydroxyapatite chromatography, and

(d) contacting the hydroxyapatite chromatography comprising a hydroxyapatite resin with the antibody preparation in load buffer comprising from 1 to 20 mM sodium phosphate and from 0.2 to 2.5 M NaCl, allowing the purified antibody to flow through the resin, while impurity HMWA binds to the resin.

16. The method of claim 15 , wherein the Protein A affinity chromatography is performed first and the hydroxyapatite chromatography is performed last.

17. The method of claim 15 , wherein the ion exchange chromatography is anion exchange chromatography.

18. A method for purifying at least one antibody monomer from an antibody preparation containing high molecular weight aggregates (HMWA) comprising:

(a) contacting the preparation with a Protein A support;

(b) allowing the antibody to adsorb to the Protein A support;

(c) washing the Protein A support and adsorbed antibody with at least one Protein A washing buffer;

(d) eluting the adsorbed antibody with at least one Protein A elution buffer;

(e) contacting the Protein A eluate with an ion exchange support;

(f) allowing the antibody to flow through the ion exchange support, while impurity HMWA binds to the ion exchange support;

(g) washing the ion exchange support with at least one ion exchange washing buffer;

(h) exchanging the ion exchange flow-through into a load buffer comprising from 1 to 20 mM sodium phosphate and from 0.2 to 2.5 M NaCl;

(i) contacting the ion exchange flow-through with a hydroxyapatite resin;

(j) allowing the antibody to flow through the hydroxyapatite resin; and

(k) washing the hydroxyapatite resin with at least one hydroxyapatite washing buffer.

19. The method of claim 18 , wherein the ion exchange chromatography is anion exchange chromatography.

20. The method of claim 18 , further comprising filtering the ion exchange flow-through before application to the hydroxyapatite resin, thereby reducing the viral contaminants.

21. The method of claim 18 , further comprising subjecting the hydroxyapatite eluate to at least one of ultrafiltration or diafiltration.