Removal of high molecular weight aggregates using hydroxyapatite chromatography

Patent metadata

Field	Value
Publication number	US-9822143-B2
Application number	US-201313803071-A
Country	US
Kind code	B2
Filing date	Mar 14, 2013
Priority date	Oct 27, 2003
Publication date	Nov 21, 2017
Grant date	Nov 21, 2017

Abstract

This invention relates to the application of hydroxyapatite chromatography to the purification of at least one antibody from a preparation containing high molecular weight aggregates. Further, this invention relates to an integration of ceramic hydroxyapatite chromatography into a combination chromatographic protocol for the removal of high molecular weight aggregates from an antibody preparation.

First claim

We claim:

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.

Assignees

• Wyeth Llc

Inventors

• Sun Shujun
• Gallo Christopher
• Kelley Brian

Classifications

• B01D15/361

  Ion-exchange · CPC title

• B01J20/048

  containing phosphorus, e.g. phosphates, apatites, hydroxyapatites · CPC title

• C07K16/18

  against material from animals or humans · CPC title

• B01J20/282

  Porous sorbents (ion exchange B01J39/00 - B01J41/00) · CPC title

• B01D15/3809

  of the antigen-antibody type, e.g. protein A, G or L chromatography · CPC title