Apatite in-situ restoration

Patent metadata

Field	Value
Publication number	US-10583371-B2
Application number	US-201816100816-A
Country	US
Kind code	B2
Filing date	Aug 10, 2018
Priority date	Jun 23, 2014
Publication date	Mar 10, 2020
Grant date	Mar 10, 2020

Abstract

The present invention discloses methods of regenerating apatite surfaces, for example after purification of a target analyte.

First claim

What is claimed is:

1. A method of purifying a target analyte with an apatite solid surface, the method comprising:
(a) contacting the apatite solid surface with the target analyte, thereby separating the target analyte from one or more contaminants;
(b) collecting the target analyte, wherein the contacting, the collecting, or both cause deterioration of resin mass or particle strength; and
(c) after the collecting and before subsequent loading of additional target analyte, regenerating the apatite solid surface, the regenerating comprising:
(i) contacting the apatite solid surface with a buffered calcium solution comprising a calcium ion at a concentration of at least 5 mM and a zwitterionic buffer, wherein the ratio of zwitterionic buffer concentration to calcium ion concentration is at least 2, and the pH of the buffered calcium solution is at least 5;
(ii) after (i), contacting the apatite solid surface with a phosphate buffered solution at a pH of at least 6.5; and
(iii) after (ii), contacting the apatite solid surface with a solution comprising an hydroxide, wherein step (c) reduces, eliminates, or reverses the deterioration of the resin mass or particle strength of said apatite solid surface.

2. The method of claim 1 , wherein (a) comprises binding the target analyte to the apatite solid surface, and (b) comprises eluting the target analyte from the apatite solid surface.

3. The method of claim 1 , wherein (a) comprises contacting the apatite solid surface with the target analyte, thereby flowing the target analyte through the apatite solid surface, and (b) comprises collecting the target analyte in the flow through.

4. The method of claim 1 , wherein the zwitterionic buffer is a sulfonic acid containing buffer.

5. The method of claim 4 , wherein the sulfonic acid containing buffer is MES, PIPES, ACES, MOPSO, MOPS, BES, TES, HEPES, DIPSO, TAPS, TAPSO, POPSO, HEPPSO, EPPS, CAPS, CAPSO, or CHES.

6. The method of claim 5 , wherein the sulfonic acid containing buffer is MES.

7. The method of claim 1 , wherein the calcium ion concentration is less than 50 mM.

8. The method of claim 1 , wherein the calcium ion concentration is at least 25 mM.

9. The method of claim 1 , wherein the ratio of zwitterionic buffer concentration to calcium ion concentration is at least 2.5.

10. The method of claim 1 , wherein the buffered calcium solution comprises calcium chloride or calcium nitrate.

11. The method of claim 1 , wherein the buffered calcium solution is at a pH of at least 5.3.

12. The method of claim 1 , wherein the buffered calcium solution is at a pH of between 5.3 and 7.

13. The method of claim 1 , wherein the phosphate buffered solution comprises a solution containing from 0.1 M to 1.0 M phosphate at a pH of from 6.5 to 8.

14. The method of claim 13 , wherein the phosphate buffered solution comprises 400 mM phosphate at a pH of 7.0.

15. The method of claim 1 , wherein the hydroxide comprises an alkaline hydroxide.

16. The method of claim 15 , wherein the alkaline hydroxide comprises sodium or potassium hydroxide.

17. The method of claim 1 , wherein the regenerating reverses or eliminates degradation of the column that occurs during protein purification or column cleaning steps.

18. The method of claim 1 , wherein the contacting of the apatite solid surface with the phosphate buffered solution comprising phosphate at a pH of at least 6.5 further comprises:
contacting the apatite solid surface with a solution comprising phosphate at a concentration of 10 mM or less at a pH of at least 6.5; and
then contacting the apatite solid surface with a solution comprising phosphate at a concentration of at least 100 mM at a pH of at least 6.5.

19. The method of claim 1 , wherein the regenerating consists of step (c)(i), followed by contacting the apatite with a wash, then steps (c)(ii) and, then (c)(iii).

Assignees

• Bio Rad Laboratories

Inventors

• Cummings Larry

Classifications

• B01J20/3433

  of sorbents or filter aids other than those covered by B01J20/3408 - B01J20/3425 · CPC title

• C01B25/455

  containing halogen {(completely halogenated alkali metal phosphates C01D, e.g. lithium hexafluorophosphate C01D15/005)} · CPC title

• B01D15/203 Primary

  Equilibration or regeneration · CPC title

• C01B25/32

  Phosphates of magnesium, calcium, strontium, or barium · CPC title

• B01J20/048

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