Apatite pretreatment

Patent metadata

Field	Value
Publication number	US-9802822-B2
Application number	US-201514747181-A
Country	US
Kind code	B2
Filing date	Jun 23, 2015
Priority date	Jun 23, 2014
Publication date	Oct 31, 2017
Grant date	Oct 31, 2017

Abstract

Apatite pretreatment methods are provided. The method is applied to the apatite solid surface prior to first chromatographic use. In one embodiment, the method may be achieved by contacting an apatite solid surface with a phosphate buffered solution at a pH of at least about 6.5 and contacting the apatite solid surface with a solution having a hydroxide.

First claim

What is claimed is:

1. A method of treating an apatite solid surface prior to first use in a chromatographic procedure, the method comprising:
(a) providing an apatite solid surface, wherein the apatite surface has not previously been used in any chromatographic procedure and has not previously been contacted with any sample;
(b) contacting the apatite solid surface with a phosphate buffered solution at a pH of at least about 6.5; and then
(c) contacting the apatite solid surface obtained in step (b) with a solution having an alkaline metal hydroxide: and then
(d) purifying protein from a sample with the apatite solid surface obtained in step (c).

2. The method of claim 1 , wherein the phosphate buffered solution is a solution having from about 0.1 M to about 1.0 M phosphate at a pH of from about 6.5 to about 10.0.

3. The method of claim 2 , wherein the phosphate buffered solution is 400 mM phosphate at a pH of 8.0.

4. The method of claim 1 ,
wherein the purifying of step (d) comprises contacting the apatite solid surface with the sample, thereby separating the protein from one or more biological compounds; then
(e) collecting the protein from the apatite solid surface; and then
(f) regenerating the apatite solid surface, the regenerating comprising,
(i) contacting the apatite solid surface with a buffered calcium solution having a calcium ion at a concentration of at least about 5 mM and a zwitterionic buffer, wherein the ratio of zwitterionic buffer concentration to calcium ion concentration is at least about 2, and the pH of the solution is at least about 6.5;
(ii) contacting the apatite solid surface with a phosphate buffered solution at a pH of at least about 6.5; and
(iii) contacting the apatite solid surface with a solution having an hydroxide.

5. The method of claim 4 , wherein (d) comprises binding the protein to the apatite solid surface, and (e) comprises eluting the protein from the apatite solid surface.

6. The method of claim 4 , wherein (d) comprises contacting the apatite solid surface to the sample, thereby flowing the protein through the apatite solid surface, and (e) comprises collecting the protein in the flow through.

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

8. The method of claim 7 , wherein the sulfonic acid containing buffer is MES, PIPES, ACES, MOPSO, MOPS, BES, TES, HEPES, DIPSO, TAPS, TAPSO, POPSO, or HEPPSO, EPPS, CAPS, CAPSO, or CHES.

9. The method of claim 7 , wherein the sulfonic acid containing buffer is MES.

10. The method of claim 4 , wherein the calcium ion is at least about 25 mM.

11. The method of claim 4 , wherein the ratio of zwitterionic buffer concentration to calcium ion concentration is at least about 2.5.

12. The method of claim 4 , wherein the buffered calcium solution comprises calcium chloride or calcium nitrate.

13. The method of claim 4 , wherein the phosphate buffered solution comprises a solution containing from about 0.1 M to about 1.0 M phosphate at a pH of from about 6.5 to about 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 4 , wherein the regenerating step reverses or eliminates degradation of a column that occurs during protein purification or column cleaning steps.

16. The method of claim 4 , wherein the regenerating step is performed before a phosphate cleaning/stripping step.

17. The method of claim 5 , wherein the regenerating step is performed after elution of the protein.

18. The method of claim 4 , wherein step (f) (ii) further comprises:
contacting the apatite solid surface with a solution comprising phosphate at a concentration of 10 mM, or less than about 10 mM, at a pH of at least about 6.5; and
then contacting the apatite solid surface with a solution comprising phosphate at a concentration of at least about 100 mM at a pH of at least about 6.5.

19. The method of claim 4 , wherein the regenerating step consists of (i), a wash, (ii), and (iii).

20. The method of claim 1 , wherein the protein is an antibody.

Assignees

• Bio Rad Laboratories Inc

Inventors

• Cummings Larry

Classifications

• B01J20/048

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

• B01D15/203

  Equilibration or regeneration · CPC title

• B01J20/281

  Sorbents specially adapted for preparative, analytical or investigative chromatography · CPC title

• B01D15/3847

  Multimodal interactions · CPC title

• C01B25/32 Primary

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