Capture purification processes for proteins expressed in a non-mammalian system

Patent metadata

Field	Value
Publication number	US-12312381-B2
Application number	US-202217874846-A
Country	US
Kind code	B2
Filing date	Jul 27, 2022
Priority date	Jun 25, 2009
Publication date	May 27, 2025
Grant date	May 27, 2025

Abstract

Methods of purifying proteins expressed in non-mammalian expression systems in a non-native soluble form directly from cell lysate are disclosed. Methods of purifying proteins expressed in non-mammalian expression systems in a non-native limited solubility form directly from a refold solution are also disclosed. Resin regeneration methods are also provided.

First claim

What is claimed is:

1. A method of cleaning a separation matrix in a matrix retaining device during purifying a protein expressed in a non-native soluble form in a non-mammalian expression system, the method comprising:
(a) lysing a non-mammalian cell in which the protein is expressed in a non-native soluble form to generate a cell lysate;
(b) contacting the cell lysate with a separation matrix under conditions suitable for the protein to associate with the separation matrix;
(c) washing the separation matrix;
(d) eluting the protein from the separation matrix; and
(e) performing a cleaning operation on the matrix retaining device with a cleaning agent.

2. The method of claim 1 , wherein the separation matrix is an affinity resin.

3. The method of claim 1 , wherein the separation matrix is a non-affinity resin.

4. The method of claim 1 , wherein the separation matrix is an affinity resin selected from the group consisting of: Protein A, Protein G, and a synthetic mimetic affinity resin.

5. The method of claim 1 , wherein the separation matrix is a non-affinity resin selected from the group consisting of: ion exchange, mixed mode, and a hydrophobic interaction resin.

6. The method of claim 1 , further comprising: repeating (a) to (d) with the same separation matrix.

7. The method of claim 1 , further comprising: repeating (a) to (d) at least 100 times with the same separation matrix.

8. The method of claim 1 , further comprising: repeating (a) to (d) at least 150 times with the same separation matrix.

9. The method of claim 1 , wherein the protein is a complex protein.

10. The method of claim 1 , wherein the cleaning agent is at least one cleaning agent selected from the group consisting of: a caustic, a detergent, a denaturant, and a reductant.

11. The method of claim 10 , wherein the caustic is sodium or potassium hydroxide.

12. The method of claim 10 , wherein the detergent is Sodium Dodecyl Sulfate or Polyethylene Glycol tert-Octyl phenyl Ether.

13. The method of claim 10 , wherein the denaturant is urea or a guanidine-derivative.

14. The method of claim 10 , wherein the denaturant is Dithiothreitol or a thioglycolate.

15. The method of claim 1 , further comprising regenerating the separation matrix after the washing.

Assignees

• Amgen Inc

Inventors

• Shultz Joseph Edward
• Hart Roger

Classifications

• C07K16/00

  Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies · CPC title

• C07K1/32

  as complexes · CPC title

• C07K1/18

  Ion-exchange chromatography · CPC title

• C07K1/145

  by extraction or solubilisation · CPC title

• C07K1/22 Primary

  Affinity chromatography or related techniques based upon selective absorption processes · CPC title