Clarification of mammalian cell culture

Patent metadata

Field	Value
Publication number	US-2017191101-A1
Application number	US-201515126790-A
Country	US
Kind code	A1
Filing date	Mar 17, 2015
Priority date	Mar 17, 2014
Publication date	Jul 6, 2017
Grant date	—

Abstract

Disclosed is an improved method for clarifying a cell culture during the manufacture of a protein. The method includes the step of transiently reducing the pH of a cell culture, followed by a holding step for a period of time, followed by the neutralization of the cell culture prior to clarification by centrifugation.

First claim

What is claimed is:

1 . A method of clarifying a cell culture comprising: (a) lowering the pH of the cell culture; (b) holding the cell culture at the lower pH; (c) increasing the pH of the cell culture; and then (d) clarifying the cell culture.

2 . The method of claim 1 , wherein the pH of the cell culture is lowered to pH 4.3±0.2 at step (a).

3 . The method of claim 2 , wherein the pH of the cell culture is lowered at step (a) by adding phosphoric acid to the cell culture at stop (a).

4 . The method of claim 3 , wherein the pH of the cell culture is lowered by adding 2M phosphoric acid to the cell culture at step (a).

5 . The method of claim 1 , wherein the cell culture is held at step (b) for 30 to 60 minutes at 15 to 20° C.

6 . The method of claim 1 , wherein the pH of the cell culture is increased at step (c) by adding tris(hydroxymethyl)aminomethane) (tris) to the cell culture to attain a pH of 6.0±0.2.

7 . The method of claim 6 , wherein the pH of the cell culture is increased at step (c) by adding 2M tris to the cell culture.

8 . The method of claim 1 , wherein the cell culture is clarified at step (d) by disk stack centrifugation, wherein a pellet fraction and a clarified supernatant fraction are formed.

9 . The method of claim 8 , wherein the cell culture is clarified by centrifugation at ≧7,000 g and at a feed flow rate of 2,000±500 L/h.

10 - 11 . (canceled)

12 . A method of manufacturing a protein comprising: (a) obtaining a cell culture, which comprises protein, cells, and media; (b) lowering the pH of the cell culture; (c) holding the cell culture at the lower pH; (d) increasing the pH of the cell culture; and then (e) forming a pellet fraction and a clarified supernatant fraction.

13 . The method of claim 12 , wherein the cells secrete the protein into the media.

14 . The method of claim 12 , wherein the protein is an antigen-binding protein.

15 . The method of claim 14 , wherein the antigen-binding protein is an antibody.

16 . The method of claim 15 , wherein the antibody is capable of binding binds more than one epitope.

17 . The method of claim 16 , wherein the antibody is binds to an epitope on one antigen and an epitope on another antigen.

18 . The method of claim 12 , wherein the cells are mammalian cells.

19 . The method of claim 18 , wherein the cells are Chinese hamster ovary (CHO) cells.

20 . The method claim 12 further comprising subjecting the clarified supernatant fraction after step (e) to depth filtration.

21 . (canceled)

22 . A protein produced according to the method of claim 12 .

23 . The protein of claim 22 , wherein said protein is an antigen-binding protein.

Assignees

• Regeneron Pharma

Inventors

• Adams Benjamin
• Tustian Andrew
• Bak Hanne

Classifications

• C12P21/00 Primary

  Preparation of peptides or proteins (single cell protein C12N1/00) · CPC title

• C12N5/0081

  Purging biological preparations of unwanted cells · CPC title