Method of culturing eukaryotic cells

Patent metadata

Field	Value
Publication number	US-10190084-B2
Application number	US-201414480366-A
Country	US
Kind code	B2
Filing date	Sep 8, 2014
Priority date	Jul 6, 2009
Publication date	Jan 29, 2019
Grant date	Jan 29, 2019

Abstract

An apparatus and method to maintain pH within a range conducive for cell growth in a bicarbonate-containing cell culture system without the addition of base. The method relies on the gas transfer characteristics of the bioreactor system to modulate the CO 2 transfer to and from the cell culture such that the pH of the cell culture can be maintained within a desired range.

First claim

What is claimed is:

1. A method for culturing Chinese hamster ovary (CHO) cells comprising:
(i) providing cell culture inoculant comprising CHO cells in about 6 L of a bicarbonate-containing culture liquid to a vessel to achieve a target cell density of about 7.5×10 5 CHO cells/mL, wherein said vessel has a volume of about 50 L and has walls that encapsulate said cell culture and a gas phase head space above said cell culture, and wherein said vessel comprises at least one port that provides an entrance and an egress of gas to and from said head space;
(ii) providing gas to said head space through said port, wherein said gas contains CO 2 in an amount of 8% (v/v) of said gas on day 1, in an amount of 5% (v/v) of said gas on day 2, and in an amount of 2% (v/v) of said gas thereafter, thereby modulating the pH of said cell culture to maintain the pH of the culture between pH 6.8 and 7.2;
(iii) providing fresh culture medium to said vessel to achieve a volume of about 20 L;
(iv) providing gas to said head space through said port, wherein said gas contains CO 2 in an amount of 8% (v/v) of said gas on day 1, in an amount of 5% (v/v) of said gas on day 2, and in an amount of 2% (v/v) of said gas thereafter, thereby modulating the pH of said cell culture to maintain the pH of the culture between pH 6.8 and 7.2;
(v) perfusing fresh culture medium into said vessel and removing spent culture medium from said vessel at a perfusion rate of about 1 volume per day; and
(vi) providing gas to said head space through said port to sweep accumulated CO 2 from the head space of the vessel, wherein said gas contains O 2 in an amount of 30% (v/v) of said gas 48 hours after the start of perfusion, thereby maintaining dissolved O 2 to a level greater than 20% of air saturation,
wherein the fresh culture medium has a pH of 7.2, the clearance rate of the head space is between 0.002 and 0.1 head space volume per minute (hvm), the vessel is agitated by rocking the vessel at a rock rate between 15 and 30 rocks per minute (rpm) at a rock angle of between 5° and 15°, and wherein the method does not require monitoring and feedback control of pH and dissolved O 2 .

2. The method of claim 1 , wherein the rock rate is between 19 and 25 rpm and the rock angle is between 8° to 12°.

3. The method of claim 1 , wherein the clearance rate is between 0.01 to 0.04 hvm.

4. The method of claim 3 wherein the clearance rate is about 0.02 hvm.

5. The method of claim 1 wherein said vessel is a rigid container.

6. The method of claim 1 wherein said vessel is a pliable container.

7. The method of claim 6 wherein said vessel is a disposable culture bag.

8. The method of claim 1 further comprising continuously or intermittently monitoring the pH of the cell culture.

9. The method of claim 1 wherein the perfusion of said fresh culture medium is performed through a perfusion device that allows retention of cells in the vessel.

10. The method of claim 1 wherein said gas is provided to said head space such that the cell culture maintains a partial pressure of dissolved CO 2 at a level of about 1 to 200 mmHg.

11. The method of claim 10 wherein said gas is provided to said head space such that the cell culture maintains a partial pressure of dissolved CO 2 at a level of about 10 to 150 mmHg.

12. The method of claim 11 wherein said gas is provided to said head space such that the cell culture maintains a partial pressure of dissolved CO 2 at a level of about 20 to 120 mmHg.

13. The method of claim 12 wherein said gas is provided to said head space such that the cell culture maintains a partial pressure of dissolved CO 2 at a level of about 20 to 80 mmHg.

14. The method of claim 1 , wherein the gas flow rate is 0.2 L/min on day 0 to day 3, 0.4 L/min on day 3 to day 6, and 0.6 L/min on day 6 and thereafter.

15. The method of claim 1 , wherein the gas flow rate is 0.6 L/min.

16. The method of claim 1 , wherein the gas flow rate is 1.0 L/min.

Assignees

• Genentech Inc

Inventors

• Baskar Dinesh
• Hsiung Jenny
• Leung Woon-Lam Susan
• Yuk Inn H

Classifications

• C12M23/14 Primary

  Bags · CPC title

• C12M41/12

  of temperature (controlling the temperature of chemical or physical processes B01J19/0013, heating or cooling apparatus for laboratory use B01L7/00) · CPC title

• C12M41/48

  Automatic or computerized control (automatic analysis G01N35/00) · CPC title

• C12M23/28

  disposable or single use · CPC title

• C12M41/34

  of gas · CPC title