	US 12,077,794 B2
	Temperature shift for high yield expression of polypeptides in yeast and other transformed cells
Gary Lesnicki, Woodinville, WA (US); and Mark Young, Boulder, CO (US)
Assigned to H. LUNDBECK A/S, Valby (DK)
Filed by H. LUNDBECK A/S, Valby (DK)
Filed on Feb. 11, 2019, as Appl. No. 16/272,079.
Application 16/272,079 is a division of application No. 14/215,406, filed on Mar. 17, 2014, granted, now 10,202,630, issued on Feb. 12, 2019.
Claims priority of provisional application 61/791,471, filed on Mar. 15, 2013.
Prior Publication US 2019/0390239 A1, Dec. 26, 2019
This patent is subject to a terminal disclaimer.
Int. Cl. C12P 21/00 (2006.01); C12N 1/16 (2006.01); C12N 15/67 (2006.01); C12N 15/90 (2006.01)

CPC C12P 21/00 (2013.01) [C12N 1/16 (2013.01); C12N 15/67 (2013.01); C12N 15/905 (2013.01); C07K 2317/14 (2013.01); C12N 2523/00 (2013.01)]

18 Claims

1. A method of producing a desired full-length antibody, wherein the expression of the desired full-length antibody occurs in both step (a) and step (b) comprising:

(a) wherein step (a) comprises culturing Pichia pastoris yeast cells comprising genes that provide for the expression of said desired full-length antibody at a first temperature which is between 27 degrees C. and 30 degrees C. inclusive; and

(b) after step (a), step (b) comprises culturing the same Pichia pastoris yeast cells which are cultured in step (a) at a second temperature which is between 1 degree C. and 3 degrees C. inclusive higher than said first temperature and allowing said same culture of Pichia pastoris yeast cells to continue to produce said desired full-length antibody;

wherein

(i) culture step (a) and step (b) are effected after an inoculum expansion step;

(ii) said desired full-length antibody is expressed under control of a non-temperature inducible promoter;

(iii) said Pichia pastoris yeast cells comprise between 1-10 copies of a gene encoding the heavy chain of said desired full-length antibody and between 1-10 copies of a gene encoding the light chain of said desired full-length antibody;

(iv) culture steps (a) and (b) comprise a batch phase followed by a fed batch phase, wherein culture step (a) is initiated during the batch phase and culture step (b) is initiated during the batch phase, during of the fed batch phase, or between the batch phase and the fed batch phase;

(v) culture step (a) and step (b) comprise detecting a concentration of dissolved oxygen in the culture of Pichia pastoris yeast cells;

(vi) said second temperature is effected after a rapid increase in the concentration of dissolved oxygen is detected in the culture; and

(vii) said method increases the yield of said desired full-length antibody and/or decreases the relative abundance of full-length antibody complexes having aberrant disulfide bonds relative to the same culture method effected without a difference between said first temperature and said second temperature.