Temperature shift for high yield expression of polypeptides in yeast and other transformed cells
View Patent ↗Methods for producing heterologous proteins are disclosed. In particular, the present disclosure provides improved methods of producing desired proteins, including multi-subunit proteins such as antibodies, with a higher yield and improved purity. In exemplary embodiments, the transformed cells are a yeast, e.g., methylotrophic yeast such as Pichia pastoris.
1. A method of producing a recombinant multi-subunit complex in yeast cells, comprising:
(a) culturing at a first temperature yeast cells comprising one or more genes that encode said multi-subunit complex; and
(b) culturing said yeast cells at a second temperature and allowing said yeast cells to produce said multi-subunit complex;
wherein said first temperature is between 27.5° C. and 28.5° C., and said second temperature is between 30° C. and 31° C.,
wherein said method increases the yield of said protein and/or decreases the relative abundance of one or more product-associated variants relative to the same method effected without a difference between said first temperature and said second temperature, and
wherein the promoter or promoters which regulate the transcription of the one or more genes that encode said multi-subunit complex are not temperature inducible.
2. The method of claim 1 , wherein said first temperature is 28° C.
3. The method of claim 1 , wherein said method decreases the relative abundance of proteins having aberrant disulfide bonds, proteins having reduced cysteines, proteins having aberrant glycosylation, and/or proteins having aberrant glycosylation relative to the same method effected without a difference between said first temperature and said second temperature.
4. The method of claim 1 , wherein said yeast cells comprise methylotrophic yeast.
5. The method of claim 1 , wherein step (a) comprises culturing said yeast cells in a culture medium comprising glycerol as a carbon source until said glycerol is exhausted.
6. The method of claim 1 , further comprising purifying said multi-subunit complex.
7. The method of claim 1 , wherein step (a) comprises a batch phase, wherein said batch phase comprises culturing the yeast cells in a medium comprising a carbon source, wherein the end of said batch phase is determined by exhaustion of the carbon source in the culture medium.
8. The method of claim 1 , wherein step (b) comprises a fed batch phase.
9. The method of claim 1 , further comprising maintaining the respiratory quotient (RQ) of the culture at a specified value or in a specified range during step (b).
10. The method of claim 1 , wherein said respiratory quotient is between 1.0 and 1.24.
11. The method of claim 4 , wherein said yeast cells comprise yeast cells of the genus Pichia.
12. The method of claim 11 , wherein said yeast cells comprise Pichia pastoris, Pichia angusta, Pichia guilliermondii, Pichia methanolica , or Pichia inositovera cells.
13. The method of claim 12 , wherein said yeast cells comprise Pichia pastoris cells.
14. The method of claim 7 , wherein said yeast cells comprise Pichia pastoris cells.
15. The method of claim 12 , wherein said yeast cells comprise Pichia pastoris cells and the recombinant multi-subunit complex comprises an antibody.
16. The method of claim 7 , wherein said yeast cells comprise Pichia pastoris cells and the recombinant multi-subunit complex comprises an antibody.