Methods of synthesizing heteromultimeric polypeptides in yeast using a haploid mating strategy

• US 8,268,582 B2
• Filed: 10/22/2004
• Issued: 09/18/2012
• Est. Priority Date: 10/22/2003
• Status: Active Grant

• Current Assignee: Keck Graduate Institute, H. Lundbeck A/S (Lundbeck Foundation)
• Original Assignee: Keck Graduate Institute, Alder Biopharmaceuticals, Inc. (Lundbeck Foundation)
• Inventors: Cregg, James M., Latham, John, Litton, Mark, Tolstorukov, Ilya I., Schatzman′, Randall
• Primary Examiner(s): VOGEL, NANCY TREPTOW
• Application Number:
  US10/577,074
• Publication Number:
  US 20080003643A1
• Time in Patent Office: 2,888 Days
• Field of Search: None
• US Class Current: 435/69.1
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C07K 16/2809 against the T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 16/2896 against molecules with a "CD"-designation, not provided for elsewhere
• CPC Class Codes: C07K 2317/14 Specific host cells or culture conditions, e.g. components, pH or temperature
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 15/815 for yeasts other than Saccharomyces

Abstract

Methods are provided for the synthesis and secretion of recombinant hetero-multimeric proteins in mating competent yeast. A first expression vector is transformed into a first haploid cell; and a second expression vector is transformed into a second haploid cell. The transformed haploid cells, each individually synthesizing a non-identical polypeptide, are identified and then genetically crossed or fused. The resulting diploid strains are utilized to produce and secrete fully assembled and biologically functional hetero-multimeric protein.

Claims

1. A method for the synthesis and recovery of a secreted, biologically active heterologous heteromultimeric polypeptide comprising at least two non-identical subunit polypeptide chains, the method comprising:

(i) producing one or more stable diploid Pichia cells by mating or fusing a first haploid Pichia cell containing a first expression construct, said first expression construct comprising nucleic acid sequences encoding for the expression of at least one subunit of said heteromultimeric polypeptide, operably linked to a first yeast promoter; and

a second haploid Pichia cell containing a second expression construct, said second expression construct comprising nucleic acid sequences encoding for the remaining subunit(s) of said heteromultimeric polypeptide, operably linked to a second yeast promoter under conditions yielding one or more diploid Pichia cells, wherein said diploid cells comprise at least one expression construct encoding for at least two non-identical subunit polypeptide chains, and which stable diploid Pichia cells are capable of the assembly, expression and secretion of said heteropolymeric polypeptide which is comprised of said at least two non-identical subunit polypeptide chains into a culture medium when cultured in said medium under appropriate culture conditions;

(ii) culturing said diploid Pichia cells, or diploid progeny thereof, in a culture medium under conditions resulting in the expression, assembly and secretion of said biologically active heteromultimeric polypeptide in the culture medium; and

(iii) recovering the resultant heteromultimeric polypeptide from the culture medium, wherein the heteromultimeric polypeptide is an antibody or an antigen binding antibody fragment.

2. The method according to claim 1, wherein said Pichia cells are selected from Pichia pastoris, Pichia methanolica, and Pichia angusta.

3. The method according to claim 2, wherein said Pichia cells are Pichia pastoris.

4. The method of claim 1, wherein said expression constructs are integrated into the genome of said diploid Pichia cells.

5. The method of claim 1, wherein said expression constructs are contained on extrachromosomal elements.

6. The method of claim 1, wherein the first or second promoters are constitutive.

7. The method of claim 1, wherein the first or second promoters are inducible.

8. The method of claim 1, wherein the diploid Pichia cells are grown in a production media.

9. The method of claim 8, wherein said production media is a minimal media.

10. The method of claim 9, wherein said minimal media lacks selective agents.

11. The method of claim 9, wherein said minimal media lacks pre-formed amino acids or other complex biomolecules.

12. The method of claim 1, wherein said diploid Pichia cells are grown to a cell density of at least about 50 g/L.

13. The method of claim 12, wherein said cell density is at least about 100 g/L.

14. The method of claim 13, wherein said cell density is at least about 300 g/L.

15. The method of claim 14, wherein said cell density is at least about 400 g/L.

16. The method of claim 15, wherein said cell density is at least about 500 g/L.

17. The method of claim 1, wherein said diploid Pichia cells are grown under conditions resulting in levels of said biologically active heteromultimeric polypeptide in the culture medium which are at least about 50 mg/L.

18. The method of claim 17, wherein said diploid Pichia cells are grown under conditions resulting in levels of said biologically active heteromultimeric polypeptide in the culture medium which are at least about 100 mg/L.

19. The method of claim 18, wherein said diploid Pichia cells are grown under conditions resulting in levels of said biologically active heteromultimeric polypeptide in the culture medium which are at least about 500 mg/L.

20. The method of claim 19, wherein said diploid Pichia cells are grown under conditions resulting in levels of said biologically active heteromultimeric polypeptide in the culture medium which are at least about 1000 mg/L.

21. The method of claim 1, wherein at least about 99% of said diploid Pichia cells comprise said expression constructs after culturing for at least 20 doublings.

22. The method of claim 21, wherein at least about 99% of said diploid Pichia cells comprise said expression constructs after culturing for at least 50 doublings.

23. The method of claim 22, wherein at least about 99% of said diploid Pichia cells comprise said expression constructs after culturing for at least 100 doublings.

24. The method of claim 1, wherein the diploid Pichia cells express the heteromultimeric polypeptide at a level of expression which is reduced by not more than about 20% relative to the starting level of expression after culturing for at least 20 doublings.

25. The method of claim 24, wherein the diploid Pichia cells express the heteromultimeric polypeptide at a level of expression which is reduced by not more than about 20% relative to the starting level of expression after culturing for at least 50 doublings.

26. The method of claim 25, wherein the diploid Pichia cells express the heteromultimeric polypeptide at a level of expression which is reduced by not more than about 20% relative to the starting level of expression after culturing for at least 100 doublings.

27. The method of claim 8, wherein the diploid Pichia cells express the heteromultimeric polypeptide at a level of expression which is reduced by not more than about 10% relative to the starting level of expression after culturing for at least 20 doublings.

28. The method of claim 8, wherein the diploid Pichia cells express the heteromultimeric polypeptide at a level of expression which is reduced by not more than about 5% relative to the starting level of expression after culturing for at least 20 doublings.

29. The method of claim 1, wherein said culture containing said diploidal Pichia cells is grown at a temperature of not more than 22°

C.

30. A culture medium containing a stable diploid Pichia culture according to claim 1, wherein the culture medium comprises expression levels of said biologically active heteromultimeric polypeptide which are at least about 50 mg/liter.

31. A culture medium containing a stable diploid Pichia culture according to claim 1, that expresses said heteromultimeric polypeptide into a culture medium, wherein the cell density of said Pichia diploid cells in said culture are at least about 50 g/L.