Multi-copy strategy for high-titer and high-purity production of multi-subunit proteins such as antibodies in transformed microbes such as Pichia pastoris

Patent metadata

Field	Value
Publication number	US-10150968-B2
Application number	US-201213589584-A
Country	US
Kind code	B2
Filing date	Aug 20, 2012
Priority date	Aug 19, 2011
Publication date	Dec 11, 2018
Grant date	Dec 11, 2018

Abstract

Methods for producing heterologous multi-subunit proteins in transformed cells are disclosed. In particular, the present disclosure provides improved methods of producing multi-subunit proteins, including antibodies and other multi-subunit proteins, which may or may not be secreted, with a higher yield and decreased production of undesired side-products. In exemplary embodiments, the transformed cells are a yeast, e.g., methylotrophic yeast such as Pichia pastoris.

First claim

What is claimed is:

1. A method of identifying a diploid yeast host cell that produces a greater purity of a desired antibody, said method comprising:
(a) providing a panel of diploid yeast host cells, said panel comprising at least two host cells that each comprise differing numbers of copies of one or more genes that provide for expression of the subunits of an antibody, wherein at least one of said genes is not contained in a polycistronic gene;
(b) culturing each of the host cells in the panel under conditions which result in the expression of the desired antibody and one or more glycovariants of the antibody, wherein the different cells in the panel produce different glycovariants comprising different glycosylated antibody chains;
(c) measuring the glycovariants produced by each of the host cells in said panel; and
(d) selecting from among the host cells of step (c) a host cell which produces substantially aglycosylated antibodies, wherein said selected host cell thereby produces antibodies of greater purity.

2. The method of claim 1 , wherein said one or more glycosylated antibody chains include a glyco-heavy variant.

3. The method of claim 1 , wherein said host cell is a methylotrophic yeast, a yeast of the genus Pichia, Pichia pastoris, Pichia angusta, Pichia guilliermondii, Pichia methanolica , or Pichia inositovera.

4. The method of claim 3 , wherein said host cell is a yeast of the genus Pichia and said genes that provide for expression of the subunits of the antibody are integrated into one or more genomic loci selected from the group consisting of the pGAP, 3′ AOX TT, PpURA5, OCH1, AOX1, HIS4, GAP, ARG, and HIS4 TT loci.

5. The method of claim 1 , wherein said genes that provide for expression of the subunits of the antibody are integrated into the genome of said host cells.

6. The method of claim 1 , wherein at least one of said genes encoding said subunits of the antibody is expressed under control of a constitutive promoter; an inducible promoter selected from the group consisting of the glyceraldehyde-3-phosphate dehydrogenase (GAP), CUP1, AOX1, and FLD1 promoters; a promoter selected from the group consisting of the AOX1, ICL1, ADH1, alcohol dehydrogenase II, GAL4, PHO3, PHO5, Pyk promoters and chimeric promoters derived therefrom; yeast promoters; mammalian promoters; insect promoters; plant promoters; reptile promoters; amphibian promoters; viral promoters; or avian promoters.

7. The method of claim 1 , wherein said at least two host cells comprise differing numbers of copies of a gene encoding a subunit of said antibody.

8. The method of claim 1 , wherein at least one host cell in said panel comprises a gene encoding at least one subunit of the antibody whose expression is driven by a different promoter than the promoter that drives the expression of the corresponding gene in a different host cell in said panel.

9. A method of recombinantly producing a desired antibody, comprising:
(a) identifying a host cell that produces a greater purity of a desired antibody by the method of claim 1 ; and
(b) culturing said host cell to produce said desired antibody.

10. The method of claim 9 , wherein said desired antibody comprises a desired humanized or human antibody.

11. The method of claim 10 , further comprising purification of said desired humanized or human antibody.

12. The method of claim 9 , wherein the copy number of the genes encoding the desired multi-subunit complex and/or the yield of said desired antibody is stable for 20, 50, 100, 500, or 1000 generations.

13. The method of claim 9 , wherein the gene copies encoding at least one of the subunits of said antibody are integrated into two or more loci or are integrated into three or more loci.

14. The method of claim 13 , wherein each locus contains no more than 5 copies of a given subunit, no more than 4 copies of a given subunit, or no more than 3 copies of a given subunit.

Assignees

• Mitchell Danielle Marie
• Garcia Martinez Leon F
• Mcneill Patricia Dianne
• Ojala Ethan Wayne
• Inan Mehmet
• Latham John
• Alderbio Holdings Llc

Inventors

• Mitchell Danielle Marie
• Garcia-Martinez Leon F
• Mcneill Patricia Dianne
• Ojala Ethan Wayne
• Inan Mehmet
• Latham John

Classifications

• C07K2319/036

  targeting to the medium outside of the cell, e.g. type III secretion · CPC title

• C07K2319/50

  containing protease site · CPC title

• C07K16/00

  Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies · CPC title

• C12N15/815 Primary

  for yeasts other than Saccharomyces · CPC title

• C12P21/005

  Glycopeptides, glycoproteins · CPC title