Multi-copy strategy for high-titer and high-purity production of multi-subunit proteins such as antibodies in transformed microbes such as
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.
1. A diploid Pichia pastoris host cell identified as a host cell that produces a desired antibody comprised of a heavy and a light chain with a greater purity by a method comprising:
(a) providing a panel of Pichia pastoris host cells, said panel comprising at least two diploid Pichia pastoris host cells that each comprise genes that provide for expression of the light and heavy chain of the desired antibody;
wherein said at least two host diploid Pichia pastoris cells are each produced by mating or fusion of a first and a second haploid Pichia pastoris parental cell, wherein the first haploid Pichia pastoris parental cell comprises one or more copies of at least one gene encoding a light chain of said desired antibody wherein at least one of said light chain genes of said desired antibody is integrated into a first locus comprised on two homologous chromosomes via homologous recombination and at least one of said light chain genes of said desired antibody is integrated into a second locus also comprised on two homologous chromosomes via homologous recombination; and a second haploid Pichia pastoris parental cell which comprises one or more copies of at least one gene encoding a heavy chain of said desired antibody wherein at least one of said heavy chain genes of said desired antibody is integrated into the same first locus comprised on two homologous chromosomes via homologous recombination and at least one of said heavy chain genes of said desired antibody is integrated into the same second locus comprised on two homologous chromosomes via homologous recombination;
(b) culturing each of said diploid Pichia pastoris host cells to express the desired antibody, wherein the genes encoding the light and heavy chains of said desired antibody which are integrated in 2 different loci of said at least two diploid Pichia pastoris host cells provide for differing levels of expression of the light and heavy chains of said desired antibody;
(c) measuring the yield of the desired antibody produced by each of said diploid Pichia pastoris host cells; and
(d) identifying the diploid Pichia pastoris host cell that produces a greater yield than another diploid Pichia pastoris host cell in said panel of diploid Pichia pastoris host cells as a host cell that produces a desired antibody with a greater purity;
and further wherein the total number of heavy chain genes integrated into said identified diploid Pichia pastoris cell ranges from 2 to 10 wherein at least 1 copy is present at a first and second gene loci and at most 7 copies are present at either of said 2 loci and the total number of integrated light chain genes ranges from 2 to 10 and at least 1 copy of said light chain genes is present at 2 different loci and at most 7 copies of said light chain genes is present at either of said 2 loci.
2. The diploid Pichia pastoris host cell of claim 1 , wherein the respective number of copies of the gene encoding the heavy chain of said desired antibody and the number of copies of the gene encoding the light chain of said desired antibody in said diploid Pichia pastoris host cell are: 2 and 2, 2 and 3, 3 and 3, 3 and 4, 3 and 5, 4 and 3, 4 and 4, 4 and 5, 4 and 6, 5 and 4, 5 and 5, 5 and 6, or 5 and 7.
3. The host cell of claim 1 , wherein the gene copies encoding the light and heavy chains of said desired antibody are respectively integrated into two loci wherein each of said two loci are on two homologous chromosomes.
4. The diploid Pichia pastoris host cell of claim 3 , wherein each locus contains no more than 5 copies of a given antibody subunit, no more than 4 copies of a given antibody subunit, or no more than 3 copies of a given antibody subunit.
5. The diploid Pichia pastoris host cell of claim 3 , wherein said 2 different gene loci comprise at least two of pGAP, HIS4 TT and AOX1 TT.
6. A culture medium containing a stable diploid Pichia pastoris yeast culture derived from the host cell of claim 1 , wherein the culture medium comprises expression levels of said desired antibody which are at least about 50 mg/liter, 100 mg/liter, 500 mg/liter or 1000 mg/liter, 1500 mg/liter or more.
7. A culture medium containing a stable diploid Pichia pastoris yeast culture derived from the host cell of claim 1 , that expresses said desired antibody into a culture medium wherein the cell density of said diploid cells in said culture are at least about 50 g/L, 100 g/L, 300 g/L, 400 g/L, 500 g/L, 700 g/L, or more.
8. The diploid Pichia pastoris host cell of claim 1 , wherein the at least one of said different gene loci are selected from pGAP, HIS4 TT and AOX1 TT.
9. The diploid Pichia pastoris host cell of claim 1 , wherein both of said different gene loci are selected from pGAP, HIS4 TT and AOX1 TT.