Patent ID: 8268582

Claim:
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.