Pichia pastoris yeast cultures comprising reduced antibody-associated variants

A Pichia pastoris yeast culture which comprises

• (i) a culture medium and
• (ii) Pichia pastoris yeast cells which have been engineered to comprise nucleic acids which encode for a full length human or humanized antibody, which nucleic acids are operably linked to at least one promoter and signal sequence that provides for the expression and secretion of the full length human or humanized antibody encoded by said nucleic acids, and said at least one promoter that provides for the expression of said full length antibody is/are not temperature inducible, wherein:
• (1) said Pichia pastoris yeast cell containing culture has been cultured by a culture method that has resulted in the expression and secretion of the full length antibody into the culture medium comprising said Pichia pastoris yeast cells,
• (2) said culture method comprises (a) culturing said Pichia pastoris yeast cells culture at a first temperature which is between 27.5° C. and 28.5° C.;
• and (b) subsequently culturing said Pichia pastoris yeast cells at a second temperature is between 30° C. and 31° C., thereby producing a Pichia pastoris yeast culture wherein the culture medium comprises the secreted full length antibody at an increased antibody yield and further comprises a decreased amount of antibody-associated variants relative to an otherwise identical culture comprising the same Pichia pastoris yeast cells which have been cultured in the same medium and under the same conditions, except that culturing is effected entirely at a temperature which is between 30° C. and 31° C. or entirely at a temperature of between 27.5° C. and 28.5° C.;
• (3) said culture steps (a) and (b) are effected after an inoculum expansion step;
• and
• (4) wherein said yeast culture medium comprising said full-length antibody and said Pichia pastoris yeast cells has not been treated by any purification method after said culture method is effected.