Methods of enhancing protein quality and quantity by yeast fed-batch fermentation

A method for producing an antibody or antigen-binding fragment thereof in Pichia pastoris comprising:

• a) providing a population of cultured Pichia pastoris cells, wherein each cell comprises a DNA segment encoding a heavy chain polypeptide and a light chain polypeptide of the antibody operably linked to a promoter and a transcription terminator;
• b) culturing the cells of step (a) under batch fermentation conditions;
• c) culturing the cells of step (b) under fed-batch fermentation conditions comprising adjusting a first respiratory quotient (RQ1) to about 0.8-1.06, to about 0.85-1.06, to about 0.90-1.06, to about 0.95-1.06 or less than 1.07 at about 32/48-100/140 hours of the fermentation process;
• d) harvesting the cells of step (c) at about 100-140 hours of the fermentation process;
• and
• e) recovering the antibody produced by the harvested cells of step (d).

A method of producing an IL-6 antibody in Pichia pastoris substantially free of cleavage comprising:

• a) providing a population of cultured Pichia pastoris cells, wherein each cell comprises a DNA segment encoding a heavy chain polypeptide and a light chain polypeptide of the antibody operably linked to a promoter and a transcription terminator;
• b) culturing the cells of step (a) under batch fermentation conditions;
• c) culturing the cells of step (b) under fed-batch fermentation conditions comprising adjusting a first respiratory quotient (RQ1) to about 0.8-1.06, to about 0.85-1.06, to about 0.90-1.06, to about 0.95-1.06 or less than 1.07 at about 32/48-100/140 hours of the fermentation process;
• d) harvesting the cells of step (c) at about 100-140 hours of the fermentation process;
• and
• e) recovering the antibody produced by the harvested cells of step (d);
• and
• wherein cleavage of the heavy chain polypeptide results in a 37 kD band and 19 kD band on a reducing SDS-PAGE gel;
• and
• wherein the heavy chain polypeptide of the produced antibody is substantially free of cleavage.

A method for producing an antibody or antigen-binding fragment thereof in Pichia pastoris comprising:

• a) providing a population of cultured Pichia pastoris cells, wherein each cell comprises a DNA segment encoding a heavy chain polypeptide and a light chain polypeptide of the antibody operably linked to a promoter and a transcription terminator;
• b) culturing the cells of step (a) under batch fermentation conditions;
• c) culturing the cells of step (b) under fed-batch fermentation conditions comprising increasing the concentration of ethanol to about 18-22 g/L or about 19-21 g/L of the cell culture at about 16/21-32/48 hour of the fermentation process, wherein the ethanol concentration of about 18-22 g/L or about 19-21 g/L is maintained for a period of up to about 5 hours, up to about 4 hours, up to about 3 hours, up to about 2 hours, up to about 1 hour, up to about 30 minutes or up to about 1 second;
• d) harvesting the cells of step (c) at about 100-140 hours of the fermentation process;
• and
• e) recovering the antibody produced by the harvested cells of step (d).

A method of producing an antibody or antigen-binding fragment in Pichia pastoris substantially free of cleavage comprising:

• a) providing a population of cultured Pichia pastoris cells, wherein each cell comprises a DNA segment encoding a heavy chain polypeptide and a light chain polypeptide of the antibody operably linked to a promoter and a transcription terminator;
• b) culturing the cells of step (a) under batch fermentation conditions;
• c) culturing the cells of step (b) under fed-batch fermentation conditions comprising adjusting a first respiratory quotient (RQ1) to about 0.8-1.06, to about 0.85-1.06, to about 0.90-1.06, to about 0.95-1.06 or less than 1.07 at about 32/48-100/140 hours of the fermentation process;
• d) harvesting the cells of step (c) at about 100-140 hours of the fermentation process;
• and
• e) recovering the antibody produced by the harvested cells of step (d);
• and
• wherein cleavage of the heavy chain polypeptide and/or light chain polypeptide is determined on a reducing SDS-PAGE gel;
• and
• wherein the heavy chain polypeptide and the light chain polypeptide of the produced antibody are substantially free of cleavage.