Asparagine feed strategies to improve cell culture performance and mitigate asparagine sequence variants

A method for preventing asparagine sequence variants in a polypeptide of interest expressed from mammalian cells in cell culture, the method comprising:

• propagating or maintaining mammalian cells in a defined cell culture medium;
• wherein the defined cell culture medium is supplemented with asparagine in an amount from about 3.6 mM to about 43.2 mM during early fed-batch cell culture and from about 3.6 mM to about 21.6 mM during late fed-batch cell culture;
• and
• maintaining said cells in said asparagine supplemented cell culture medium for at least a portion of the early and late fed-batch cell culture under conditions sufficient for expression of the polypeptide of interest;
• wherein extracellular asparagine levels are maintained in the cell culture medium during at least early stage fed-batch cell culture above a depletion limit of about 0.1 mM such that the polypeptide of interest expressed by the mammalian cells comprise less than 0.30% of asparagine sequence variants at all individual sequence variant loci.

A method for detecting asparagine sequence variants in a polypeptide of interest expressed from mammalian cells in cell culture, the method comprising:

• propagating or maintaining mammalian cells in a defined cell culture medium;
• expressing a recombinant protein of interest from the eukaryotic cells;
• measuring intracellular and/or extracellular concentrations of asparagine or one or more asparagine related amino acids in the cell culture or defined cell culture medium;
• and
• correlating the measured concentrations of asparagine or one or more asparagine related amino acids to the presence of asparagine sequence variants in the expressed polypeptide of interest.

A method for monitoring and controlling one or more cell culture parameters, the method comprising:

• measuring one or more cell culture parameters in a cell culture in situ using one or more of freezing point depression, electrochemistry, digital imaging, photometry, or Raman spectroscopy;
• comparing the measured one or more cell culture parameters to a predetermined set point value for the cell culture parameter to determine if the one or more cell culture parameters are within a predetermined threshold range;
• and
• adjusting one or more of the cell culture parameters if a cell culture parameter is determined to be out of the predetermined threshold range.