Rationally designed media for cell culture

Abstract

This invention relates to methods for rationally designing cell culture media for use in cell cultures, e.g., cell cultures employed in polypeptide production; cell culture media designed with the disclosed methods; methods of producing a polypeptide of interest, e.g., an antibody, using such media; polypeptides produced using the methods and media disclosed herein; and pharmaceuticals compositions containing such polypeptides. The rationally designed media contain a concentration of an amino acid that is calculated for use in cell mass, a concentration of the amino acid that is calculated for use in cell maintenance, and a concentration of the amino acid that is calculated for incorporation into the polypeptide of interest.

1 . A method of producing a polypeptide in a cell culture comprising:
 (1) providing a cell culture, comprising:
 a. cells, comprising a nucleic acid encoding a polypeptide of interest; and 
 b. a desired cell culture medium, comprising a concentration of an amino acid that is calculated for use in cell mass, a concentration of the amino acid that is calculated for use in cell maintenance, and a concentration of the amino acid that is calculated for incorporation into the polypeptide of interest; and 
   (2) maintaining the cell culture under conditions that allow expression of the polypeptide of interest.   
     
     
         2 . The method of  claim 1 , wherein both the concentration of
 an amino acid that is calculated for use in cell mass and the concentration of the amino acid that is calculated for incorporation into the polypeptide of interest are multiplied by a baseline factor.   
     
     
         3 . The method of  claim 1 , wherein the desired cell culture medium, comprises a baseline adjusted
 amino acid concentration, A, according to the formula A=[(M*X)+(N*P)+(Y*M*X)]*F, wherein X is a concentration of the amino acid that is used per unit of cell mass, P is a concentration of the amino acid that is used for incorporation into the polypeptide of interest per unit of polypeptide titer, M is a multiplier for a desired peak cell density of the cell culture, N is a multiplier for a desired concentration of the polypeptide of interest, Y is a cell maintenance factor, and F is a baseline factor.   
     
     
         4 - 5 . (canceled) 
     
     
         6 . The method of  claim 1 , wherein the method comprises providing a starting cell culture medium, wherein the volume of the
 starting cell culture medium is about 60-99% of the volume of a desired cell culture medium volume; and   providing a feeding cell culture medium to the cell culture, wherein the volume of the feeding cell culture medium is about 1-40% of the desired cell culture medium volume, and wherein the resulting desired cell culture medium comprises the concentration of an amino acid that is calculated for use in cell mass, the concentration of the amino acid that is calculated for use in cell maintenance, and the concentration of the amino acid that is calculated for incorporation into the polypeptide of interest.   
     
     
         7 . The method of  claim 6 , wherein the resulting desired cell culture medium comprises a
 baseline-adjusted amino acid concentration, A, according to the formula A=[(M*X)+(N*P)+(Y*M*X)]*F, wherein X is a concentration of the amino acid that is used per unit of cell mass, P is a concentration of the amino acid that is used for incorporation into the polypeptide of interest per unit of polypeptide titer, M is a multiplier for a desired peak cell density of the cell culture, N is a multiplier for a desired concentration of the polypeptide of interest, Y is a cell maintenance factor, and F is a baseline factor.   
     
     
         8 . The method of  claim 7 , wherein the starting cell culture medium comprises a concentration, B, of the amino acid according to the formula B=[A−(Z*V)]/(1−V), wherein Z is a concentration of the amino acid in the feeding cell culture medium, and V is a volume of the feeding culture medium as a proportion of the desired cell culture medium volume. 
     
     
         9 . The method of  claim 3 , wherein Y is 0 to about 1.5. 
     
     
         10 . The method of  claim 3 , wherein F is about 1 to about 1.5. 
     
     
         11 - 18 . (canceled) 
     
     
         19 . The method of  claim 3 , wherein the combined concentration of leucine, lysine, threonine, and valine in the desired cell culture medium is between about 60% and about 80% of the concentration of the total essential amino acids in the desired cell culture medium. 
     
     
         20 . The method of  claim 3 , wherein the combined concentration of the essential amino acids in the desired cell culture medium is between about 30% and about 50% of the concentration of the total amino acids in the desired cell culture medium. 
     
     
         21 . The method of  claim 3 , wherein the concentration of amino acids in the desired cell culture medium is between about 120 mM and about 350 mM. 
     
     
         22 . The method of  claim 3 , wherein the concentration of proline in the cell culture is maintained at greater than about 1 mM. 
     
     
         23 . The method of  claim 3 , wherein the concentration of proline in the cell culture is maintained at greater than about 2 mM. 
     
     
         24 - 29 . (canceled) 
     
     
         30 . A method of cell culture comprising:
 (1) providing a cell culture, comprising:
 a. cells; and 
 b. a desired cell culture medium, comprising a concentration of an amino acid that is calculated for use in cell mass and a concentration of the amino acid that is calculated for use in cell maintenance; and 
   (2) maintaining the cell culture under conditions that allow growth and replication of the cells in the cell culture.   
     
     
         31 . The method of  claim 30 , wherein the desired cell culture medium, comprises a baseline-adjusted
 amino acid concentration, A′, according to the formula A′=[(M*X)+(Y*M*X)]*F, wherein X is a concentration of the amino acid that is used per unit of cell mass, M is a multiplier for a desired peak cell density of the cell culture, Y is a cell maintenance factor, and F is a baseline factor.   
     
     
         32 - 33 . (canceled) 
     
     
         34 . The cell culture medium of  claim 36 , comprising a total concentration of amino acids from between about 120 mM and about 350 mM. 
     
     
         35 . (canceled) 
     
     
         36 . A cell culture medium for use in the production of a polypeptide of interest, comprising a concentration of an amino acid that is calculated for use in cell mass, a concentration of the amino acid that is calculated for use in cell maintenance, and a concentration of the amino acid that is calculated for incorporation into the polypeptide of interest. 
     
     
         37 . The cell culture medium of  claim 36 , comprising a baseline-adjusted amino acid concentration, AT, according to the formula A′=[(M*X)+(Y*M*X)]*F, wherein X is a concentration of the amino acid that is used per unit of cell mass, M is a multiplier for desired peak cell density of the cell culture, Y is a cell maintenance factor, and F is a baseline factor. 
     
     
         38 . The cell culture medium of  claim 36  for use in the production of a polypeptide of interest, comprising a baseline-adjusted amino acid concentration, A, according to the formula A=[(M*X)+(N*P)+(Y*M*X)]*F, wherein X is a concentration of the amino acid that is used per unit of cell mass, P is a concentration of the amino acid that is used for incorporation into the polypeptide of interest per unit of polypeptide titer, M is a multiplier for desired peak cell density of the cell culture, N is a multiplier for desired concentration of the polypeptide of interest, Y is a cell maintenance factor, and F is a baseline factor. 
     
     
         39 - 41 . (canceled) 
     
     
         42 . A method for determining an optimized concentration of an amino acid used in a cell culture medium for the production of a polypeptide of interest in a cell culture, comprising:
 (1) determining the amino acid concentration required for the cell mass of the cells at a target cell density;   (2) determining the amino acid concentration required to produce the polypeptide of interest at a target polypeptide titer;   (3) determining the amino acid concentration required for cell maintenance of the cells; and   (4) adding the concentrations obtained from step (1), step (2), and step (3) to provide an optimized concentration of the amino acid used in the cell culture medium for the production of the polypeptide of interest.   
     
     
         43 - 45 . (canceled)