Cell culture methods and media comprising n-acetylcysteine

Abstract

This application provides improved cell culture media and cell culture methods comprising N-acetylcysteine. These improved cell culture media and cell culture methods increase cell viability, cellular growth rate and/or reduce cell doubling time of cholesterol auxotrophic cells, myeloma cells, and hybridoma cells.

What is claimed is: 
     
         1 . A cell culture method comprising:
 a. providing a cell culture medium sufficient to support cell growth, wherein the cell culture medium comprises N-acetylcysteine; and
 culturing a cell in the cell culture medium, wherein the cell is a cholesterol auxotroph, a myeloma, or a hybridoma. 
   
     
     
         2 . A method of increasing cell viability comprising:
 a. providing a cell culture medium sufficient to support cell growth, wherein the cell culture medium comprises N-acetylcysteine; and   b. culturing a cell in the cell culture medium, wherein the cell is a cholesterol auxotroph, a myeloma, or a hybridoma.   
     
     
         3 . A method of increasing cell growth rate comprising:
 a. providing a cell culture medium sufficient to support cell growth, wherein the cell culture medium comprises N-acetylcysteine; and   b. culturing a cell in the cell culture medium, wherein the cell is a cholesterol auxotroph, a myeloma, or a hybridoma.   
     
     
         4 . A method of reducing cell doubling time comprising:
 a. providing a cell culture medium sufficient to support cell growth, wherein the cell culture medium comprises N-acetylcysteine; and   b. culturing a cell in the cell culture medium, wherein the cell is a cholesterol auxotroph, a myeloma, or a hybridoma.   
     
     
         5 . The method of any one of  claims 1 - 4 , wherein the cells are thawed from a frozen stock. 
     
     
         6 . The method of any one of  claims 1 - 5 , wherein the cells are in an expansion phase. 
     
     
         7 . The method of any one of  claims 1 - 6 , wherein the cell culture medium is a serum free and animal-protein free medium. 
     
     
         8 . The method of any one of  claims 1 - 7 , wherein the cell culture medium is a chemically-defined medium. 
     
     
         9 . The method of any one of  claims 1 - 8 , wherein the medium comprises lipids. 
     
     
         10 . The method of any one of  claims 1 - 9 , wherein the cells are derived from a mammal. 
     
     
         11 . The method of  claim 10 , wherein the mammalian cells are murine, hamster, rat, monkey, or human. 
     
     
         12 . The method of  claim 10 , wherein the cells are selected from the group consisting of: NS0, NS1, U937, M19, SRD-12B, SRD-13A, CHO-215, X63Ag8, Sp2/0, J558L, U266, P3U1, XG-1, XG-2, XG-3, XG-4, XG-5, XG-6, XG-7, XG-8, XG-9, U266, RPM1-8226, LP1, L363, OPM1, OPM2, and NCLH929 cells. 
     
     
         13 . The method of  claim 12 , wherein the cells are derived from NS0, NS1, U937, M19, SRD-12B, SRD-13A, CHO-215, X63Ag8, Sp2/0, J558L, U266, P3U1, XG-1, XG-2, XG-3, XG-4, XG-5, XG-6, XG-7, XG-8, XG-9, U266, RPM1-8226, LP1, L363, OPM1, OPM2, or NCLH929 cells. 
     
     
         14 . The method of  claim 12 , wherein the cells are NS0 cells. 
     
     
         15 . The method of any of  claims 1 - 12 , wherein the cells are engineered to be a cholesterol auxotroph. 
     
     
         16 . The method of  claim 1 - 15 , wherein the cell culture medium comprises: a carbon source, essential and non-essential amino acid sources, vitamins, inorganic salts, trace metals, pH buffers, surfactants, antioxidants, lipids, and cholesterol. 
     
     
         17 . The method of any one of  claims 1 - 16 , wherein the cell culture medium comprises N-acetylcysteine at a concentration of from about 0.25 mM to about 3 mM. 
     
     
         18 . The method of any one of  claims 1 - 17 , wherein the cell culture medium comprises N-acetylcysteine at a concentration of from about 0.5 to about 2.5 mM 
     
     
         19 . The method of any one of  claims 1 - 18 , wherein the cell culture medium comprises N-acetylcysteine at a concentration of from about 1.0 to about 1.5 mM. 
     
     
         20 . The method of any one of  claims 1 - 19 , wherein the cell culture medium comprises N-acetylcysteine at a concentration about 1 mM or about 1.5 mM. 
     
     
         21 . The method of any one of  claims 1 - 20 , wherein the cell culture medium comprises yeastolate. 
     
     
         22 . The method of  claim 21 , wherein the cell culture medium comprises 1 g/L of yeastolate. 
     
     
         23 . The method of any one of  claims 1 - 22 , wherein the average doubling time is shorter than in a cell culture with a control medium excluding N-acetylcysteine. 
     
     
         24 . The method of  claim 23 , wherein the average doubling time is reduced by at least about 10% compared to a cell culture medium without N-acetylcysteine. 
     
     
         25 . The method of  claim 23 , wherein the average doubling time is reduced by at least 15% compared to a cell culture medium without N-acetylcysteine. 
     
     
         26 . The method of  claim 23 , wherein the average doubling time is reduced by at least 20% compared to a cell culture medium without N-acetylcysteine. 
     
     
         27 . The method of  claim 23 , wherein the average doubling time is reduced by at least 25% compared to a cell culture medium without N-acetylcysteine. 
     
     
         28 . The method of  claim 23 , wherein the average doubling time is reduced by at least 50% compared to a cell culture medium without N-acetylcysteine. 
     
     
         29 . The method of  claim 23 , wherein the average cell doubling time is 60 hours or less. 
     
     
         30 . The method of  claim 23 , wherein the average doubling time is 42 hours or less. 
     
     
         31 . The method of  claim 23 , wherein the average doubling time is 34 hours or less. 
     
     
         32 . The method of  claim 23 , wherein the average doubling time is 30 hours or less 
     
     
         33 . The method of  claim 23 , wherein the average doubling time is about 29 hours or less. 
     
     
         34 . The method of any one of  claims 1 - 33 , wherein the cell viability is increased over a cell culture with a control medium excluding N-acetylcysteine. 
     
     
         35 . The method of  claim 34 , wherein the cell viability is increased by at least about 5% compared to a cell culture medium without N-acetylcysteine. 
     
     
         36 . The method of  claim 34 , wherein the cell viability is increased by at least 7% compared to a cell culture medium without N-acetylcysteine. 
     
     
         37 . The method of  claim 34 , wherein the cell viability is increased by at least 10% compared to a cell culture medium without N-acetylcysteine. 
     
     
         38 . The method of  claim 34 , wherein the cell viability is at least about 90%. 
     
     
         39 . The method of  claim 34 , wherein the cell viability is at least 92%. 
     
     
         40 . The method of  claim 34 , wherein the cell viability is at least 93%. 
     
     
         41 . The method of any one of  claims 1 - 40 , wherein the cells do not express a heterologous protein. 
     
     
         42 . The method of any one of  claims 1 - 40 , wherein the cells express a heterologous protein. 
     
     
         43 . The method of any one of  claims 1 - 40  and  42 , wherein the cells are transformed with a heterologous nucleic acid. 
     
     
         44 . The method of  claim 43 , wherein the heterologous nucleic acid is cDNA, a vector, a plasmid, a nucleic acid operably linked to a promoter, and/or a nucleic acid that incorporates into the genome. 
     
     
         45 . The method of  claim 42 , wherein the heterologous protein is transiently expressed. 
     
     
         46 . The method of  claim 42 , wherein the heterologous protein is stably expressed. 
     
     
         47 . The method of any one of  claim 42 ,  45 , or  46 , wherein the heterologous protein is an antibody or antigen-binding fragment thereof. 
     
     
         48 . The method of  claim 47 , wherein the antibody or antigen-binding fragment thereof is an IL-13 antibody. 
     
     
         49 . The method of  claim 48 , wherein the antibody is BAK502G9 or an antigen-binding fragment thereof. 
     
     
         50 . The method of  claim 48 , wherein the antibody or antigen binding fragment has a heavy chain variable region comprising a sequence that is at least about 80% identical to any one of SEQ ID NOs: 1, 9, 17, or 25 and a light chain variable region comprising a sequence that is at least about 80% identical to any one of SEQ ID NOs: 2, 10, 18, or 26. 
     
     
         51 . The method of  claim 48 , wherein the antibody or antigen binding fragment comprising:
 a. a heavy chain variable region comprising:
 i. a HC CDR1 that has one mutation compared to a sequence chosen from SEQ ID NOs: 3, 11, 19, or 27; 
 ii. a HC CDR2 that has one or two mutations compared to a sequence chosen from SEQ ID NOs: 4, 12, 20, and 28; and 
 iii. a HC CDR3 that has one or two mutations compared to a sequence chosen from SEQ ID NOs: 5, 13, 21, and 29; and 
   b. a light chain variable region comprising:
 i. a LC CDR1 that has one mutation compared to a sequence chosen from SEQ ID NOs: 6, 14, 22, and 30; 
 ii. a LC CDR2 that has one or two mutations compared to a sequence chosen from SEQ ID NOs: 7, 15, 23, and 31; and 
 iii. a LC CDR3 that has one or two mutations compared to a sequence chosen from SEQ ID NOs: 8, 16, 24, and 32. 
   
     
     
         52 . The method of any one of  claims 1 - 52 , wherein the cell is a cholesterol auxotroph. 
     
     
         53 . The method of any one of  claims 1 - 53 , wherein the cell is a myeloma cell. 
     
     
         54 . The method of any one of  claims 1 - 53 , wherein the cell is a hybridoma cell. 
     
     
         55 . A cell culture medium comprising N-acetylcysteine, a carbohydrate source, an amino acid source, and a cholesterol source. 
     
     
         56 . The cell culture medium of  claim 55 , wherein the carbohydrate source and the amino acid source are different. 
     
     
         57 . The cell culture medium of any one of  claims 55 - 56 , wherein the cell culture medium is a serum free and animal-protein free medium. 
     
     
         58 . The cell culture medium of any one of  claims 55 - 57 , wherein the cell culture medium is a chemically-defined medium. 
     
     
         59 . The cell culture medium of any one of  claims 55 - 58 , wherein the medium further comprises lipids. 
     
     
         60 . The cell culture medium of any one of  claims 55 - 59 , wherein the cell culture medium comprises N-acetylcysteine at a concentration of from about 0.25 mM to about 3 mM. 
     
     
         61 . The cell culture medium of any one of  claims 55 - 59 , wherein the cell culture medium comprises N-acetylcysteine at a concentration of from about 0.5 to about 2.5 mM 
     
     
         62 . The cell culture medium of any one of  claims 55 - 59 , wherein the cell culture medium comprises N-acetylcysteine at a concentration of from about 1.0 to about 1.5 mM. 
     
     
         63 . The cell culture medium of any one of  claims 55 - 59 , wherein the cell culture medium comprises N-acetylcysteine at a concentration of about 1 mM or 1.5 mM. 
     
     
         64 . The cell culture medium of any one of  claims 55 - 63 , wherein the cell culture medium further comprises yeastolate. 
     
     
         65 . The cell culture medium of  claim 64 , wherein the cell culture medium comprises 1 g/L of yeastolate.