Methods for Improving Viability and Productivity in Cell Culture

Abstract

Methods for increasing viability and production of secreted proteins in fed batch eukaryotic cell culture are disclosed.

1 . A method of increasing cell viability in a fed batch mammalian cell culture, comprising culturing a mammalian cell line over-expressing MDM2 D300A . 
     
     
         2 . A method of increasing cell viability in a fed batch mammalian cell culture, comprising culturing a mammalian cell line over-expressing MDM2 D300A  and E1B19K. 
     
     
         3 . The method of  claim 1  or  2  wherein the mammalian cell line is a Chinese Hamster Ovary (CHO) cell line. 
     
     
         4 . The method of  claim 3  wherein the CHO cell line is CHO-K1. 
     
     
         5 . The method of  claim 3  wherein the CHO cell line is CHO-K1SV. 
     
     
         6 . The method of  claim 1  or  2  wherein the mammalian cell line is a myeloma cell line. 
     
     
         7 . The method of  claim 6  wherein the myeloma cell line is NS0. 
     
     
         8 . The method of  claim 6  wherein the myeloma cell line is Sp2/0. 
     
     
         9 . The method of  claim 1  or  2  wherein the mammalian cell line is a hybridoma cell line. 
     
     
         10 . The method of  claim 1  or  2  wherein the cell viability is at least 50% at 14 days of the fed batch cell culture. 
     
     
         11 . The method of  claim 1  or  2  wherein the cell viability is at least 40% at 20 days of the fed batch cell culture. 
     
     
         12 . The method of  claim 1  or  2  wherein the viable cell density is at least 4×10 6  cells/mL at 15 days of the fed batch cell culture. 
     
     
         13 . A method of increasing production of a secreted protein in a Chinese Hamster Ovary (CHO) fed batch cell culture, comprising culturing a CHO cell line over-expressing MDM2 D300A  and one or more genes encoding the secreted protein. 
     
     
         14 . A method of increasing production of a secreted protein in a Chinese Hamster Ovary (CHO) fed batch cell culture, comprising culturing a CHO cell line over-expressing MDM2 D300A  and E1B19K and one or more genes encoding the secreted protein. 
     
     
         15 . A method of increasing production of a secreted protein in a Chinese Hamster Ovary (CHO) fed batch cell culture, comprising culturing a CHO cell line over-expressing MDM2 and E1B19K and one or more genes encoding the secreted protein. 
     
     
         16 . The method of  claims 13  to  15  wherein the titer of the secreted protein is at least 600 mg/L. 
     
     
         17 . The method of  claims 13  to  15  wherein the CHO cell line is CHO-K1. 
     
     
         18 . The method of  claims 13  to  15  wherein the CHO cell line is CHO-K1SV. 
     
     
         19 . The method of  claim 13  to  15  wherein the secreted protein is an antibody heavy chain and an antibody light chain. 
     
     
         20 . An isolated polynucleotide encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4. 
     
     
         21 . The isolated polynucleotide of  claim 19  having the sequence shown in SEQ ID NO: 3 or a complementary sequence thereof. 
     
     
         22 . A vector comprising an isolated polynucleotide having the sequence shown in SEQ ID NO: 3. 
     
     
         23 . The vector of  claim 21  that is an expression vector. 
     
     
         24 . An isolated host cell comprising the vector of  claim 21 . 
     
     
         25 . An isolated polypeptide comprising a polypeptide having the sequence shown in SEQ ID NO: 4. 
     
     
         26 . A method for expressing a polypeptide comprising the steps of:
 a. providing the host cell of  claim 23 ; and   b. culturing the host cell under conditions sufficient for the expression of the polypeptide comprising the sequence shown in SEQ ID NO: 4.