Methods for Protein Expression in Mammalian Cells in Serum-Free Medium

Abstract

Disclosed are compositions and methods for increasing the longevity of a cell culture and permitting the increased production of proteins, preferably recombinant proteins, such as antibodies, peptides, enzymes, growth factors, interleukins, interferons, hormones, and vaccines. Cells transfected with an apoptosis-inhibiting gene or vector, such as a triple mutant Bcl-2 gene, can survive longer in culture, resulting in extension of the state and yield of protein biosynthesis. Such transfected cells exhibit maximal cell densities that equal or exceed the maximal density achieved by the parent cell lines. Transfected cells can also be pre-adapted for growth in serum-free medium, greatly decreasing the time required to obtain protein production in serum-free medium. In certain methods, the pre-adapted cells can be used for protein production following transfection under serum-free conditions. In preferred embodiments, the cells of use are SpESF or SpESF-X cells.

1 . A mammalian cell line comprising a gene encoding a mutant Bcl-2 protein, said protein comprising T69E, S70E and S87E amino acid substitutions, the cell line pre-adapted to grow in serum-free medium, the cell line capable of transfection under serum-free conditions with one or more expression vectors expressing a protein of interest and of producing said protein of interest under serum-free conditions. 
     
     
         2 . The cell line of  claim 1 , wherein said cell line is transfected with one or more expression vectors expressing a protein of interest. 
     
     
         3 . The cell line of  claim 2 , wherein the transfected cell line is capable of producing said protein of interest under serum-free conditions without the need for further adaptation to serum-free conditions after transfection. 
     
     
         4 . The cell line of  claim 2 , wherein the protein of interest is selected from the group consisting of an antibody, a humanized antibody, a chimeric antibody, a human antibody, a bispecific antibody, a multispecific antibody, a multivalent antibody and an antigen-binding antibody fragment. 
     
     
         5 . The cell line of  claim 1 , wherein the cell line is frozen. 
     
     
         6 . The cell line of  claim 1 , wherein the pre-adapted cell line may be frozen and thawed prior to transfection with one or more expression vectors expressing a protein of interest. 
     
     
         7 . The cell line of  claim 2 , wherein said transfected cell line produces the protein of interest at a yield of at least 150 mg protein/mL of growth medium. 
     
     
         8 . The cell line of  claim 7 , wherein said transfected cell line produces the protein of interest at a yield of at least 200 mg protein/mL of growth medium. 
     
     
         9 . The cell line of  claim 2 , wherein said cell line is stably transfected with the one or more expression vectors. 
     
     
         10 . The cell line of  claim 1 , wherein said cell line is a myeloma cell line. 
     
     
         11 . The cell line according to  claim 10 , wherein said myeloma cell line is an Sp2/0 cell line or derivative thereof. 
     
     
         12 . A protein produced by a cell line comprising a gene encoding a mutant Bcl-2 protein, said mutant Bcl-2 protein comprising T69E, S70E and S87E amino acid substitutions, the cell line pre-adapted to grow in serum-free medium, the cell line capable of transfection under serum-free conditions with one or more expression vectors expressing a protein of interest and of producing said protein of interest under serum-free conditions, wherein said cell line is transfected with one or more expression vectors expressing a protein of interest according to  claim 2 . 
     
     
         13 . The protein of  claim 12 , wherein the protein is selected from the group consisting of an antibody, a humanized antibody, a chimeric antibody, a human antibody, a bispecific antibody, a multispecific antibody, a multivalent antibody and an antigen-binding antibody fragment. 
     
     
         14 . The protein of  claim 12 , wherein the protein is selected from the group consisting of a growth factor, a hormone, a cytokine, a chemokine, an interleukin, an interferon, a vaccine and an enzyme. 
     
     
         15 . The protein of  claim 14 , wherein the protein is selected from the group consisting of EPO, G-CSF, GM-CSF, EGF, VEGF, thrombopoietin, IL-1 through IL-31, interferon-alpha, interferon-beta and interferon-gamma. 
     
     
         18 . The protein of  claim 12 , wherein the protein is produced in serum-free medium. 
     
     
         19 . The protein of  claim 18 , wherein the cell line is transfected under serum-free conditions and the protein is produced in serum-free medium, without further adaptation to serum-free conditions after transfection. 
     
     
         20 . The protein of  claim 12 , wherein the protein is produced by the transfected cell line at a yield of at least 150 mg protein/mL of growth medium. 
     
     
         21 . The protein of  claim 12 , wherein the protein is produced by the transfected cell line at a yield of at least 200 mg protein/mL of growth medium. 
     
     
         22 . The protein of  claim 12 , wherein said cell line is a myeloma cell line. 
     
     
         23 . The protein of  claim 22 , wherein said myeloma cell line is an Sp2/0 cell line or derivative thereof.