Method for producing recombinant proteins

Abstract

A method for producing recombinant proteins, preferably fusion proteins comprising an Fc portion of an immunoglobulin molecule, more preferably fusion proteins comprising an extracellular domain of a tumor necrosis factor receptor fused to the Fc portion of an immunoglobulin molecule, is disclosed. The method of the present invention allows a reduction of misfolding of the protein, thereby giving rise to a higher yield of the desired protein.

1 . A method for production of recombinant protein comprising culturing a recombinant mammalian host cell which encodes and expresses said recombinant protein so as to produce said recombinant protein, and obtaining the recombinant protein so produced, wherein during a production phase in serum-free medium, said host cell is cultured at a temperature of 25-34° C., wherein said method results in reduction in disulfide scrambling in the recombinant protein produced compared to when carrying out the production phase at a temperature of 37° C., wherein said recombinant protein is a fusion protein of a first protein selected from the group consisting of FLT3L, CD40L, TRAIL and IL-1R type II, and a second protein comprising an Fc portion of an immunoglobulin molecule.  
     
     
         2 . The method of  claim 1 , wherein said immunoglobulin molecule is selected from the group consisting of IgG, IgM, IgA and IgE.  
     
     
         3 . The method of  claim 2 , wherein said immunoglobulin molecule is IgG.  
     
     
         4 . The method of  claim 3 , wherein said immunoglobulin molecule is IgG 1  or IgG 3 .  
     
     
         5 . The method of  claim 1 , wherein during the production phase, said host cell is cultured at a temperature of 25-30° C.  
     
     
         6 . The method of  claim 5 , wherein during the production phase, said host cell is cultured at a temperature of 26-29° C.  
     
     
         7 . The method of  claim 6 , wherein during the production phase, said host cell is cultured at a temperature of 27-28° C.  
     
     
         8 . The method of  claim 7 , wherein during the production phase, said host cell is cultured at a temperature of 28° C.  
     
     
         9 . The method of  claim 1 , wherein said production phase is carried out in the presence of an alkanoic acid or salt thereof.  
     
     
         10 . The method of  claim 9 , wherein said alkanoic acid or salt thereof is selected from the group consisting of butyric acid, sodium butyrate or dibutyl cAMP.  
     
     
         11 . The method of  claim 10 , wherein said alkanoic acid or salt thereof is sodium butyrate.  
     
     
         12 . The method of  claim 9 , wherein said alkanoic acid is employed in a concentration of 0.1-5.0 mM.  
     
     
         13 . The method of  claim 12 , wherein said alkanoic acid is employed in a concentration of 0.25-2.0 mM.  
     
     
         14 . The method of  claim 1 , wherein said production phase is carried out at an osmolality of 200-400 mOsm.  
     
     
         15 . The method of  claim 14 , wherein said osmolality is 240-350 mOsm.  
     
     
         16 . The method of  claim 1 , wherein said production phase is carried out at a pH of about 6.6-7.4.  
     
     
         17 . The method of  claim 16 , wherein said pH is about 6.8-7.0.  
     
     
         18 . The method of  claim 1 , wherein said host cell is a CHO cell.  
     
     
         19 . The method of  claim 18 , wherein said CHO cell is a dhfr −  CHO cell.  
     
     
         20 . The method of  claim 1 , wherein said production phase is preceded by a transition phase where the temperature is lowered from about 37° C. to about 28-34° C.  
     
     
         21 . The method of  claim 1 , wherein said production phase is preceded by a transition phase where an alkanoic acid is added to the culture.  
     
     
         22 . The method of  claim 1 , wherein said production phase is carried out as a batch or fed batch.  
     
     
         23 . The method of  claim 22 , wherein said production phase is carried out in the presence of 0.1-5.0 mM of an alkanoic acid or salt thereof and an osmolality of 200-400 mOsm.  
     
     
         24 . The method of  claim 22 , wherein said production phase is preceded by a transition phase where the temperature is lowered from about 37° C. to about 28-34° C.  
     
     
         25 . The method of  claim 24 , wherein said transition phase is cultured in serum-free medium.  
     
     
         26 . The method of  claim 1 , wherein said production phase is preceded by a growth phase, wherein said growth phase is cultured in serum-free medium.  
     
     
         27 . The method of  claim 1 , wherein said culturing is carried out in the absence of growth hormone.