Flocculation method

Abstract

The present invention relates to a method for harvesting recombinant proteins from mammalian cell culture fluid. The method makes use of cationic polymers, non-ionic polymers and non-ionic surfactants.

What is claimed is: 
     
         1 . A mammalian cell culture harvest method comprising culturing mammalian cells expressing a recombinant protein in a cell culture medium for a predetermined time or until a desired cell density and/or packed cell volume is achieved,
 adding a cationic polymer and a non-ionic polymer to the cell culture medium initiating flocculation,   mixing the cell culture medium during flocculation,   allowing the flocculent to settle for a primary settle, and   recovering the primary clarified supernatant.   
     
     
         2 . The mammalian cell culture harvest method according to  claim 1 , wherein the cationic polymer is poly diallyldimethyammonium chloride. 
     
     
         3 . The mammalian cell culture harvest method according to  claim 1 , wherein the non2-ionic polymer is selected from poly ethyleneglycol and dextran. 
     
     
         4 . The mammalian cell culture harvest method according to  claim 1 , wherein the non-ionic polymer is selected from PEG 3,000 and PEG 6,000. 
     
     
         5 . The mammalian cell culture harvest method according to  claim 1 , further comprising adding a non-ionic surfactant to the cell culture medium. 
     
     
         6 . The mammalian cell culture harvest method according to  claim 5 , wherein the non-ionic surfactant is Triton X-100. 
     
     
         7 . The mammalian cell culture harvest method according to  claim 1 , further comprising
 washing the primary settle flocculent,   allowing the washed flocculent to settle for a secondary settle, and   recovering the secondary clarified supernatant.   
     
     
         8 . The method according to  claim 1 , where the cationic polymer and the non-ionic polymer are added simultaneously. 
     
     
         9 . The method according to  claim 1 , where the cationic polymer is added first and mixed for at least 30 seconds followed by addition of the non-ionic polymer. 
     
     
         10 . The method according to  claim 1 , where the cationic polymer, the non-ionic polymer and a non-ionic surfactant are added simultaneously. 
     
     
         11 . The method according to  claim 1 , where the cationic polymer is added first and mixed for at least 30 seconds followed by addition of the non-ionic polymer and a non-ionic surfactant. 
     
     
         12 . The method according to  claim 1 , wherein the cationic polymer is a polymer of diallyldimethylammonium chloride, poly diallyldimethyammonium chloride, polyethyleneimine, polyacrylamide or chitosan. 
     
     
         13 . The method according to  claim 1 , wherein the non-ionic polymer is polyethylene glycol or a dextran. 
     
     
         14 . The method according to  claim 5 , wherein the non-ionic surfactant is Sapoin or Triton X100. 
     
     
         15 . The method according to  claim 2 , wherein the poly diallyldimethyammonium chloride is added at a concentration of at or about 20 to at or about 90 pg/total cell density. 
     
     
         16 . The method according to  claim 2 , wherein the poly diallyldimethyammonium chloride is added at a concentration of at or about 25 pg/total cell density wherein the mammalian cells originate from a diploid cell line. 
     
     
         17 . The method according to  claim 2 , wherein the poly diallyldimethyammonium chloride is added between 43 pg/total cell density and 57 pg/total cell density wherein the mammalian cells originate from a tetraploid cell line. 
     
     
         18 . The method according to  claim 4 , wherein the concentration of PEG 3,000 is at or about 3% to at or about 4.5%. 
     
     
         19 . The method according to  claim 4 , wherein the concentration of PEG 6,000 is at or about 2.5% to at or about 3.5%. 
     
     
         20 . The method according to  claim 6 , wherein the concentration of Triton X100 is 0.05% (w/v). 
     
     
         21 . The method according to  claim 1 , wherein the mammalian cell culture medium is between 36° C. and 20° C. 
     
     
         22 . The method according to  claim 1 , wherein the mammalian cell culture medium is at or above 20° C. 
     
     
         23 . The method according to  claim 7 , wherein the flocculent from the primary settle is washed in a 9% sucrose solution.