pH ADJUSTMENT TO IMPROVE THAW RECOVERY OF CELL BANKS

Abstract

Provided herein are methods of freezing mammalian cells for storage or improving thaw recovery of cell banks comprising freezing mammalian cells in a freezing medium having a pH of 6.7 to 8.5.

1 - 48 . (canceled) 
     
     
         49 : A method of freezing mammalian cells for storage comprising (a) adjusting pH of a freezing medium to a pH of 7.5 to 8.5, wherein the freezing medium comprises a buffered solution and a cryoprotective agent; (b) combining the mammalian cells with the freezing medium to form a cell pool; and (c) freezing the mammalian cells in the cell pool. 
     
     
         50 . (canceled) 
     
     
         51 : The method of  claim 49 , wherein the pH is adjusted to a pH of 7.5. 
     
     
         52 : The method of  claim 49 , wherein the adjusted pH is a target pH or a measured pH. 
     
     
         53 : The method of  claim 52 , wherein the target pH is 7.7 to 8.3. 
     
     
         54 : The method of  claim 52 , wherein the measured pH is 7.7 to 8.3. 
     
     
         55 : The method of  claim 49 , further comprising a step of measuring the adjusted pH of the freezing medium. 
     
     
         56 : The method of  claim 55 , wherein if the measured pH of the freezing medium is below a target pH, repeating the adjusting step and measuring step until the adjusted pH of the freezing medium is 7.5 to 8.5. 
     
     
         57 : The method of  claim 49 , wherein the pH is adjusted by adding a base. 
     
     
         58 : The method of  claim 57 , wherein the base is selected from the group consisting of sodium carbonate, sodium bicarbonate, HEPES sodium salt, sodium hydroxide, and potassium hydroxide. 
     
     
         59 : The method of  claim 49 , wherein the mammalian cells are in a medium having a pH of about 6.2 to about 6.6 before the mammalian cells are combined with the freezing medium. 
     
     
         60 : The method of  claim 49 , wherein the cryoprotective agent is DMSO, glycerol, propanediol, ethylene glycol, or a sugar. 
     
     
         61 : The method of  claim 60 , wherein the cryoprotective agent is DMSO or glycerol, and wherein the DMSO or glycerol in the cell pool is at a concentration of about 5% to about 12.5% by volume. 
     
     
         62 : The method of  claim 61 , wherein the DMSO or glycerol in the cell pool is at a concentration of about 5% to about 10% by volume. 
     
     
         63 : The method of  claim 49 , wherein the cell pool contains the mammalian cells at a cell density of 8% to 28% packed cell volume (PCV). 
     
     
         64 : The method of  claim 49 , further comprising a step of cooling cell culture fluid during cell harvest and concentration process before the mammalian cells are combined with a freezing medium. 
     
     
         65 : The method of  claim 64 , wherein the cell culture fluid is cooled to a temperature at or below about 20° C. 
     
     
         66 : The method of  claim 64 , wherein the cell culture fluid is cooled to a temperature at or below about 10° C. 
     
     
         67 - 81 . (canceled) 
     
     
         82 : The method of  claim 49 , wherein the pH of the freezing medium is adjusted to a pH of 7.7 to 8.3 in step a. 
     
     
         83 : The method of  claim 49 , wherein the pH of the freezing medium is adjusted to a pH of 7.8 to 8.3 in step a. 
     
     
         84 : The method of  claim 49 , wherein the mammalian cell is a CHO cell.