Use of flow-cytrometric analysis to optimize cell banking strategies for cho cells

Abstract

Production of biopharmaceuticals from CHO cells requires generation of master-, working- and post-production cell banks of high quality, partly under GMP conditions. An optimal cryopreservation strategy is needed for each new production cell line, particularly with regard to the desire to establish production processes that are completely devoid of serum or even any animal components and to ensure robust thaw performance for reliable production. Here we describe a novel strategy employing flow cytometric (FC) analysis of Annexin V-stained cells for high-throughput characterization of CHO cell banks. Our data show that this method enables evaluation of a cryopreservation procedure just 6 h after thaw.

1 - 8 . (canceled) 
   
   
       9 . A method for establishing the vitality rate of a cryopreserved CHO cell bank, the method comprising staining thawed CHO cells from the cryopreserved CHO cell bank with Annexin V. 
   
   
       10 . The method of  claim 9 , further comprising:
 quantifying the number of thawed CHO cells that binds to Annexin V;   quantifying the number of thawed CHO cells that do not bind to Annexin V; and   calculating the ratio of Annexin V-binding thawed CHO cells to Annexin V-non-binding thawed CHO cells.   
   
   
       11 . The method of  claim 10 , wherein Annexin V is incubated with the thawed CHO cells within 24 hours post-thaw. 
   
   
       12 . The method of  claim 10 , wherein Annexin V is incubated with the thawed CHO cells within 6 hours post-thaw. 
   
   
       13 . The method of  claim 10 , wherein the Annexin V is labeled with fluorescein isothiocyanate (FITC). 
   
   
       14 . The method of  claim 10 , wherein the cryopreserved cell bank of CHO cells comprises CHO cells that are cryopreserved into parts in a liquid medium. 
   
   
       15 . The method of  claim 10 , wherein the thawed CHO cells are thawed from a portion of the cryopreserved cell bank. 
   
   
       16 . The method of  claim 10 , wherein the thawed CHO cells are cultured before being incubated with Annexin V. 
   
   
       17 . (canceled) 
   
   
       18 . (canceled)