Method for the Massive Culture of Cells Producing Recombinant Human Erythropoietin

Abstract

The present invention relates, in general, to a method for the massive culture of recombinant mammalian cells for the production of recombinant human erythropoietin (EPO) in culture medium containing insulin. The present invention also refers to a method of producing EPO and to the EPO thus produced.

1 - 12 . (canceled) 
     
     
         13 . A method for preparing a lyophilized recombinant human erythropoietin composition comprising:
 (i) providing a composition comprising:
 (a) recombinant human erythropoietin; 
 (b) mannitol; 
 (c) two or more salts selected from the group consisting of: NaCl, NaH 2 PO 4 , and Na 2 HPO 4 , and mixtures thereof; and 
 (d) human albumin; 
   (ii) incubating the composition at a temperature equal to or less than −30° C. and at atmospheric pressure;   (iii) exposing the composition to a pressure equal to or less than 30 microns; and   (iv) raising the temperature of the composition at a rate equal to or less than 3° C. per hour until reaching a temperature of at least 25° C. while maintaining a pressure equal to or less than 5 microns.   
     
     
         14 . The method of  claim 13 , wherein the composition comprises:
 (a) recombinant human erythropoietin;   (b) mannitol;   (c) NaCl, NaH 2 PO 4 , and Na 2 HPO 4 ; and   (d) human albumin.   
     
     
         15 . The method of  claim 13 , wherein the composition of (ii) is incubated for a time equal to or greater than 4 hours. 
     
     
         16 . The method of  claim 13 , wherein the composition of (iii) is exposed for a time equal to or greater than 1 hour. 
     
     
         17 . The method of  claim 13 , wherein the composition of (ii) is incubated for a time equal to or greater than 4 hours, and wherein the composition of (iii) is exposed for a time equal to or greater than 1 hour. 
     
     
         18 . A pharmaceutical composition made by the method of  claim 13 . 
     
     
         19 . A pharmaceutical composition for lyophilization comprising:
 (a) recombinant human erythropoietin;   (b) mannitol;   (c) two or more salts selected from the group consisting of: NaCl, NaH 2 PO 4 , and Na 2 HPO 4 , and mixtures thereof; and   (d) human albumin.   
     
     
         20 . The pharmaceutical composition of  claim 19 , wherein the composition comprises NaCl, NaH 2 PO 4 , and Na 2 HPO 4 . 
     
     
         21 . A pharmaceutical composition for lyophilization comprising:
 (a) recombinant human erythropoietin;   (b) mannitol;   (c) NaCl, NaH 2 PO 4 , and Na 2 HPO 4 ; and   (d) human albumin.