Cho-k1 cell line

Abstract

The current invention reports a CHO-K1 cell, characterized in that said CHO-K 1 cell is derived from CHO-K1 cell deposited as ATCC CCL-61, grows in suspension, requires no glutamine, no insulin, and no growth factors in the cultivation medium for growth, whereby said CHO-K1 cell is not modified compared to the deposited CHO-K1 cell ATCC CCL-61 cell line by the introduction, deletion, or inactivation of a nucleic acid. Also reported are a method for obtaining said CHO-K1 cell and a method for the production of a heterologous polypeptide using such a CHO-K1 cell according to the invention.

1 .- 11 . (canceled) 
   
   
       12 . A CHO-K1-Gln(−) cell, derived from the CHO-K1 cell deposited as ATCC CCL-61. 
   
   
       13 . The CHO-K1-Gln(−) cell of  claim 12 , wherein the CHO-K1-Gln(−) cell requires no glutamine, no insulin, and no growth factors in the cultivation medium for growth, and wherein further said CHO-K1-Gln(−) cell is not genetically modified by molecular biological methods compared to the parent CHO-K1 cell (ATCC CCL-61) by the introduction, deletion, or inactivation of a nucleic acid encoding an enzyme not required for the expression, secondary modification, or section of a heterologous polypeptide. 
   
   
       14 . A method for the generation of the CHO-K1-Gln(−) cell according to claim  1 , comprising:
 a) providing a CHO-K1 cell (ATCC CCL-61),   b) adapting said CHO-K1 cell to growth in suspension in a polypeptide-free, chemical defined medium supplemented with glutamine hypoxanthine and thymidine,   c) adapting said CHO-K1 cell adapted in step b) to growth in suspension in a polypeptide-free, chemical defined medium supplemented with hypoxanthine and thymidine, thereby obtaining said CHO-K1-Gln(−) cell,
 whereby the CHO-K1-Gln(−) cell grows on a cultivation medium that is a polypeptide-free, chemical defined medium not containing glutamine, insulin or growth factors, and 
 whereby said CHO-K1-Gln(−) cell is not genetically modified by the introduction, deletion or inactivation of a nucleic acid via molecular biological methods. 
   
   
   
       15 . The CHO-K1-Gln(−) cell obtained by the method of  claim 14 . 
   
   
       16 . A method for the recombinant production of an immunoglobulin in large scale comprising:
 a) providing a CHO-K1 cell according to  claim 12 ,   b) providing one or more nucleic acids encoding said immunoglobulin,   c) transfecting said CHO-K1 cell of a) with said one or more nucleic acids,   d) cultivating said transfected CHO-K1 cell of step c) in a polypeptide-free, chemical defined medium not containing glutamine, insulin or a growth factor in large scale, wherein further the ammonium concentration in the cultivation medium is below 0.12 mmol/L during the cultivation,   e) recovering said heterologous polypeptide from the cultivation medium of said CHO-k1 cell or the CHO-K1 cell, and optionally   f) purifying said heterologous polypeptide by one or more chromatographic steps.   
   
   
       17 . The method according to  claim 16 , characterized in that a Protein A affinity chromatography is followed by one or two additional ion exchange chromatography steps. 
   
   
       18 . The method of  claim 17 , characterized in that the method beginning with the first transfection and ending with the recovery of the heterologous polypeptide performed in the same medium. 
   
   
       19 . The method of  claim 16 , characterized in that the large scale cultivation is performed in a volume of at least 500 l. 
   
   
       20 . The method according to  claim 19 , characterized in that the cultivation of said transfected CHO cell is performed in the presence of eukaryotic selection agent(s) in a volume of less than 500 liter and the cultivation of said transfected CHO cell is performed in the absence of eukaryotic selection agent(s) in a volume of 500 liter or more and the recovering of the heterologous polypeptide is from the cultivation medium without said eukaryotic selection agents.