Protein expression from multiple nucleic acids

Abstract

The current invention reports a method for the recombinant production of a secreted heterologous immunoglobulin in a CHO cell comprising the following steps: i) providing a CHO cell, which is adapted to growth in suspension culture, adapted to growth in serum-free medium, mycoplasma free, and virus free, ii) providing a vector comprising a prokaryotic origin of replication, a first nucleic add conferring resistance to a prokaryotic selection agent, a second nucleic acid encoding the heavy chain of said heterologous immunoglobulin, a third nucleic acid encoding the light chain of said heterologous immunoglobulin, a fourth nucleic acid conferring resistance to a eukaryotic selection agent, iii) transfecting said CHO cell, wherein said transfecting comprises a) transfecting said CHO cell with said vector comprising a fourth nucleic acid conferring resistance to a first eukaryotic selection agent, h) selecting a CHO cell by growth in cultivation medium containing said first eukaryotic selection agent, c) transfecting said selected CHO cell with said vector comprising a fourth nucleic acid conferring resistance to a second eukaryotic selection agent different to said first eukaryotic selection agent, d) selecting a CHO cell by selected growth in cultivation medium containing said first and said second eukaryotic selection agent, iv) cultivating said transfected CHO cell in a medium in the presence of said first and second eukaryotic selection agent, under conditions suitable for the expression of said second, and third nucleic acid, and v) recovering said secreted heterologous immunoglobulin from the cultivation medium.

1 . A method for the recombinant production of a heterologous immunoglobulin in a CHO cell which is secreted to the cultivation medium comprising:
 a) providing a CHO cell, which is
 adapted to growth in suspension culture, 
 adapted to growth in serum-free medium, 
 mycoplasma free, 
   b) providing a nucleic acid comprising
 a prokaryotic origin of replication, 
 a first nucleic acid sequence conferring resistance to a prokaryotic selection agent, 
 a second nucleic acid sequence encoding the heavy chain of said heterologous immunoglobulin, and a third nucleic acid sequence encoding the light chain of said heterologous immunoglobulin, 
   whereby a first transfection vector is provided which comprises said provided nucleic acid and an additional fourth nucleic acid sequence conferring resistance to a first eukaryotic selection agent,   whereby a second transfection vector is provided which comprises said provided nucleic acid and an additional fourth nucleic acid sequence conferring resistance to a second eukaryotic selection agent, whereby said second eukaryotic selection agent is different to said first eukaryotic selection agent,   c) transfecting said CHO cell, wherein said transfecting comprises the following steps in the following order:
 (i) transfecting said CHO cell with said first transfection vector, 
 (ii) selecting a CHO cell transfected in (i) by selected growth in cultivation medium containing a first eukaryotic selection agent to which the first transfection vector confers resistance, 
 (iii) transfecting said selected CHO cell in (ii) with said second transfection vector, 
 (iv) selecting a CHO cell transfected in (iii) by selected growth in cultivation medium containing said first eukaryotic selection agent to which the first transfection vector confers resistance and said second eukaryotic selection agent to which the second transfection vector confers resistance, 
   d) cultivating said transfected CHO cell in a medium in the presence of said first and said second eukaryotic selection agent, under conditions suitable for the expression of said second, and/or third nucleic acid, and   e) recovering said secreted heterologous immunoglobulin from the cultivation medium and thereby producing a heterologous immunoglobulin in a CHO cell which is secreted to the cultivation medium.   
     
     
         2 . The method of  claim 1 , characterized in that said CHO cell is a CHO K1 cell, or a CHO DG44 cell, or a CHO XL99 cell, a CHO DXB11 cell, and ora CHO DP12 cell. 
     
     
         3 . The method of  claim 1 , characterized in that said second and/or third nucleic acid contains hybrid intronic nucleic acid sequence. 
     
     
         4 . The method of  claim 1 , characterized in that said first transfection vector and said second transfection vector differ only in the nucleic acid conferring resistance to said eukaryotic selection agent. 
     
     
         5 . The method of  claim 1 , characterized in that said method further comprises:
 after step b) a step b1):   b1) providing a nucleic acid comprising
 a prokaryotic origin of replication, 
 a first nucleic acid sequence conferring resistance to a prokaryotic selection agent, 
 a second nucleic acid sequence encoding the heavy chain of said heterologous immunoglobulin, and/or a third nucleic acid sequence encoding the light chain of said heterologous immunoglobulin, 
   whereby a third transfection vector is provided which comprises said provided nucleic acid and an additional fourth nucleic acid sequence conferring resistance to a third eukaryotic selection agent, whereby said third eukaryotic selection agent is different to said first eukaryotic selection agent and is also different to said second eukaryotic selection agent,   and further comprises after step c) (iv) the following steps (v) and (vi):
 (v) transfecting said CHO cell selected in (iv) with said third transfection vector, 
 (vi) selecting a CHO cell transfected in (v) by selected growth in a cultivation medium containing said first eukaryotic selection agent to which the first transfection vector confers resistance and said second eukaryotic selection agent to which the second transfection vector confers resistance and said third eukaryotic selection agent to which the third transfection vector confers resistance, 
   and further comprises in step d) that said medium for cultivating said transfected CHO cell further comprises a third eukaryotic selection agent.   
     
     
         6 . The method of  claim 1 , characterized in that step c) and step d) are performed in the same medium. 
     
     
         7 . The method of  claim 6 , characterized in that said medium is a serum-free medium, or a serum-free medium supplemented with defined animal-derived components, or a animal-derived component free medium, a protein-free medium, a protein-free medium supplemented with defined animal-derived components or a defined protein-free medium, or a chemically defined medium. 
     
     
         8 . The method of  claim 1 , characterized in that in said step d) said cultivating is in the presence of the eukaryotic selection agents in a volume of less than 500 liter and that said cultivating is in the absence of said eukaryotic selection agents in a volume of 500 liter or more, and that said recovering of the secreted heterologous immunoglobulin is from the cultivation medium without said eukaryotic selection agents. 
     
     
         9 . The method of  claim 1 , characterized in that the productivity of said CHO cells is over 40 generations not less than 70% and not more than 130% of the productivity after 10 generations of cultivation as split-batch cultivation. 
     
     
         10 . The method of  claim 1 , characterized in that the productivity of said CHO cell is at least 1.5 g/l of said heterologous immunoglobulin within 21 days as fed-batch cultivation. 
     
     
         11 . The method of  claim 1 , characterized in that said method further comprises:
 f) purifying said heterologous immunoglobulin with one or more chromatographic steps.   
     
     
         12 . The method of  claim 1 , characterized in that in that said transfected CHO cell of step c) has
 a doubling time of 150% or less of the doubling time of the CHO cell selected in substep (ii),   a volumetric yield of at least 125% compared to the volumetric yield of the CHO cell   selected in (ii).   
     
     
         13 . The method of  claim 1 , characterized in that said heterologous immunoglobulin is an anti-Aβ antibody. 
     
     
         14 . The method of  claim 1 , characterized in that said heterologous immunoglobulin is an anti-P-selection antibody. 
     
     
         15 . The method of  claim 1 , characterized in that said heterologous immunoglobulin is an anti-IL-13Rα antibody. 
     
     
         16 . The method of  claim 1 , characterized in that said heterologous immunoglobulin is an anti-CD4 antibody conjugate. 
     
     
         17 . A CHO cell obtainable with the following method:
 a) providing a CHO cell, which is
 adapted to growth in suspension culture, 
 adapted to growth in serum-free medium, 
 mycoplasma free, 
   b) providing a nucleic acid comprising
 a prokaryotic origin of replication, 
 a first nucleic acid sequence conferring resistance to a prokaryotic selection agent, 
 a second nucleic acid sequence encoding the heavy chain of said heterologous immunoglobulin, and a third nucleic acid sequence encoding the light chain of said heterologous immunoglobulin, 
   whereby a first transfection vector is provided which comprises said provided nucleic acid and an additional fourth nucleic acid sequence conferring resistance to a first eukaryotic selection agent,   whereby a second transfection vector is provided which comprises said provided nucleic acid and an additional fourth nucleic acid sequence conferring resistance to a second eukaryotic selection agent, whereby said second eukaryotic selection agent is different to said first eukaryotic selection agent,   c) transfecting said CHO cell, wherein said transfecting comprises the following steps in the following order:
 (i) transfecting said CHO cell with said first transfection vector, 
 (ii) selecting a CHO cell transfected in (i) by selected growth in cultivation medium containing a first eukaryotic selection agent to which the first transfection vector confers resistance, 
 (iii) transfecting said selected CHO cell in (ii) with said second transfection vector, 
 (iv) selecting a CHO cell transfected in (iii) by selected growth in cultivation medium containing said first eukaryotic selection agent to which the first transfection vector confers resistance and said second eukaryotic selection agent to which the second transfection vector confers resistance. 
   
     
     
         18 . An anti-Aβ antibody produced with a method of  claim 1 . 
     
     
         19 . An anti-P-Selection antibody produced with a method of  claim 1 . 
     
     
         20 . An anti-IL-13Rα antibody produced with a method of  claim 1 . 
     
     
         21 . An anti-CD4 antibody conjugate produced with a method of  claim 1 .