Expression system for factor VIII

Abstract

This invention describes a protein-free production process for proteins having factor VIII procoagulant activity. The process includes the derivation of stable human cell clones with high productivity for B-domain deleted Factor VIII, and (2) the adaptation of cells to grow in a medium free of plasma-derived proteins.

We claim:  
     
         1 . A method of producing cells which express a protein having factor VIII procoagulant activity comprising the sequential steps of: 
 a) obtaining cells which are solely of human origin,    b) contacting the cells of step a) with a vector under conditions sufficient to allow the vector to enter the cells, wherein the vector comprises a selectable marker and a sequence coding for the protein having factor VIII procoagulant activity operably linked to a promoter,    c) selecting the cells from step b) with a selection agent, and    d) isolating individual clones which express high levels of the protein having factor VIII activity from the cells obtained from step c).    
     
     
         2 . The method of  claim 1 , further comprising the step 
 e) adapting the clones of step d) to growth in a plasma derived protein-free medium.    
     
     
         3 . The method of  claim 1 , wherein the cells of step a) are hybrids of human lymphoma cells and 293S cells.  
     
     
         4 . The method of  claim 1 , wherein the cells of step a) are hybrids of 2B8 cells (ATCC CRL-12569) and 293S cells.  
     
     
         5 . The method of  claim 1 , wherein the cells of step a) are HKB11 cells (ATCC CRL-12568).  
     
     
         6 . The method of  claim 1 , wherein the steps c) and d) are performed more than once.  
     
     
         7 . The method of  claim 1 , wherein the sequence of step b) codes for the sequence shown in FIG. 1 (SEQ ID NO:1).  
     
     
         8 . The method of  claim 1 , wherein the sequence of step b) codes for human factor VIII.  
     
     
         9 . The method of  claim 1 , wherein the selectable marker of step b) is dhfr and the selection agent of step c) is methotrexate.  
     
     
         10 . The method of  claim 1 , wherein the selectable marker of step b) is gs and the selection agent of step c) is methionine sulfoximine.  
     
     
         11 . The method of  claim 1 , wherein the selectable marker of step b) is mdr and the selection agent of step c) is colchicine.  
     
     
         12 . A method of producing a protein having factor VIII activity comprising growing the cells produced by the method of  claim 1  in a growth medium and then isolating the protein having factor VIII activity from the medium.  
     
     
         13 . The method of  claim 11  wherein the protein is human factor VIII.  
     
     
         14 . The method of  claim 11  wherein the protein has the amino acid sequence shown in FIG. 1 (SEQ ID NO:1]).  
     
     
         15 . A human cell line derived from human lymphoma cells and 293S cells which expresses high levels of a protein having factor VIII activity.  
     
     
         16 . The human cell line of  claim 14 , wherein the human cell line is derived from HKB11 cells (ATCC CRL-12568).  
     
     
         17 . A human cell line derived from human lymphoma cells and 293S cells which expresses high levels of a protein having factor VIII activity when grown in plasma derived protein-free medium.  
     
     
         18 . The cell line of  claim 16 , wherein the cell line is derived from HKB11 cells (ATCC CRL-12568).  
     
     
         19 . A cell line designated 20B8 (ATCC CRL-12582).