METHOD OF PRODUCING RECOMBINANT HIGH MOLECULAR WEIGHT vWF IN CELL CULTURE

Abstract

Among other aspects, the present invention relates to cell culture conditions for producing high molecular weight vWF, in particular, highly multimericWF with a high specific activity and ADAMTS13 with a high specific activity. The cell culture conditions of the present invention can include, for example, a cell culture medium with an increased copper concentration and/or cell culture supernatant with a low ammonium (NH 4 + ) concentration. The present invention also provides methods for cultivating cells in the cell culture conditions to express high molecular weight vWF and rA13 having high specific activities.

1 . A method for producing a recombinant Von Willebrand Factor (rVWF) composition, the method comprising the steps of:
 (a) providing a basal cell culture media;   (b) supplementing the basal cell culture media with copper to provide a final copper concentration of at least 2.4 μg/L;   (c) providing one or more cells comprising a nucleic acid encoding a rVWF protein;   (d) culturing the one or more cells in the copper supplemented cell culture media such that rVWF is expressed and excreted from the cells into a culture supernatant; and   (e) recovering at least a portion of the culture supernatant,   wherein the recovered supernatant has a rVWF specific ristocetin cofactor activity of at least 30 mU/μg rVWF.   
     
     
         2 . The method of  claim 1 , further comprising the step of supplementing the basal cell culture media with a hydrolysate prior to culturing the one or more cells. 
     
     
         3 . The method of  claim 2 , wherein the hydrolysate is a plant hydrolysate. 
     
     
         4 . The method of  claim 3 , wherein the hydrolysate is a soy hydrolysate. 
     
     
         5 . The method of  claim 1 , wherein the basal cell culture media is an animal protein free culture media. 
     
     
         6 . The method of  claim 1 , wherein the basal cell culture media is a protein free culture media. 
     
     
         7 . The method of  claim 1 , wherein the basal cell culture media is a chemically defined culture media. 
     
     
         8 . The method of  claim 1 , wherein the final copper concentration of the copper supplemented basal cell culture media is at least 4 μg/L copper. 
     
     
         9 . The method of  claim 1 , wherein the final copper concentration of the copper supplemented basal cell culture media is between 2.4 μg/L and 20 μg/L copper. 
     
     
         10 . The method of  claim 1 , wherein the copper supplementing the basal cell culture media is provided as a copper salt, a copper chelate, or a combination thereof. 
     
     
         11 . The method of  claim 10 , wherein the copper salt is selected from the group consisting of copper sulfate, copper acetate, copper carbonate, copper chloride, copper hydroxide, copper nitrate, and copper oxide. 
     
     
         12 . The method of  claim 1 , wherein the one or more cells are mammalian cells. 
     
     
         13 . The method of  claim 12 , wherein the mammalian cells are CHO cells. 
     
     
         14 . The method of  claim 1 , wherein culturing the one or more cells comprises batch cultivation of the cells. 
     
     
         15 . The method of  claim 1 , wherein culturing the one or more cells comprises continuous cultivation of the cells. 
     
     
         16 . The method of  claim 15 , wherein the continuous cultivation of cells is performed in chemostatic mode. 
     
     
         17 . The method of  claim 15 , wherein the continuous cultivation of cells is performed in perfusion mode. 
     
     
         18 . The method of  claim 1 , wherein the one or more cells is cultured in at least 100 L of the supplemented basal cell culture media. 
     
     
         19 . The method of  claim 1 , wherein the cell density is maintained at less than 2.5×10 6  cells per mL during the step of culturing the one or more cells. 
     
     
         20 - 45 . (canceled) 
     
     
         46 . A method for producing a recombinant ADAMTS13 (rA13) composition, the method comprising the steps of:
 (a) providing a basal cell culture media;   (b) supplementing the basal cell culture media with copper to provide a final copper concentration of from 1.0 μg/L to 20.0 μg/L;   (c) providing one or more cells comprising a nucleic acid encoding a rA13 protein;   (d) culturing the one or more cells in the copper supplemented cell culture media such that rA13 is expressed and excreted from the cells into a culture supernatant, wherein the NH 4   +  content of the cell culture supernatant is maintained at a concentration below 10 mM; and   (e) recovering at least a portion of the culture supernatant,   wherein at least 1500 Units FRETS-VWF73 activity per liter supplemented basal cell culture media per day is present in the recovered culture supernatant.