Host cells comprising alpha 1, 2 mannosidase and culture methods thereof

Abstract

Improved host cells and culture methods involving overexpression of MAN1C1 activity to improve protein production are provided.

We claim: 
     
         1 . A host cell engineered to overexpress (a) alpha 1,2 mannosidase (MAN1C1) native to the host cell and (b) a glycoprotein of interest. 
     
     
         2 . The host cell of  claim 1  wherein said host cell has been transfected with a vector comprising a nucleic acid encoding MAN1C1 operably linked to a heterologous expression control sequence. 
     
     
         3 . The host cell of  claim 1  wherein said host cell comprises a heterologous expression control sequence operably linked to a nucleic acid encoding MAN1C1. 
     
     
         4 . The host cell of any of  claims 1 - 3  wherein said host cell has been transfected with a vector comprising a nucleic acid encoding said glycoprotein of interest operably linked to a heterologous expression control sequence. 
     
     
         5 . The host cell of any of  claims 1 - 3  wherein said host cell comprises a heterologous expression control sequence operably linked to a nucleic acid encoding said glycoprotein of interest. 
     
     
         6 . The host cell any of  claims 1 - 5  wherein the glycoprotein of interest is an erythropoiesis-stimulating molecule. 
     
     
         7 . The host cell of  claim 6  wherein the glycoprotein of interest is erythropoietin of SEQ ID NO: 3. 
     
     
         8 . The host cell of  claim 6  wherein the glycoprotein of interest is darbepoetin of SEQ ID NO: 5. 
     
     
         9 . The host cell of any of  claims 1 - 8  which is a mammalian cell. 
     
     
         10 . The host cell of any of  claims 1 - 8  which is a CHO cell. 
     
     
         11 . The host cell of any of  claims 1 - 8  which is a human cell. 
     
     
         12 . The host cell of any of  claims 1 - 8  which is a BHK cell. 
     
     
         13 . The host cell of any of  claims 1 - 8  which is an NS/0 cell. 
     
     
         14 . The host cell of any of  claims 1 - 8  which is an HT-1080 cell. 
     
     
         15 . A method of producing a glycoprotein of interest comprising the steps of:
 culturing the host cell of any of  claims 1 - 14  in culture medium; and recovering the glycoprotein of interest from the host cell or culture medium.   
     
     
         16 . The method of  claim 15  wherein said host is cultured under conditions that induce increased MAN1C1 expression. 
     
     
         17 . The method of  claim 15  wherein said host cell expresses MAN1C1 protein at a level that increases specific productivity (pg/cell/day) of glycoprotein of interest produced. 
     
     
         18 . The method of  claim 17  wherein at least a 2-fold increase in specific productivity is achieved by overexpression of MAN1C1. 
     
     
         19 . A method of increasing production of a glycoprotein of interest comprising the steps of:
 culturing the host cell of any of  claims 1 - 14  in culture medium under conditions that permit expression of MAN1C1 at levels that improve production of the glycoprotein of interest; and recovering the glycoprotein of interest from the host cell or culture medium.   
     
     
         20 . The method of  claim 15  comprising the step of further adding an inducer of protein production to the culture medium. 
     
     
         21 . A method of screening for an inducer of protein production comprising the steps of:
 (a) contacting a host cell with a candidate compound,   (b) determining expression level of MAN1C1, and   (c) identifying said candidate compound as an inducer of protein production if the expression level of MAN1C1 increases.   
     
     
         22 . The method of  claim 21  wherein MAN1C1 mRNA expression level is determined. 
     
     
         23 . The method of  claim 21  wherein MAN1C1 protein expression level is determined.