Glycosylation of proteins

Abstract

The present invention relates to the finding of methods to shift the glycosylation profile of recombinant produced serum glycoproteins to the predominant bi-antennary form found in human plasma. This is accomplished by providing a mammalian cell line according to the invention with a series of gene disruptions and/or gene insertions that facilitate this shift.

1 .- 15 . (canceled) 
     
     
         16 . A recombinant human glycoprotein of interest produced according to a method comprising the steps of:
 a) culturing a population of cells of a recombinant mammalian cell line in a suitable cell culture medium; and   b) harvesting said human protein of interest from the cell culture or cell culture medium,   wherein the recombinant mammalian cell line has   i) one or more of the endogenous genes Mgat4A, Mgat4B, Mgat5, St3Gal3, St3Gal4, St3Gal6, SPPL3, and FUT8 inactivated and/or downregulated; and   ii) optionally a gene ST6Gal1 inserted,   and wherein the recombinant mammalian cell line has been further modified to express the recombinant human glycoprotein.   
     
     
         17 . The recombinant human glycoprotein produced according to  claim 16 , wherein the recombinant mammalian cell is a CHO-S cell line wherein the genes FUT8, MGAT4a, MGAT4b, MGAT5, ST3GAL3, ST3GAL4, ST3GAL6, B3GNT2 and SPPL3 are inactivated and/or downregulated and the human gene ST6GAL1 is inserted. 
     
     
         18 . The recombinant human glycoprotein according to  claim 17 , which is a human serum protein. 
     
     
         19 . The recombinant human glycoprotein according to  claim 18 , which is a human serpin. 
     
     
         20 . The recombinant human glycoprotein according to  claim 19 , wherein the human serpin is selected from the group consisting of SERPINA1 (AAT), SERPINA2, SERPINA3, SERPINA4, SERPINAS, SERPINA6, SERPINA7, SERPINA8, SERPINA9, SERPINA10, SERPINA11, SERPINA12, SERPINA13, SERPINB1, SERPINB2, SERPINB3, SERPINB4, SERPINB5, SERPINB6, SERPINB7, SERPINB8, SERPINB9, SERPINB10, SERPINB11, SERPINB12, SERPINB13, SERPINC1, SERPIND1, SERPINE1, SERPINE2, SERPINE3, SERPINF1, SERPINF2, SERPING1, SERPINH1, SERPINI1, and SERPINI2. 
     
     
         21 . The recombinant human glycoprotein according to  claim 17 , which has a primary n-glycan structure that is a fully sialylated bi-antennary structure without core fucosylation. 
     
     
         22 . The recombinant human glycoprotein according to  claim 21 , which glycan structure is according to the structure A2G2S2 with the following pictorial representation: 
       
         
           
           
               
               
           
         
       
     
     
         23 . The recombinant human glycoprotein according to  claim 22 , which a glycan structure is according to the following pictorial representation: 
       
         
           
           
               
               
           
         
       
     
     
         24 . The recombinant human glycoprotein according to  claim 22 , which is selected from the group consisting of
 Plasma protease C1 inhibitor (C1Inh; SERPING1) glycosylated at one or more positions selected from Asn3, Asn47, Asn59, Asn216, Asn231, Asn250, and Asn330;   Antithrombin-III (ATIII; SERPINC1) glycosylated at one or more positions selected from Asn96, Asn135, Asn155 and Asn192; and   Human alpha-1-antitrypsin (AAT; SERPINA1) glycosylated at one or more, such as two or three of the positions Asn46, Asn83, and Asn247.   
     
     
         25 . A preparation of a recombinant human glycoprotein, wherein more than 80% of the glycan structures of the recombinant human glycoprotein preparation is a fully sialylated bi-antennary structure without core fucosylation. 
     
     
         26 . The preparation according to  claim 25 , wherein the recombinant human glycoprotein is a human serum protein. 
     
     
         27 . The preparation according to  claim 26 , wherein the recombinant human glycoprotein is a human serpin. 
     
     
         28 . The preparation according to  claim 27 , wherein the human serpin is selected from the group consisting of SERPINA1 (AAT), SERPINA2, SERPINA3, SERPINA4, SERPINAS, SERPINA6, SERPINA7, SERPINA8, SERPINA9, SERPINA10, SERPINA11, SERPINA12, SERPINA13, SERPINB1, SERPINB2, SERPINB3, SERPINB4, SERPINBS, SERPINB6, SERPINB7, SERPINB8, SERPINB9, SERPINB10, SERPINB11, SERPINB12, SERPINB13, SERPINC1, SERPIND1, SERPINE1, SERPINE2, SERPINE3, SERPINF1, SERPINF2, SERPING1, SERPINH1, SERPINI1, and SERPINI2. 
     
     
         29 . The preparation according to  claim 25 , wherein more than 80% of the glycan structures of the recombinant human glycoprotein preparation comprises the structure A2G2S2 with the following pictorial representation: 
       
         
           
           
               
               
           
         
       
     
     
         30 . The preparation according to  claim 29 , wherein more than 80% of the glycan structures of the recombinant human glycoprotein preparation comprises the structure A2G2S2 with the following pictorial representation: 
       
         
           
           
               
               
           
         
       
     
     
         31 . The preparation according to  claim 29 , wherein the recombinant human glycoprotein is selected from the group consisting of
 Plasma protease C1 inhibitor (C1Inh; SERPING1) glycosylated at one or more positions selected from Asn3, Asn47, Asn59, Asn216, Asn231, Asn250, and Asn330;   Antithrombin-III (ATIII; SERPINC1) glycosylated at one or more positions selected from Asn96, Asn135, Asn155 and Asn192; and   Human alpha-1-antitrypsin (AAT; SERPINA1) glycosylated at one or more, such as two or three of the positions Asn46, Asn83, and Asn247.