O-Linked Glycoforms Of Polypeptides And Method To Manufacture Them

Abstract

The present invention relates to compositions comprising glycoproteins having altered patterns of O-linked glycosylation, in particular Factor VII, Factor IX, and methods for making these.

1 . A preparation of a glycoprotein containing a Cys-X1-Ser/Thr-X2-Pro-Cys motif, wherein the serine/threonine is linked to a sugar chain by a Glc-O-Ser/Thr covalent bond, X1 and X2 each represent any amino acid residue and the preparation has a substantially uniform serine/threonine-linked glycosylation pattern. 
     
     
         2 . The preparation according to  claim 1 , wherein the glycosylation pattern is at least 80% uniform. 
     
     
         3 . The preparation according to  claim 1 , wherein the serine/threonine-linked sugar chain is Xyl-Xyl-Glc-. 
     
     
         4 . The preparation according to  claim 1 , wherein the serine/threonine-linked sugar chain is Xyl-Glc-. 
     
     
         5 . The preparation according to  claim 1 , wherein the serine/threonine-linked sugar chain is Glc-. 
     
     
         6 . The preparation according to  claim 1 , wherein the glycoprotein is selected from the group consisting of Factor VII polypeptides, Factor VII-related polypeptides, Factor IX polypeptides, Factor IX-related polypeptides, Factor X polypeptides, Factor X-related polypeptides, Factor XII polypeptides, and protein Z polypeptides. 
     
     
         7 . The preparation according to  claim 6 , wherein the glycoprotein is human Factor VII. 
     
     
         8 . A preparation according to  claim 6 , wherein the glycoprotein is a variant of Factor VII and wherein the ratio between the activity of the Factor VII-variant and the activity of native human factor VIIa (wild-type FVIIa) is at least about 1.25 when tested in the “In Vitro Hydrolysis Assay” or in the “In vitro Proteolysis Assay”. 
     
     
         9 . The preparation according to  claim 6 , wherein the glycoprotein is human Factor IX or a human Factor IX sequence variant. 
     
     
         10 . A method for making a preparation according to  claim 5  comprising:
 (a) obtaining a preparation of a precursor glycoprotein containing a Cys-X1-Ser/Thr-X2-Pro-Cys motif, wherein the serine/threonine is linked to a sugar chain by Glc-O-Ser/Thr covalent bond and X1 and X2 each represent any amino acid residue; and   (b) contacting the preparation obtained in step (a) with an α-xylosidase under conditions appropriate for removing xylose residues from the precursor glycoprotein, thereby producing the glycoprotein.   
     
     
         11 . The method according to  claim 10 , further including the step of isolating the glycoprotein prepared in step (b). 
     
     
         12 . The method according to  claim 10 , wherein the glycosylation is a serine glycosylation. 
     
     
         13 . The method according to  claim 10   10 , further including the step of analysing the structure of the sugar chain linked to the glycoprotein to determine a glycoform pattern, and, optionally, repeating step (b) until the desired glycoform pattern is achieved. 
     
     
         14 . A method for making a preparation according to in  claim 6  comprising:
 (a) obtaining a preparation of a polypeptide containing a Cys-X1-Ser/Thr-X2-Pro-Cys motif, wherein X1 and X2 each represent any amino acid residue; and   (b) contacting the preparation obtained in step (a) with a O-glucosyltransferase and an activated glucose donor under conditions appropriate for transferring a glucose residue from the glucose donor moiety to the serine/threonine thereby producing the glycoprotein.   
     
     
         15 . The method according to  claim 14 , further including the step of isolating the glycoprotein prepared in step (b). 
     
     
         16 . The method according to  claim 14 , wherein the glycosylation is a serine glycosylation. 
     
     
         17 . The method according to  claim 14 , further including the step of analyzing the structure of the sugar chain linked to glycoproteins in the preparation to determine if the glycoproteins have a desired glycoform pattern, and, optionally, repeating step (b) until the desired glycoform pattern is achieved. 
     
     
         18 . A method for making a preparation according to  claim 4  comprising:
 (a) obtaining a preparation of a precursor glycoprotein containing a Cys-X1-Ser/Thr-X2-Pro-Cys motif, wherein the serine/threonine is linked to a sugar chain by a Glc-O-Ser/Thr covalent bond and X1 and X2 each represent any amino acid residue; and   (b) contacting the preparation obtained in step (a) with (i) UDP-D-xylose: β-D-glucoside α-1,3-D-xylosyltransferase and (ii) an activated xylosyl donor under conditions appropriate for transferring a xylose residue from an xylose donor moiety to an acceptor moiety, thereby producing the glycoprotein.   
     
     
         19 . The method according to  claim 18 , further including the step of isolating the glycoprotein prepared in step (b). 
     
     
         20 . The method according to  claim 18 , wherein the glycosylation is a serine glycosylation. 
     
     
         21 . The method according to  claim 18 , further including the step of analyzing the structure of the sugar chain linked to glycoproteins in the preparation to determine if the glycoproteins have a desired glycoform pattern, and, optionally, repeating step (b) until the desired glycoform pattern is achieved. 
     
     
         22 . The method according to  claim 18 , further including the step of removing terminal xylose-residues by subjecting the preparation obtained in step (a) to the method described in  claim 10  prior to step (b). 
     
     
         23 . A method for making a preparation according to  claim 3  comprising:
 (a) obtaining a preparation of a precursor glycoprotein containing a Cys-X1-Ser/Thr-X2-Pro-Cys motif, wherein the serine/threonine is linked to a sugar chain by a Glc-O-Ser/Thr covalent bond and X1 and X2 each represent any amino acid residue;   (b) contacting the preparation obtained in step (a) with UDP-D-xylose: β-D-glucoside α-1,3-D-xylosyltransferase and an activated xylosyl donor under conditions appropriate for transferring a xylose residue from a xylose donor moiety to an acceptor moiety; and   (c) contacting the preparation obtained in step (b) with UDP-D-xylose: α-D-xyloside α-1,3-xylosyltransferase and an activated xylosyl donor under conditions appropriate for transferring a xylose residue from a xylose donor moiety to an acceptor moiety, thereby producing the glycoprotein.   
     
     
         24 . The method according to  claim 23 , further including the step of isolating the preparation obtained in step (b) prior to subjecting the preparation to step (c). 
     
     
         25 . The method according to  claim 23 , further including the step of isolating the glycoprotein prepared in step (c). 
     
     
         26 . The method according to  claim 23 , wherein the glycosylation is a serine glycosylation. 
     
     
         27 . The method according to  claim 23 , further including the step of analyzing the structure of the sugar chain linked to the glycoproteins in the preparation to determine if the glycoproteins have a desired glycoform pattern, and, optionally, repeating step (b) and/or step (c) until the desired glycoform pattern is achieved. 
     
     
         28 . The method according to  claim 23 , further including the step of removing terminal xylose-residues by subjecting the preparation obtained in step (a) to the method described in  claim 10  prior to step (b). 
     
     
         29 . The preparation according to  claim 2 , wherein the glycosylation pattern is at least 90% uniform. 
     
     
         30 . The preparation according to  claim 29 , wherein the glycosylation pattern is at least 98% uniform.