O-Linked Glycoforms Of Polypeptides And Method To Manufacture Them
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.