US2008070275A1PendingUtilityA1
Factor VIII: Remodeling and glycoconjugation of factor VIII
Est. expiryOct 10, 2021(expired)· nominal 20-yr term from priority
A61K 38/00C07K 14/755A61K 47/60C07K 9/00C07K 1/006C07K 1/13C07K 14/505C07K 1/1077
68
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Claims
Abstract
The invention includes methods and compositions for remodeling a peptide molecule, including the addition or deletion of one or more glycosyl groups to a peptide, and/or the addition of a modifying group to a peptide.
Claims
exact text as granted — not AI-modified1 - 115 . (canceled)
116 . A cell-free, in vitro method of forming a covalent conjugate of a Factor VIII peptide, said peptide having the formula:
wherein
AA is a terminal or internal amino acid residue of said peptide;
X 1 -X 2 is a saccharide covalently linked to said AA, wherein
X 1 is a first glycosyl residue; and
X 2 is a second glycosyl residue covalently linked to X 1 , wherein X 1 and X 2 are selected from monosaccharyl and oligosaccharyl residues;
said method comprising:
(a) removing X 2 or a saccharyl subunit thereof from said peptide, thereby forming a truncated glycan; and
(b) contacting said truncated glycan with at least one glycosyltransferase and at least one modified sugar donor under conditions suitable for said at least one glycosyltransferase to transfer a modified sugar moiety of said at least one modified sugar donor to said truncated glycan, wherein said modified sugar moiety comprises at least one modifying group which is a polymeric modifying group,
thereby forming said covalent conjugate of said Factor VIII peptide.
117 . The method of claim 116 , wherein said Factor VIII peptide is a Factor VIII variant wherein the Factor VIII variant further comprises a B-domain deletion.
118 . The method of claim 117 , wherein said Factor VIII variant further comprises amino acids 1-740 linked to amino acids 1649 to 2332 by at least 3 amino acid residues.
119 . The method of claim 116 , further comprising:
(c) prior to step (b), removing a group added to said saccharide during post-translational modification.
120 . The method of claim 119 , wherein said group is a member selected from phosphate, sulfate, carboxylate and esters thereof.
121 . The method of claim 116 , wherein said peptide has the formula:
wherein
Z is a member selected from O, S, NH or a crosslinker.
122 . The method of claim 116 , wherein said polymeric modifying group is a water-soluble polymer.
123 . The method of claim 122 , wherein said water-soluble polymer comprises poly(ethylene glycol).
124 . The method of claim 123 , wherein said poly(ethylene glycol) is a monomethoxy-poly(ethylene glycol).
125 . The method of claim 123 , wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol).
126 . The method of claim 123 , wherein said poly(ethylene glycol) has a molecular weight distribution that is essentially homodisperse.
127 . The method of claim 116 , wherein said glycosyltransferase is selected from the group consisting of ST3Gal1, ST3Gal3 and CST-I.
128 . The method of claim 116 , wherein, following said forming said covalent conjugate, said Factor VIII peptide is contacted with a sialic acid donor and a sialyltransferase under conditions suitable for said sialyltransferase to transfer a sialic acid residue onto said Factor VIII peptide, thereby transferring a sialic acid moiety onto said Factor VIII peptide.
129 . The method of claim 116 , wherein said peptide has the formula:
wherein
X 9 and X 10 are independently selected from monosaccharyl and oligosaccharyl residues; and
m, n and f are integers selected from 0 and 1.
130 . The method of claim 116 , wherein said peptide has the formula:
wherein
X 11 and X 12 are independently selected glycosyl moieties; and
r and x are integers independently selected from 0 and 1.
131 . The method of claim 130 , wherein X 11 and X 12 are (mannose) q , wherein
q is selected from the integers between 1 and 20, and when q is three or greater (mannose) q is selected from linear and branched structures.
132 . The method of claim 116 , wherein said peptide has the formula:
wherein
X 13 , X 14 , and X 15 are independently selected glycosyl residues; and
g, h, i, j, k, and p are independently selected from the integers 0 and 1, with the proviso that at least one of g, h, i, j, k and p is 1.
133 . The method of claim 132 , wherein
X 14 and X 15 are members independently selected from GlcNAc and Sia; and i and k are independently selected from the integers 0 and 1, with the proviso that at least one of i and k is 1, and when k is 1, g, h, and j are 0.
134 . The method of claim 116 , wherein said peptide has the formula:
wherein
X 16 is a member selected from:
wherein
s and i are integers independently selected from 0 and 1.
135 . The method of claim 116 , wherein said removing utilizes a glycosidase.
136 . A cell-free, in vitro method of forming a covalent conjugate of a Factor VIII peptide, said peptide having the formula:
wherein
AA is a terminal or internal amino acid residue of said peptide;
X 1 is a glycosyl residue covalently linked to said AA, selected from monosaccharyl and oligosaccharyl residues; and
u is an integer selected from 0 and 1,
said method comprising:
contacting said peptide with at least one glycosyltransferase and at least one modified sugar donor under conditions suitable for said at least one glycosyltransferase to transfer a modified sugar moiety of said at least one modified sugar donor to said peptide, wherein said modified sugar moiety comprises at least one modifying group which is a polymeric modifying group,
thereby forming said covalent conjugate of said Factor VIII peptide.
137 . The method of claim 136 , wherein said Factor VIII peptide is a Factor VIII variant wherein the Factor VIII variant further comprises a B-domain deletion.
138 . The method of claim 137 , wherein said Factor VIII variant further comprises amino acids 1-740 linked to amino acids 1649 to 2332 by at least 3 amino acid residues.
139 . The method of claim 136 , wherein said polymeric modifying group is a water-soluble polymer.
140 . The method of claim 139 , wherein said water-soluble polymer comprises poly(ethylene glycol).
141 . The method of claim 140 , wherein said poly(ethylene glycol) is a monomethoxy-poly(ethylene glycol).
142 . The method of claim 140 , wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol).
143 . The method of claim 140 , wherein said poly(ethylene glycol) has a molecular weight distribution that is essentially homodisperse.
144 . The method of claim 136 , wherein said glycosyltransferase is selected from the group consisting of ST3Gal1, ST3Gal3, CST-I and CST-II.
145 . The method of claim 136 , wherein, following said forming said covalent conjugate, said Factor VIII peptide is contacted with a sialic acid donor and a sialyltransferase under conditions suitable for said sialyltransferase to transfer a sialic acid residue onto said Factor VIII peptide, thereby transferring a sialic acid moiety onto said Factor VIII peptide.
146 . A cell-free, in vitro method of forming a covalent conjugate of a Factor VIII peptide, said peptide having the formula:
wherein
r, s, and t are integers independently selected from 0 and 1,
said method comprising:
(a) contacting said peptide with at least one glycosyltransferase and at least one modified sugar donor under conditions suitable for said at least one glycosyltransferase to transfer a modified sugar moiety of said at least one modified sugar donor to said peptide, wherein said modified sugar moiety comprises at least one modifying group which is a polymeric modifying group,
thereby forming said covalent conjugate of said Factor VIII peptide.
147 . The method of claim 146 , wherein said Factor VIII peptide is a Factor VIII variant wherein the Factor VIII variant further comprises a B-domain deletion.
148 . The method of claim 147 , wherein said Factor VIII variant further comprises amino acids 1-740 linked to amino acids 1649 to 2332 by at least 3 amino acid residues.
149 . The method of claim 146 , wherein, following said forming said covalent conjugate, said Factor VIII peptide is contacted with a sialic acid donor and a sialyltransferase under conditions suitable for said sialyltransferase to transfer a sialic acid residue onto said Factor VIII peptide, thereby transferring a sialic acid moiety onto said Factor VIII peptide.
150 . The method according to claim 146 , wherein said polymeric modifying group is a water-soluble polymer.
151 . The method according to claim 150 , wherein said water-soluble polymer is poly(ethylene glycol).
152 . The method according to claim 151 , wherein said poly(ethylene glycol) has a molecular weight that is essentially homodisperse.
153 . The method of claim 151 , wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol).
154 . The method of claim 151 , wherein said poly(ethylene glycol) is monomethoxy-poly(ethylene glycol).
155 . The method of claim 146 , wherein said glycosyltransferase is a member selected from the group consisting of GalT, ST3Gal3, CST-II and combinations thereof.
156 . The method of claim 155 , wherein said at least one glycosyltransferase is GalT, followed by a member selected from ST3Gal3, CST-II and combinations thereof.
157 . The method of claim 146 , wherein said glycosyltransferase comprises:
(i) GalT1; and (ii) ST3Gal3.
158 . A cell-free, in vitro method of forming a covalent conjugate of a Factor VIII peptide, said peptide having the formula:
wherein
AA is a terminal or internal amino acid residue of said peptide;
X 1 -X 2 is a saccharide covalently linked to said AA, wherein
X 1 is a first glycosyl residue; and
X 2 is a second glycosyl residue covalently linked to X 1 , wherein X 1 and X 2 are selected from monosaccharyl and oligosaccharyl residues;
said method comprising:
(a) removing X 1 and X 2 , exposing said AA; and
(b) contacting said peptide with at least one glycosyltransferase and at least one modified sugar donor under conditions suitable for said at least one glycosyltransferase to transfer a modified sugar moiety of said at least one modified sugar donor to said peptide, wherein said modified sugar moiety comprises at least one modifying group which is a polymeric modifying group,
thereby forming said covalent conjugate of said Factor VIII peptide.
159 . The method of claim 158 , wherein said Factor VIII peptide is a Factor VIII variant wherein the Factor VIII variant further comprises a B-domain deletion.
160 . The method of claim 159 , wherein said Factor VIII variant further comprises amino acids 1-740 linked to amino acids 1649 to 2332 by at least 3 amino acid residues.
161 . The method of claim 158 , wherein, following said forming said covalent conjugate, said Factor VIII peptide is contacted with a sialic acid donor and a sialyltransferase under conditions suitable for said sialyltransferase to transfer a sialic acid residue onto said Factor VIII peptide, thereby transferring a sialic acid moiety onto said Factor VIII peptide.
162 . The method according to claim 158 , wherein said polymeric modifying group is a water-soluble polymer.
163 . The method according to claim 162 , wherein said water-soluble polymer is poly(ethylene glycol).
164 . The method according to claim 163 , wherein said poly(ethylene glycol) has a molecular weight that is essentially homodisperse.
165 . The method of claim 163 , wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol).
166 . The method of claim 163 , wherein said poly(ethylene glycol), is monomethoxy-poly(ethylene glycol).
167 . The method of claim 158 , wherein said glycosyltransferase is selected from the group consisting of ST3Gal3, ST3Gal1, CST-I and CST-II.
168 . A cell-free, in vitro method of forming a covalent conjugate of a Factor VIII peptide, said peptide having the formula:
wherein
X 3 , X 4 , X 5 , X 6 , X 7 , and X 17 are independently selected from monosaccharyl and oligosaccharyl residues; and
a, b, c, d, e and x are independently selected from the integers 0, 1 and 2, with the proviso that at least one member selected from a, b, c, d, and e and x is 1 or 2; said method comprising:
(a) removing at least one of X 3 , X 4 , X 5 , X 6 , X 7 , or X 17 , or a saccharyl subunit thereof from said peptide, thereby forming a truncated glycan; and
(b) contacting said truncated glycan with at least one glycosyltransferase and at least one glycosyl donor under conditions suitable to transfer said at least one glycosyl donor to said truncated glycan, one modified sugar donor under conditions suitable for said at least one glycosyltransferase to transfer a modified sugar moiety of said at least one modified sugar donor to said truncated glycan, wherein said modified sugar moiety comprises at least one modifying group which is a polymeric modifying group,
thereby forming said covalent conjugate of said Factor VIII peptide.
169 . The method of claim 168 , wherein said Factor VIII peptide is a Factor VIII variant wherein the Factor VIII variant further comprises a B-domain deletion.
170 . The method of claim 169 , wherein said Factor VIII variant further comprises amino acids 1-740 linked to amino acids 1649 to 2332 by at least 3 amino acid residues.
171 . The method of claim 168 , wherein said removing of step (a) produces a truncated glycan in which a, b, c, e and x are each 0.
172 . The method of claim 168 , wherein X 3 , X 5 , and X 7 , are selected from the group consisting of (mannose) z and (mannose) z -(X 8 ) y wherein
X 8 is a glycosyl moiety selected from mono- and oligo-saccharides;
y is an integer selected from 0 and 1; and
z is an integer between 1 and 20, wherein
when z is 3 or greater, (mannose) z is selected from linear and branched structures.
173 . The method of claim 168 , wherein X 4 is selected from the group consisting of GlcNAc and xylose.
174 . The method of claim 168 , wherein X 3 , X 5 , and X 7 are (mannose) u , wherein
u is selected from the integers between 1 and 20, and when u is 3 or greater, (mannose) u is selected from linear and branched structures.
175 . The method of claim 168 , wherein, following said forming said covalent conjugate, said Factor VIII peptide is contacted with a sialic acid donor and a sialyltransferase under conditions suitable for said sialyltransferase to transfer a sialic acid residue onto said Factor VIII peptide, thereby transferring a sialic acid moiety onto said Factor VIII peptide.
176 . The method according to claim 168 , wherein said polymeric modifying group is a water-soluble polymer.
177 . The method of claim 176 , wherein said water-soluble polymer comprises poly(ethylene glycol).
178 . The method of claim 177 , wherein said poly(ethylene glycol) has a molecular weight distribution that is essentially homodisperse.
179 . The method of claim 177 , wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol).
180 . The method of claim 177 , wherein said poly(ethylene glycol) is monomethoxy-poly(ethylene glycol).
181 . The method of claim 168 , wherein said glycosyltransferase is ST3Gal3.
182 . A cell-free, in vitro method of forming a covalent conjugate between a polymeric modifying group and a glycosylated or non-glycosylated Factor VIII peptide, wherein said polymeric modifying group is conjugated to said Factor VIII peptide via an intact glycosyl linking group interposed between and covalently linked to both said Factor VIII peptide and said polymeric modifying group, said method comprising:
contacting said Factor VIII peptide with a mixture comprising a nucleotide sugar covalently linked to said polymeric modifying group, and a glycosyltransferase for which said nucleotide sugar is a substrate under conditions suitable for said at least one glycosyltransferase to transfer a modified sugar moiety of said nucleotide sugar to said Factor VIII peptide, wherein said modified sugar moiety comprises at least one modifying group which is a polymeric modifying group, thereby forming said covalent conjugate of said Factor VIII peptide.
183 . The method of claim 182 , wherein said Factor VIII peptide is a Factor VIII variant wherein the Factor VIII variant further comprises a B-domain deletion.
184 . The method of claim 183 , wherein said Factor VIII variant further comprises amino acids 1-740 linked to amino acids 1649 to 2332 by at least 3 amino acid residues.
185 . The method of claim 182 , wherein said glycosyl linking group is covalently attached to a glycosyl residue covalently attached to said peptide.
186 . The method of claim 182 , wherein said glycosyl linking group is covalently attached to an amino acid residue of said peptide.
187 . The method of claim 182 , wherein said polymeric modifying group is a water-soluble polymer.
188 . The method of claim 187 , wherein said water-soluble polymer comprises polyalkylene oxide.
189 . The method of claim 188 , wherein said polyalkylene oxide is poly(ethylene glycol).
190 . The method of claim 189 , wherein said poly(ethylene glycol) has a degree of polymerization from about 1 to about 40,000.
191 . The method of claim 189 , wherein said poly(ethylene glycol) has a degree of polymerization from about 1 to about 80,000, and further wherein said poly(ethylene glycol) is a branched structure.
192 . The method of claim 189 , wherein said poly(ethylene glycol) has a mass from about 10 kDa to about 40 kDa.
193 . The method of claim 189 , wherein said poly(ethylene glycol) has a degree of polymerization from about 1 to about 30,000, and further wherein said poly(ethylene glycol) is a linear structure.
194 . The method of claim 190 , wherein said poly(ethylene glycol) has a degree of polymerization from about 1 to about 5,000.
195 . The method of claim 194 , wherein said poly(ethylene glycol) has a degree of polymerization from about 1 to about 1,000.
196 . The method of claim 189 , wherein said poly(ethylene glycol) is monomethoxy-poly(ethylene glycol).
197 . The method of claim 189 , wherein said poly(ethylene glycol) has a molecular weight distribution that is essentially homodisperse.
198 . The method of claim 189 , wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol).
199 . The method of claim 182 , wherein said glycosyltransferase is selected from the group consisting of ST3Gal1, ST3Gal3 and CST-II.
200 . The method of claim 182 , wherein, following said forming said covalent conjugate, said Factor VIII peptide is contacted with a sialic acid donor and a sialyltransferase under conditions suitable for said sialyltransferase to transfer a sialic acid residue onto said Factor VIII peptide, thereby transferring a sialic acid moiety onto said Factor VIII peptide.
201 . The method of claim 182 , wherein said glycosyltransferase is selected from the group consisting of sialyltransferase, galactosyltransferase, glucosyltransferase, GalNAc transferase, GlcNAc transferase, fucosyltransferase, and mannosyltransferase.
202 . The method of claim 182 , wherein said glycosyltransferase is recombinantly produced.
203 . The method of claim 202 , wherein said glycosyltransferase is a recombinant prokaryotic enzyme.
204 . The method of claim 202 , wherein said glycosyltransferase is a recombinant eukaryotic enzyme.
205 . The method of claim 182 , wherein said glycosylated peptide is partially deglycosylated prior to said contacting.
206 . The method of claim 182 , wherein said intact glycosyl linking group is a sialic acid residue.
207 . The method of claim 182 , wherein said method is performed in a cell-free environment.
208 . The method of claim 182 , wherein said covalent conjugate is isolated.
209 . The method of claim 208 , wherein said covalent conjugate is isolated by membrane filtration.
210 . A cell-free, in vitro method of forming a covalent conjugate of a Factor VIII peptide, said peptide having the formula:
wherein
AA is a terminal or internal amino acid residue of said peptide, said method comprising:
contacting said peptide with at least one glycosyltransferase and at least one modified sugar donor under conditions suitable for said at least one glycosyltransferase to transfer a modified sugar moiety of said at least one modified sugar donor to said amino acid residue, wherein said modified sugar moiety comprises at least one modifying group which is a polymeric modifying group,
thereby forming said covalent conjugate of said Factor VIII peptide.
211 . The method of claim 210 , wherein said Factor VIII peptide is a Factor VIII variant wherein the Factor VIII variant further comprises a B-domain deletion.
212 . The method of claim 211 , wherein said Factor VIII variant further comprises amino acids 1-740 linked to amino acids 1649 to 2332 by at least 3 amino acid residues.
213 . The method of claim 210 , wherein, following said forming said covalent conjugate, said Factor VIII peptide is contacted with a sialic acid donor and a sialyltransferase under conditions suitable for said sialyltransferase to transfer a sialic acid residue onto said Factor VIII peptide, thereby transferring a sialic acid moiety onto said Factor VIII peptide.
214 . The method according to claim 210 , wherein said polymeric modifying group is a water-soluble polymer.
215 . The method according to claim 214 , wherein said water-soluble polymer is poly(ethylene glycol).
216 . The method according to claim 215 , wherein said poly(ethylene glycol) has a molecular weight that is essentially homodisperse.
217 . The method of claim 215 , wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol).
218 . The method of claim 215 , wherein said poly(ethylene glycol) is monomethoxy-poly(ethylene glycol).
219 . The method of claim 210 , wherein said glycosyltransferase is selected from the group consisting of GalNAcT2, ST3Gal1, ST3Gal3, ST6GalNAcI, CST-I, CST-II and combinations thereof.
220 . The method of claim 219 , wherein said at least one glycosyltransferase is GalNAcT2, followed by a member selected from ST3Gal1, ST3Gal3, ST6GalNAcI, CST-I, CST-II and combinations thereof.
221 . A cell-free, in vitro method of forming a covalent conjugate between a Factor VIII peptide and a modifying group, wherein said modifying group is covalently attached to said Factor VIII peptide through an intact glycosyl linking group, said Factor VIII peptide comprising a glycosyl residue having a formula which is a member selected from:
and
wherein
a, b, c, d, i, n, o, p, q, r, s, t, u, aa, cc, and dd are members independently selected from 0 and 1;
e, f, g, and h are members independently selected from the integers between 0 and 6;
j, k, l, and m are members independently selected from the integers between 0 and 20;
v, w, x, y and z are 0; and
each R is a polymeric modifying group; and
R′ is a member selected from H, a glycosyl residue, a modifying group and a glycoconjugate,
said method comprising:
(a) contacting said Factor VIII peptide with at least one glycosyltransferase and at least one modified sugar donor under conditions suitable for said at least one glycosyltransferase to transfer a modified sugar moiety of said at least one modified sugar donor to said Factor VIII peptide, wherein said modified sugar moiety comprises at least one modifying group which is a polymeric modifying group, such that, following said contacting, at least one of v, w, x, or y is 1,
thereby forming intact glycosyl linking group.
222 . The method of claim 221 , wherein said Factor VIII peptide is a Factor VIII variant wherein the Factor VIII variant further comprises a B-domain deletion.
223 . The method of claim 222 , wherein said Factor VIII variant further comprises amino acids 1-740 linked to amino acids 1649 to 2332 by at least 3 amino acid residues.
224 . The method of claim 221 , wherein, following said forming said covalent conjugate, said Factor VIII peptide is contacted with a sialic acid donor and a sialyltransferase under conditions suitable for said sialyltransferase to transfer a sialic acid residue onto said Factor VIII peptide, thereby transferring a sialic acid moiety onto said Factor VIII peptide.
225 . The method according to claim 221 , wherein said polymeric modifying group is a water-soluble polymer.
226 . The method according to claim 225 , wherein said water-soluble polymer is poly(ethylene glycol).
227 . The method according to claim 226 , wherein said poly(ethylene glycol) has a molecular weight that is essentially homodisperse.
228 . The method of claim 226 , wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol).
229 . The method of claim 226 , wherein said poly(ethylene glycol) is monomethoxy-poly(ethylene glycol).
230 . The method of claim 221 , wherein said glycosyltransferase is selected from the group consisting of ST3Gal1, ST3Gal3, CST-I and CST-II.
231 . The method of claim 221 , further comprising:
(b) prior to step (a), contacting said Factor VIII peptide with a sialidase under conditions appropriate to remove sialic acid from said Factor VIII peptide.
232 . The method of claim 221 , further comprising:
(c) contacting the product of step (a) with a sialyltransferase and a sialic acid donor under conditions appropriate to transfer sialic acid to said product.
233 . The method of claim 221 , further comprising:
(d) prior to step (a), contacting said Factor VIII peptide with a galactosyl transferase and a galactose donor under conditions appropriate to transfer said galactose to said Factor VIII peptide.
234 . The method of claim 221 , further comprising:
(e) contacting the product from step (a) with a moiety that reacts with said modifying group, thereby forming a conjugate between said intact glycosyl linking group and said moiety.
235 . The method of claim 221 , further comprising:
(f) prior to step (a), contacting said Factor VIII peptide with N-acetylglucosamine transferase and a GlcNAc donor under conditions appropriate to transfer GlcNAc to said Factor VIII peptide.
236 . The method of claim 221 , further comprising:
(g) prior to step (a), contacting said Factor VIII peptide with a sialidase under conditions appropriate to remove sialic acid from said Factor VIII peptide.
237 . The method of claim 221 , further comprising:
(h) prior to step (a), contacting said Factor VIII peptide with N-acetylglucosamine transferase and a GlcNAc donor under conditions appropriate to transfer GlcNAc to said Factor VIII peptide.
238 . The method of claim 235 , further comprising contacting said Factor VIII peptide with galactosyl transferase and a galactose donor under conditions appropriate to transfer galactose to said Factor VIII peptide.
239 . The method of claim 221 , further comprising:
(i) prior to step (a), contacting said Factor VIII peptide with endoglycanase under conditions appropriate to cleave a glycosyl moiety from said Factor VIII peptide.
240 . The method of claim 239 , further comprising contacting said Factor VIII peptide with galactosyl transferase and a galactose donor under conditions appropriate to transfer galactose to said Factor VIII peptide.
241 . The method of claim 221 , further comprising:
(j) prior to step (a), contacting said Factor VIII peptide with ST3Gal3 and a sialic acid donor under conditions appropriate to transfer sialic acid to said product.
242 . The method of claim 221 , further comprising:
(k) prior to step (a), contacting said Factor VIII peptide with a mannosidase under conditions appropriate to remove mannose from said Factor VIII peptide.
243 . The method of claim 242 , further comprising contacting said Factor VIII peptide with N-acetylglucosamine transferase and a GlcNAc donor under conditions appropriate to transfer GlcNAc to said Factor VIII peptide.
244 . The method of claim 243 , further comprising contacting said Factor VIII peptide with galactosyl transferase and a galactose donor under conditions appropriate to transfer galactose to said Factor VIII peptide.
245 . The method of claim 221 , wherein
e, f, g, and h are members independently selected from the integers between 1 and 4; a, b, c, d, i, j, k, l, m, n, o, p, q, r, s, t, u, aa, and cc are members independently selected from 0 and 1; and v, w, x, y, z, and dd are 0.
246 . A method of treating a mammal having a bleeding disorder, said method comprising administering to said mammal a Factor VIII peptide, having one or glycans having a polymeric modifying group molecule attached to said peptide.
247 . The method according to claim 246 , wherein said bleeding disorder is a member selected from the group consisting of hemophilia A, hemophilia B, von Willebrand's disease, Factor VIII:C deficiency and fibrinogen deficiency.
248 . The method according to claim 246 , wherein said polymeric modifying group is a water-soluble polymer.
249 . The method according to claim 248 , wherein said water-soluble polymer is poly(ethylene glycol).
250 . The method according to claim 249 , wherein said poly(ethylene glycol) has a molecular weight that is essentially homodisperse.
251 . The method of claim 249 , wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol).
252 . The method of claim 249 , wherein said poly(ethylene glycol) is monomethoxy-poly(ethylene glycol).Join the waitlist — get patent alerts
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