التفاصيل البيبلوغرافية
| العنوان: |
CHARACTERIZATION OF N-GLYCANS USING EXOGLYCOSIDASES |
| Document Number: |
20100129843 |
| تاريخ النشر: |
May 27, 2010 |
| Appl. No: |
12/595925 |
| Application Filed: |
April 15, 2008 |
| مستخلص: |
The present disclosure provides methods for analyzing structure and/or composition of N-glycans. Such methods often involve digestion of N-glycans with multiple exoglycosidases. In some embodiments, N-glycans are digested with multiple exoglycosidases simultaneously. In some embodiments, N-glycans are digested with multiple exoglycosidases sequentially. In some embodiments, methods in accordance with the present disclosure involve comparison of cleavage products of N-glycans that have been digested with multiple exoglycosidases simultaneously to N-glycans that have been digested with multiple exoglycosidases sequentially. |
| Inventors: |
Parsons, Ian Christopher (Belmont, MA, US); Bulik, Dorota A. (Winchester, MA, US); Bosques, Carlos J. (Arlington, MA, US); Thiruneelakantapillai, Lakshmanan (Boston, MA, US); Collins, Brian Edward (Arlington, MA, US) |
| Assignees: |
MOMENTA PHARMACEUTICALS, INC. (Cambridge, MA, US) |
| Claim: |
1. A method comprising steps of: providing a glycan preparation; subjecting a first sample of the glycan preparation to a first exoglycosidase procedure; subjecting a second sample of the glycan preparation to a second exoglycosidase procedure, wherein the second exoglycosidase procedure differs from the first exoglycosidase procedure and further wherein the first and second exoglycosidase procedures are selected from the group consisting of: no treatment; sequential treatment; and simultaneous treatment; characterizing the cleavage products of the first and second exoglycosidase procedure; and comparing the characterization of the cleavage products of the first exoglycosidase procedure to the characterization cleavage products of the second exoglycosidase procedure so that at least one glycosylation feature of the glycan preparation is determined. |
| Claim: |
2. The method of claim 1, wherein the step of comparing comprises comparing the characterization of the cleavage products of the first or second exoglycosidase procedure to the cleavage products of a reference sample. |
| Claim: |
3. The method of claim 1, wherein the first or second exoglycosidase procedure comprises treating the glycan preparation sample with one exoglycosidase selected from the group consisting of a sialidase, a galactosidase, a hexosaminidase, a fucosidase, a mannosidase, and combinations thereof. |
| Claim: |
4. The method of claim 1, wherein the first or second exoglycosidase procedure comprises treating the glycan preparation sample with at least two exoglycosidases selected from the group consisting of a sialidase, a galactosidase, a hexosaminidase, a fucosidase, a mannosidase, and combinations thereof. |
| Claim: |
5-12. (canceled) |
| Claim: |
13. The method of claim 1, wherein one of the first or second exoglycosidase procedure comprises sequential treatment of the glycan preparation sample with at least one exoglycosidase; and the other of the first or second exoglycosidase procedure comprises simultaneous treatment of the glycan preparation sample with at least two exoglycosidases. |
| Claim: |
14-15. (canceled) |
| Claim: |
16. The method of claim 1, wherein at least one cleavage product comprises a modification selected from the group consisting of a sulfated glycan, a phosphorylated glycan, a sialic acid linked to an N-acetylglucosamine, an acetylated glycan, an antennary fucosylation, a lactosamine extension, and a pentasaccharide core comprising three mannose and 2 N-acetylglucosamine residues. |
| Claim: |
17-19. (canceled) |
| Claim: |
20. The method of claim 1, wherein the step of providing a glycan preparation comprises: a) providing a plurality of glycoproteins, each glycoprotein comprising at least one N-glycan, which N-glycan is linked to a glycoprotein of the plurality; b) removing at least one N-glycan from at least one glycoprotein; c) optionally, isolating removed N-glycans. |
| Claim: |
21. The method of claim 20, wherein the step of removing comprises subjecting a glycoprotein or plurality of glycoproteins to a glycanase, to chemical cleavage, or to hydrazine. |
| Claim: |
22-24. (canceled) |
| Claim: |
25. The method of claim 20, further comprising a step of labeling removed N-glycans with a fluorescent or radioactive label prior to or after the isolating step. |
| Claim: |
26-29. (canceled) |
| Claim: |
30. The method of claim 1, wherein the step of characterization comprises subjecting the cleavage products to a procedure selected from the group consisting of chromatographic methods, liquid chromatography (LC), high performance liquid chromatography (HPLC), ultra performance liquid chromatography (UPLC), thin layer chromatography (TLC), amide column chromatography, mass spectrometry (MS), tandem MS, LC-MS, LC-MS/MS, matrix assisted laser desorption ionisation mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation with mass spectrometry (IMS-MS), electron transfer dissociation (ETD-MS), electrophoretic methods, capillary electrophoresis (CE), CE-MS, gel electrophoresis, agarose gel electrophoresis, acrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting using antibodies that recognize specific glycan structures, nuclear magnetic resonance (NMR) one-dimensional NMR (ID-NMR), two-dimensional NMR (2D-NMR), correlation spectroscopy magnetic-angle spinning NMR (COSY-NMR), total correlated spectroscopy NMR (TOCSY-NMR), heteronuclear single-quantum coherence NMR (HSQC-NMR), heteronuclear multiple quantum coherence (HMQC-NMR), rotational nuclear overhauser effect spectroscopy NMR (ROESY-NMR), nuclear overhauser effect spectroscopy (NOESY-NMR), and combinations thereof. |
| Claim: |
31-37. (canceled) |
| Claim: |
38. The method of claim 1, further comprising evaluating the presence of modifications in the N-glycan preparation selected from the group consisting of phosphorylation, sulfation, acetylation, lactosamine extensions, and antennary fucosylation. |
| Claim: |
39. (canceled) |
| Claim: |
40. A method of characterizing a plurality of protein-linked N-glycans comprising: a) providing a plurality of glycoproteins, each glycoprotein comprising at least one N-glycan, which N-glycan is linked to a glycoprotein of the plurality; wherein the glycosylation pattern of at least one member of the plurality differs from the glycosylation pattern of at least one other member of the plurality; b) removing at least one N-glycan from at least one glycoprotein; c) optionally, isolating removed N-glycans; d) cleaving at least one terminal glycosidic bond from the non-reducing end of at least one removed N-glycan by subjecting the at least one removed N-glycan to at least one exoglycosidase enzyme to generate a plurality of cleavage products; e) characterizing the cleavage products; and f) optionally, comparing the characterization of the cleavage products to a reference sample. |
| Claim: |
41-72. (canceled) |
| Claim: |
73. A method comprising steps of: subjecting a glycan preparation to an exoglycosidase procedure, wherein the exoglycosidase procedure comprises sequential administration of a plurality of exoglycosidases, and wherein the exoglycosidase procedure results in cleavage of glycans within the glycan preparation; obtaining at least a first sample from the glycan preparation during the exoglycosidase procedure; and analyzing cleaved glycans within the at least one obtained sample. |
| Claim: |
74. The method of claim 73, wherein: the step of obtaining comprises removing a plurality of samples, separated in time, from the glycan preparation; and the step of analyzing comprises characterizing cleaved glycans in one or more of the removed samples. |
| Claim: |
75-86. (canceled) |
| Claim: |
87. The method of claim 73, wherein: the exoglycosidase treatment comprises sequential administration of at least a first and second exoglycosidase; and the step of obtaining comprises: removing at least a first sample after administration of the first exoglycosidase but before administration of the second exoglycosidase; and removing at least a second sample after administration of the second exoglycosidase. |
| Claim: |
88. A method for comparing batches of a glycoprotein, the method comprising steps of: providing a first glycan preparation from a first batch of the glycoprotein; providing a second glycan preparation from a second batch of the glycoprotein; subjecting each of the first and second glycan preparations to an exoglycosidase procedure, to specifically cleave monosaccharides from the non-reducing ends of N-linked glycans in the glycan preparations; and comparing the cleavage products obtained from the first glycan preparation with the cleavage products obtained from the second preparation so that consistency of the two batches is assessed. |
| Claim: |
89-99. (canceled) |
| Claim: |
100. The method of claim 88, wherein the step of providing a glycan preparation comprises: a) removing at least one N-glycan from at least one glycoprotein from the first or second batch; and b) optionally, isolating removed N-glycans. |
| Claim: |
101-104. (canceled) |
| Claim: |
105. A method for monitoring production of a glycoprotein, the method comprising steps of: during production of a glycoprotein, removing at least first and second glycan-containing samples from the production system; subjecting each of the first and second glycan-containing samples to an exoglycosidase procedure, to specifically cleave monosaccharides from the non-reducing ends of N-linked glycans in the glycan-containing samples; and comparing the cleavage products obtained from the first glycan-containing sample with those obtained from the second glycan-containing sample so that differences are determined and therefore progress of glycoprotein production is monitored. |
| Claim: |
106-124. (canceled) |
| Claim: |
125. The method of claim 105, wherein at least one glycan-containing sample is removed when the exoglycosidase procedure is about 10% complete. |
| Claim: |
126-130. (canceled) |
| Claim: |
131. The method of claim 105, wherein at least one glycan-containing sample is removed before the exoglycosidase procedure is 100% complete. |
| Claim: |
132. The method of claim 105, wherein at least one glycan-containing sample is removed before the glycans are 100% cleaved. |
| Current U.S. Class: |
435/18 |
| Current International Class: |
12 |
| رقم الانضمام: |
edspap.20100129843 |
| قاعدة البيانات: |
USPTO Patent Applications |
