Multi-dimensional chromatographic methods for separating n-glycans

Patent metadata

Field	Value
Publication number	US-2016356783-A1
Application number	US-201615241717-A
Country	US
Kind code	A1
Filing date	Aug 19, 2016
Priority date	Apr 16, 2007
Publication date	Dec 8, 2016
Grant date	—

Abstract

A multi-dimensional chromatographic method for the separation of N-glycans. The method comprises providing a glycan preparation that includes at least one negatively charged N-glycan. The glycan preparation is then separated by anion-exchange chromatography and at least one secondary chromatographic technique.

First claim

1 . A method of characterizing individual species within a mixture of N-glycans, said method comprising steps of:
(i) providing a glycan preparation, wherein the glycan preparation includes at least one negatively charged N-glycan; and (ii) separating the glycan preparation by anion-exchange chromatography and at least one secondary chromatographic technique, wherein the secondary chromatographic technique is selected to resolve species that co-elute on the anion-exchange chromatography, thereby characterizing the individual species.

2 . A method of characterizing individual species a mixture of N-glycans, said method comprising steps of:
(i) cleaving N-linked glycans from a glycoprotein preparation to provide a glycan preparation that includes a mixture of N-glycans, wherein at least one of the N-glycans in the glycan preparation is negatively charged; and (ii) separating the glycan preparation by anion-exchange chromatography such that at least two species co-elute; (iii) separating the co-eluting species via at least one secondary chromatographic technique, thereby characterizing individual glycan species within the mixture of N-glycans.

3 . A method of characterizing individual species within a mixture of N-glycans, said method comprising steps of:
(i) cleaving N-linked glycans from a glycoprotein preparation to provide a glycan preparation that includes a mixture of N-glycans, wherein at least one of the N-glycans in the glycan preparation is negatively charged; and (ii) reacting the glycan preparation with a labeling agent to provide a glycan preparation that includes a mixture of labeled N-glycans; and then (iii) separating the glycan preparation by anion-exchange chromatography and at least one secondary chromatographic technique, wherein the secondary chromatographic technique is selected to resolve species that co-elute on the anion-exchange chromatography, thereby characterizing the individual species.

4 . The method of any one of claims 1 - 3 , wherein the at least one secondary chromatographic technique is selected from the group consisting of reversed phase liquid chromatography (RP), normal phase liquid chromatography (NP), ion-pairing reverse phase chromatography (IP-RP), size exclusion chromatography, affinity chromatography (AC), capillary electrophoresis (CE); fluorophore-assisted carbohydrate electrophoresis (FACE); electrochromatography, and micellar electrokinetic chromatography (MEKC).

5 . The method of any one of claims 1 - 3 , wherein the at least one secondary chromatographic technique is a reversed phase chromatographic technique.

6 . The method of claim 5 , wherein the reversed-phase chromatographic technique employs a C18 reverse phase resin.

7 . The method of claim 5 , wherein the reversed-phase chromatographic technique employs a porous graphitized-carbon (PGC) resin.

8 . The method of any one of claims 1 - 3 , wherein the at least one secondary chromatographic technique is a normal phase chromatographic technique.

9 . The method of claim 8 , wherein the normal phase chromatographic technique employs a modified silica gel.

10 . The method of claim 9 , wherein the modified silica gel is selected from the group consisting of cyano-modified silica, amine-modified silica, and amide-modified silica.

11 . The method of claim 10 , wherein the modified silica gel is an amide-modified silica.

12 . A method of characterizing a mixture of N-glycans, said method comprising steps of:
(i) providing a glycan preparation, wherein the glycan preparation includes at least one negatively charged N-glycan and a known quantity of a reference N-glycan, wherein the reference N-glycan is labeled with a labeling agent; (ii) separating the glycan preparation by anion-exchange chromatography and at least one secondary chromatographic technique; and (iii) quantifying at least one N-glycan in the glycan preparation relative to the reference N-glycan.

13 . The method of claim 12 , wherein the at least one secondary chromatographic technique is selected from the group consisting of reversed phase liquid chromatography (RP), normal phase liquid chromatography (NP), ion-pairing reverse phase chromatography (IP-RP), size exclusion chromatography, affinity chromatography (AC), capillary electrophoresis (CE); fluorophore assisted carbohydrate electrophoresis (FACE); electrochromatography, and micellar electrokinetic chromatography (MEKC).

14 . The method of claim 12 , wherein the at least one secondary chromatographic technique is a reversed phase chromatographic technique.

15 . The method of claim 12 , wherein the reversed-phase chromatographic technique employs a C18 reverse phase resin.

16 . The method of claim 12 , wherein the reversed-phase chromatographic technique employs a porous graphitized-carbon (PGC) resin.

17 . The method of claim 16 wherein the at least one secondary chromatographic technique is a normal phase chromatographic technique.

18 . The method of claim 17 , wherein the normal phase chromatographic technique employs a modified silica gel.

19 . The method of claim 18 , wherein the modified silica gel is selected from the group consisting of cyano-modified silica, amine-modified silica, and amide-modified silica.

20 . The method of claim 18 , wherein the modified silica gel is an amide-modified silica.

Assignees

• Momenta Pharmaceuticals Inc

Inventors

• Parsons Ian Christopher
• Zheng Ting
• Gunay Nur Sibel
• Bosques Carlos J

Classifications

• G01N2440/38

  addition of carbohydrates, e.g. glycosylation, glycation · CPC title

• B01D15/1878

  for multi-dimensional chromatography · CPC title

• G01N30/463

  for multidimensional chromatography · CPC title

• B01D15/166

  Fluid composition conditioning, e.g. gradient · CPC title

• G01N2400/00

  Assays, e.g. immunoassays or enzyme assays, involving carbohydrates · CPC title