Separation of glycans by mixed-mode liquid chromatography
AssigneeDionex Corporation
InventorsXiaodong LIU, Udayanath AICH, Christopher A. POHL
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Abstract
An exemplary multimodal chromatographic medium of the invention includes one or more strong anion exchange, weak anion exchange, strong cation exchange and/or weak cation exchange binding sites in combination with one or more reverse phase and/or hydrophilic interaction chromatography binding site. In an exemplary embodiment, the sites interact with one or more glycans in a mixture of glycans in a manner that allows separation of glycans in the mixture and analysis of the glycan mixture. The media are incorporated into devices and systems for chromatographic analysis. Also provided are methods of using the multimodal media of the invention to analyze glycans.
Claims41
A multimodal chromatographic method of separating a first glycan component from a second glycan component of a glycan mixture, said method comprising:
- (a) contacting said glycan mixture with a multimodal chromatographic medium comprising an ion exchange chromatographic moiety bound to a first substrate and an uncharged chromatographic moiety bound to a second substrate, said uncharged chromatographic moiety selected from a reverse phase chromatographic moiety, a hydrophilic interaction chromatographic moiety and a combination thereof, and an aqueous eluent comprising an electrolyte and an organic solvent under conditions effective to achieve said separating, thereby separating said first glycan component and said second glycan component.
A method of improving the chromatographic separation of an unsialylated first glycan and an unsialylated second glycan on a multimodal chromatographic medium, said medium comprising an ion exchange chromatographic moiety bound to a first substrate and an uncharged chromatographic moiety bound to a second substrate, said uncharged chromatographic moiety selected from a reverse phase chromatographic moiety, a hydrophilic interaction chromatographic moiety and a combination thereof, said method comprising:
- (a) derivatizing said first unsialylated glycan and said second unsialylated glycan with a detectable label comprising a charged moiety, forming a labeled first glycan and a labeled second glycan;
- and
- (b) contacting said multimodal chromatographic medium with said labeled first glycan and said labeled second glycan under conditions effective to separate said first and second labeled glycans, wherein said separation is improved relative to a separation of said first unsialylated glycan and said second unsialylated glycan on said multimodal chromatographic medium.
A multimodal chromatographic medium, comprising:
- (a) a particulate silica substrate;
- (b) a first ion exchange binding site bound to said substrate;
- (c) an uncharged binding site which is a member selected from a hydrophilic interaction chromatographic binding site, and a reverse phase chromatographic binding site also bound to said substrate;
- and
- (d) a member selected from a second ion exchange binding site and a second uncharged binding site which is a member selected from a hydrophilic interaction chromatographic binding site, and a reverse phase chromatographic binding site also bound to said substrate.
Dependencies are inferred from the claim text.