Microfluidic glycan analysis
AssigneeAgilent Technologies, Inc.
InventorsMagdalena Anna Ostrowski, Rudolf Grimm, Kevin P. Killeen, Karla M. Robotti
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Abstract
Microfluidic devices and methods for analyzing glycan profiles of glycoproteins are provided. Some embodiments of the devices comprise a deglycosylation column for cleaving glycans, an optional cleaning column for removing proteins, a trapping column for enriching glycans, and a separation column for resolving glycans. The devices and methods significantly improve the speed and sensitivity of glycan analysis.
Claims17
A method for analyzing the carbohydrate moieties of glycoproteins, comprising:
- applying a sample comprising glycoproteins to a deglycosylation column, wherein the glycoproteins in the deglycosylation column are digested to result in cleaved carbohydrates and at least some of the cleaved carbohydrates remain in amino glycan forms;
- without precipitating the cleaved carbohydrates, binding the cleaved carbohydrates to a trapping column;
- eluting the cleaved carbohydrates from the trapping column;
- and
- separating amino glycan forms of the cleaved carbohydrates from hydroxyl for of the cleaved carbohydrates using a separation column to resolve the cleaved carbohydrates, wherein the separating of the cleaved carbohydrates comprises separating isomers of the cleaved carbohydrates;
- and
- separately analyzing the resolved cleaved carbohydrates.
A method for separating the carbohydrate moieties of glycoproteins, comprising:
- obtaining a microfluidic device comprising:
- a deglycosylation column comprising a solid support and an enzyme immobilized to the solid support, wherein the enzyme is capable of cleaving carbohydrates from glycoproteins;
- a trapping column that is capable of binding carbohydrates;
- a separation column capable of separating carbohydrates;
- a plurality of inlet/outlet ports;
- and
- a switching element comprising at least one channel;
- applying a sample that may comprise glycoproteins to the deglycosylation column to result in cleaved carbohydrates, wherein at least some of the cleaved carbohydrates remain in amino glycan forms;
- switching the device to a first state that allows fluid communication between the deglycosylation column and the trapping column to bind the cleaved carbohydrates to the trapping column, without precipitating the cleaved carbohydrates;
- switching the device to a second state that allows fluid communication between the trapping column and the separation column to elute the cleaved carbohydrates to the separating column;
- and
- separating amino glycan forms of the cleaved carbohydrates from hydroxyl forms of the cleaved carbohydrates using the separation column to resolve the cleaved carbohydrates.
Dependencies are inferred from the claim text.