Isolation and deglycosylation of glycoproteins
AssigneeProZyme, Inc.
InventorsScott P. Fulton, Steven M. Murphy
Life of this patent
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Abstract
The invention provides more rapid and cost-effective methods of deglycosylating target glycoproteins. In methods of the invention, the target glycoprotein is isolated from initial samples, which may contain multiple other glycoproteins, by subjecting the initial sample to a solid phase containing an affinity ligand, such as a deglycosylated antibody, that interacts specifically with the target glycoprotein. Once separated from the sample, the target glycoprotein can be deglycosylated in situ, or eluted from the solid phase, quantitated, and then deglycosylated.
Claims25
A method of releasing glycans from a target glycoprotein in a biological sample, said method comprising performing the following steps in the following order:
- (a) adding said biological sample to a solid phase support comprising (i) a solid phase and (ii) an affinity ligand which binds to said target glycoprotein, which molecule is immobilized on said solid phase, thereby binding said target glycoprotein to said molecule on said solid phase;
- (b) washing said solid phase with a buffer to wash away any unbound glycoprotein;
- and,
- (c) contacting said bound target glycoprotein on said solid phase with an enzyme that releases glycans from glycoproteins,
- thereby releasing said glycans from said target glycoprotein.
A method of releasing glycans from a target glycoprotein in a biological sample, said method comprising performing the following steps in the following order:
- (a) adding said biological sample to a solid phase support comprising (i) a solid phase and (ii) an affinity ligand, which affinity ligand is immobilized on said solid phase, thereby binding said target glycoprotein to said molecule on said solid phase;
- (b) washing said bound target glycoprotein with a wash buffer to wash away any unbound glycoprotein;
- (c) releasing said bound target glycoprotein, and eluting said target glycoprotein from said solid phase support;
- (d) optionally, quantitating the amount of said target glycoprotein eluted from said solid phase support;
- and
- (e) contacting said target glycoprotein with an enzyme that releases glycans from glycoproteins,
- thereby releasing said glycans from said target glycoprotein.
Dependencies are inferred from the claim text.