Deglycosylation reagents and methods
AssigneeNew England Biolabs, Inc.
InventorsPaula Magnelli, Ellen Guthrie, Christopher H. Taron, Ming-Qun Xu, John Buswell
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Abstract
Compositions and methods are provided for efficiently preparing a completely deglycosylated antibody where efficiency is measured in relative amounts of reagents in soluble or lyophilized form, and time and temperature of the reaction. Compositions and methods are also provided for separating substantially all N-linked glycans from a glycosylated antibody and for preserving functionality of the antibody. The methods are compatible with glycan labeling and protease digestion without the need for prior purification steps.
Claims26
A composition, comprising:
- (i) a bile salt or a dialyzable non-cleavable carboxylated anionic surfactant;
- (ii) peptide-N-glycosidase F (PNGase F);
- (iii) a deglycosylated antibody wherein the deglycosylated antibody is an N-glycosylated antibody from which at least 90% of the N-glycans have been removed;
- and
- (iv) glycan cleavage products;
- wherein the composition does not comprise sodium dodecyl sulfate (SDS).
A method for removing N-glycans from an antibody, comprising incubating a composition lacking SDS comprising:
- i. a bile salt or a dialyzable non-cleavable carboxylated anionic surfactant,
- ii. peptide-N-glycosidase F (PNGase F), and
- iii. an N-glycosylated antibody,
- for less than 60 minutes, to remove at least 90% of the N-glycans from the N-glycosylated antibody to produce-a deglycosylated antibody.
Dependencies are inferred from the claim text.