Rapid and efficient de-glycosylation of glycoproteins
AssigneeBiocon Limited
InventorsAnushikha Thakur, Seija Rohil, Shrivardhan Patil, Meenakshi Sudhakaran, Laxmi Adhikary
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Abstract
The present invention discloses rapid and cost-effective method of de-glycosyation of a glycoprotein, wherein, glycoprotein is combined with anionic surfactant and reducing agent and non-ionic surfactant in order to obtain stable denatured glycoprotein. An endoglycosidase is further added to denatured glycoprotein to cleave N-linked glycans in order to obtain de-glycosylated protein. A rapid tool for assessing the protein conformation by partial de-glycosylation is also presented wherein the partial de-glycosylated protein is analysed using capillary electrophoresis (CE-SDS).
Claims17
A method of complete N-glycan de-glycosylation of a glycoprotein, wherein, the method comprising steps of:
- (a) providing a glycoprotein;
- (b) combining the glycoprotein with an anionic surfactant and reducing agent, wherein the reducing agent is in a sufficient amount to denature the glycoprotein and the anionic surfactant is in a concentration of 0.1% to 0.8%;
- (c) incubating components of step (b) at a temperature from 90° C. to 100° C. for 2 minutes to 5 minutes to provide for a denatured glycoprotein;
- (d) cooling the denatured glycoprotein;
- (e) combining the denatured glycoprotein with a non-ionic surfactant in an amount to counter the inhibitory effects of the anionic surfactant, where the non-ionic surfactant is in a concentration of 0.6% to 1.2%;
- (f) introducing an endoglyrosidase to cleave N-linked glycans in an amount from 0.66 unit to 10 units per 1 mg of denatured glycoprotein;
- (g) incubating components of step (f) at 37° C. for 1 to 15 minutes at atmospheric pressure to provide for a completely de-glycosylated protein;
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- (h) separating the completely N-glycan de-glycosylated protein from released glycans.
A method of partial de-glycosylation of a glycoprotein for comparing tertiary structure, wherein, the method comprising steps of:
- (a) providing a glycoprotein;
- (b) combining the glycoprotein with endoglycosidase to partially cleave N-linked glycans in an amount from 1 unit to 10 units per 1 mg of glycoprotein;
- (c) incubating components of step (b) at 37° C. for 45 minutes to 8 hours to provide for a partially de-glycosylated protein.
Dependencies are inferred from the claim text.