Deglycosylation methods for electrophoresis of glycosylated proteins

Patent metadata

Field	Value
Publication number	US-12480955-B2
Application number	US-202318495290-A
Country	US
Kind code	B2
Filing date	Oct 26, 2023
Priority date	Jan 21, 2020
Publication date	Nov 25, 2025
Grant date	Nov 25, 2025

Abstract

The disclosure relates to methods of analyzing a post-translationally modified protein of interest using electrophoresis, the methods comprising deglycosylating the protein of interest after labeling.

First claim

What is claimed is:

1 . A method of analyzing a sample comprising a protein of interest, wherein the protein of interest comprises an Fc domain, the method comprising:
a. denaturing the sample; b. labeling the denatured sample with a fluorescent label to produce a labeled sample; c. quenching un-reacted fluorescent label in the labeled sample; d. deglycosylating the labeled sample with an endoglycosidase; and e. performing electrophoresis on the labeled sample after deglycosylating; wherein the sample is denatured, labeled and quenched in steps (a) through (c) prior to deglycosylation in step (d).

2 . The method of claim 1 , wherein the Fc domain is a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, or a human IgG4 Fc domain.

3 . The method of claim 1 , wherein the protein of interest comprises at least one glycosylation site.

4 . The method of claim 3 , wherein the protein of interest comprises between one and eight N-linked glycosylation sites.

5 . The method of claim 1 , wherein the protein of interest is a glycosylated protein comprising at least one attached glycan.

6 . The method of claim 1 , wherein the protein of interest comprises an antigen binding domain.

7 . The method of claim 1 , wherein the protein of interest comprises a receptor-Fc-fusion protein or an antibody.

8 . The method of claim 7 , wherein the receptor-Fc-fusion protein is a trap protein or a mini trap protein.

9 . The method of claim 8 , wherein the trap protein is an IL-1 trap protein, a VEGF trap protein or a TNF trap protein.

10 . The method of claim 8 , wherein the trap protein is selected from the group consisting of rilonacept, aflibercept, and etanercept.

11 . The method of claim 7 , wherein the antibody is a bispecific antibody.

12 . The method of claim 5 , wherein the at least one attached glycan is N-linked or O-linked.

13 . The method of claim 12 , wherein the endoglycosidase catalyzes deglycosylation of N-linked glycans.

14 . The method of claim 1 , wherein the endoglycosidase catalyzes deglycosylation of N-linked glycans, and wherein the endoglycosidase is selected from the group consisting of Peptide-N-Glycosidase F (PNGase F), Endoglycosidase H (Endo H), Endoglycosidase S (Endo S), Endoglycosidase D, Endoglycosidase F1, Endoglycosidase F2 and Endoglycosidase F4.

15 . The method of claim 14 , wherein the PNGase F is Rapid PNGase F.

16 . The method of claim 15 , wherein the Rapid PNGase F is non-reducing.

17 . The method of claim 15 , wherein deglycosylating the sample comprises heating the sample to about 50° C. for 10 minutes.

18 . The method of claim 15 , wherein deglycosylating the sample comprises a reaction mixture comprising between 0.2-1.5 mg labeled protein of interest, and between 1-5 μL Rapid PNGase F in a 10 μL reaction volume, excluding the volume of the Rapid PNGase F.

19 . The method of claim 12 , wherein the endoglycosidase catalyzes deglycosylation of O-linked glycans, and wherein the endoglycosidase comprises Endo-α-N-acetylgalactosaminidase (O-glycosidase).

20 . The method of claim 1 , wherein labeling the sample with the fluorescent label comprises heating the sample to about 35° C. for 10-30 minutes.

21 . The method of claim 1 , wherein the sample is denatured using a reducing solution or a non-reducing solution.

22 . The method of claim 21 , wherein the reducing solution comprises dithiothreitol (DTT), and wherein the non-reducing solution comprises iodoacetamide (IAM).

23 . The method of claim 1 , wherein denaturing the sample comprises heating the sample to between 50° C. and 99° C. for between 1 to 60 minutes.

24 . The method of claim 1 , wherein quenching the un-reacted fluorescent label comprises adding a stop solution.

25 . The method of claim 1 , wherein the electrophoresis is selected from the group consisting of gel electrophoresis, isoelectric focusing, capillary electrophoresis (CE) and microchip capillary electrophoresis (MCE).

26 . The method of claim 1 , wherein the method results in reduced free dye interference in the less than 20 kDa range and a reduced or absent endoglycosidase peak in an electropherogram when compared to an electropherogram generated using a sample labeled after deglycosylation.

Assignees

• Regeneron Pharma

Inventors

• Zhao Yiming
• Chen Hunter
• Wang shao-chun
• Riehlman Timothy
• Carreau Gabriel
• Wang Ying

Classifications

• G01N27/041

  of a solid body · CPC title

• G01N2570/00

  Omics, e.g. proteomics, glycomics or lipidomics; Methods of analysis focusing on the entire complement of classes of biological molecules or subsets thereof, i.e. focusing on proteomes, glycomes or lipidomes · CPC title

• G01N2440/38

  addition of carbohydrates, e.g. glycosylation, glycation · CPC title

• G01N33/582 Primary

  with fluorescent label · CPC title

• G01N27/44791

  Microapparatus (sample containers with integrated microfluidic structures B01L3/5027) · CPC title