Deglycosylation methods for electrophoresis of glycosylated proteins

Patent metadata

Field	Value
Publication number	US-11835527-B2
Application number	US-202117153150-A
Country	US
Kind code	B2
Filing date	Jan 20, 2021
Priority date	Jan 21, 2020
Publication date	Dec 5, 2023
Grant date	Dec 5, 2023

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, 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 protein of interest comprises at least one glycosylation site.

3. The method of claim 1 , wherein the protein of interest is a glycosylated protein.

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

5. The method of claim 4 , wherein the protein of interest comprises an antibody, an antibody fragment or an scFv.

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

7. The method of claim 1 , wherein the protein of interest comprises a receptor fusion protein.

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

9. The method of claim 1 , wherein the protein of interest is a recombinant human protein.

10. The method of claim 1 , wherein the protein of interest comprises at least one attached glycan that is N-linked.

11. The method of claim 1 , wherein the endoglycosidase catalyzes deglycosylation of N-linked glycans.

12. The method of claim 11 , 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.

13. The method of claim 11 , wherein the endoglycosidase is PNGase F.

14. The method of claim 13 , wherein the PNGase F is Rapid PNGase F.

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

16. The method of claim 13 , wherein deglycosylating the labeled sample comprises heating the sample to about 50° C. for 10 minutes.

17. The method of claim 16 , wherein deglycosylating the labeled 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.

18. The method of claim 1 , wherein the protein of interest comprises at least one glycan that is an O-linked glycan.

19. The method of claim 18 , wherein the endoglycosidase catalyzes deglycosylation of O-linked glycans.

20. The method of claim 19 , wherein the endoglycosidase comprises Endo-α-N acetylgalactosaminidase (O-glycosidase).

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

22. The method of claim 1 , wherein the sample is denatured using a reducing solution.

23. The method of claim 22 , wherein the reducing solution comprises dithiothreitol (DTT).

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

25. The method of claim 24 , wherein the non-reducing solution comprises iodoacetamide (IAM).

26. The method of claim 1 , wherein denaturing the sample comprises heating the sample to between 40° C. and 99° C. for between 1 minute and 60 minutes.

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

28. The method of claim 1 , further comprising analyzing a reference standard in parallel to the labeled sample after deglycosylating.

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

30. 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.

31. A method of determining stability of a protein of interest comprising:
a. stressing a sample comprising the protein of interest;
b. denaturing the stressed sample and a non-stressed sample comprising the protein of interest;
c. labeling the stressed sample and the non-stressed sample with a fluorescent label to produce a labeled stressed sample and a labeled non-stressed sample;
d. quenching un-reacted fluorescent label in the labeled stressed sample and the labeled non-stressed sample;
e. deglycosylating the labeled stressed sample and the labeled non-stressed sample with an endoglycosidase;
f. performing microchip capillary electrophoresis (MCE) on the labeled stressed sample and the labeled non-stressed sample after deglycosylating to generate electropherograms for the stressed sample and the non-stressed sample; and
g. comparing the electropherograms from the stressed sample and the nonstressed sample after deglycosylating, thereby determining the stability of the protein of interest;
wherein the stressed sample and the non-stressed sample are denatured, labeled and quenched in steps (b) through (d) prior to deglycosylation in step (e).

Assignees

• Regeneron Pharma

Inventors

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

Classifications

• 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

• G01N2400/00

  Assays, e.g. immunoassays or enzyme assays, involving carbohydrates · CPC title

• G01N21/6486

  Measuring fluorescence of biological material, e.g. DNA, RNA, cells (G01N21/6428 takes precedence) · CPC title

• C07K1/26

  Electrophoresis · CPC title