Method and System of Identifying and Quantifying A Protein

• US 20200132697A1
• Filed: 10/30/2019
• Published: 04/30/2020
• Est. Priority Date: 10/31/2018
• Status: Active Grant

• Current Assignee: Regeneron Pharmaceuticals Incorporated
• Original Assignee: Regeneron Pharmaceuticals Incorporated
• Inventors: Yan, Yuetian, Wang, Shunhai
• Granted Patent: US 11,385,239 B2
• Time in Patent Office: Days
• CPC Class Codes: G01N 2030/8831 involving peptides or proteins
• CPC Class Codes: G01N 2440/00 Post-translational modifications [PTMs] in chemical analysis of biological material
• CPC Class Codes: G01N 30/06 Preparation
• CPC Class Codes: G01N 30/34 of fluid composition, e.g. gradient G01N30/36 takes precedence
• CPC Class Codes: G01N 30/72 Mass spectrometers mass spectrometers per se H01J49/00
• CPC Class Codes: G01N 30/7233 interfaced to liquid or supercritical fluid chromatograph interfaces in general for introducing or extracting samples to be analysed with specially adapted mass spectrometer, see H01J49/04
• CPC Class Codes: G01N 33/6848 Methods of protein analysis involving mass spectrometry
• CPC Class Codes: G01N 33/6854 Immunoglobulins

Abstract

Methods and system for identifying and/or quantifying a protein are provided herein.

Claims

1. A method for quantifying an impurity in a sample, said method comprising:

contacting said sample to a chromatographic system having a mixed-mode size exclusion chromatography resin with an additional functionality;

washing said mixed-mode size exclusion chromatography resin using a mobile phase to provide an eluent including the impurity; and

quantifying an amount of the impurity in said eluent using a mass spectrometer.

2. The method of claim 1, wherein the mobile phase used to elute the impurity has ammonium acetate, ammonium bicarbonate, or ammonium formate, or combinations thereof

3. The method of claim 1, wherein the mobile phase used to elute the impurity has a total concentration of less than about 600 mM of ammonium acetate and ammonium bicarbonate.

4. The method of claim 1, wherein the mobile phase used to elute the impurity has a flow rate of about 0.2 ml/min-about 0.4 ml/min.

5. The method of claim 1, wherein the amount of the sample loaded onto the mixed-mode size exclusion chromatography resin is about 10 μ

g to about 100 μ

g.

6. The method of claim 1, wherein the impurity is a product-related impurity.

7. The method of claim 1, wherein the impurity is a homodimer.

8. The method of claim 1, wherein the sample includes the impurity and at least one target protein that are separated when eluted.

9. The method of claim 8, wherein the target protein is an antibody.

10. The method of claim 9, wherein the antibody is a bispecific antibody.

11. The method of claim 8, wherein the target protein is a therapeutic antibody.

12. The method of claim 1, wherein the mass spectrometer is coupled to the chromatographic system.

13. The method of claim 1, wherein the additional functionality is a hydrophobic interaction functionality.

14. The method of claim 1, wherein the additional functionality is a charge-charge interaction functionality.

15. The method of claim 1, wherein the mass spectrometer is a native mass spectrometer.

16. A method for detecting an impurity in a sample, said method comprising:

contacting said sample to a chromatographic system having a mixed-mode size exclusion chromatography resin with an additional functionality;

washing said mixed-mode size exclusion chromatography resin using a mobile phase to provide an eluent including the impurity; and

detecting the impurity in said eluent using a mass spectrometer.

17. (canceled)

18. (canceled)

19. A mixed-mode chromatography system comprising:

a chromatographic column having a mixed-mode size exclusion chromatography resin with an additional functionality, wherein the column is capable of receiving a mobile phase and a sample having a target protein, and

a mass spectrometer coupled to said chromatographic column for detecting the target protein.

20. The mixed-mode chromatography system of claim 19, wherein the additional functionality is a hydrophobic interaction functionality.

21. The mixed-mode chromatography system of claim 19, wherein the additional functionality is a charge-charge interaction functionality.

22. The mixed-mode chromatography system of claim 19, wherein the system is further capable of quantifying a target protein.