IP Library › Granted Patent US 12,247,983
Granted Patent B2
US 12,247,983 · App. 16/751,706 · Granted Mar 11, 2025

Protein A chromatography-electrospray ionization mass spectrometer

Inventor: Shunhai Wang (Scarsdale, NY)
Assignee: Regeneron Pharmaceuticals, Inc.
G01N33/6848G01N30/14G01N30/7206G01N30/7233G01N30/7266G01N33/6854H01J49/165G01N2560/00
View Patent ↗
Loading inventors, assignments & file history…
Monitor This Case
Get email alerts when status or documents change.
Order Certified Copies
Most orders are placed with the USPTO same day — all within 24 business hours.
Order via The Patent Place →
Pre-filled with this patent's details
Quick Facts
Patent No.
US 12,247,983
App. No.
16/751,706
Granted
Mar 11, 2025
Kind
B2
Abstract

Methods and system for characterizing a protein using a chromatographic system having a protein A chromatography resin and electrospray ionization mass spectrometer run under native conditions are provided.

Claims (16)

1. A method for identifying at least one protein, said method comprising:

contacting a sample including the protein to a chromatographic system having a protein A chromatography resin;

washing said protein A chromatography resin using a mobile phase to provide an eluate including the protein, wherein said eluate has a more acidic pH after washing; and

identifying the protein in said eluate using an electrospray ionization mass spectrometer run under native conditions,

wherein at least one splitter with at least three paths is used to couple said chromatographic system with said mass spectrometer and with an ultraviolet detector.

2. The method of claim 1 , wherein the electrospray ionization mass spectrometer is coupled online to the chromatographic system having the protein A chromatography resin.

3. The method of claim 1 , wherein the electrospray ionization mass spectrometer is a nano-electrospray ionization mass spectrometer.

4. The method of claim 1 , wherein the mobile phase used to wash the protein A chromatography resin comprises a volatile salt.

5. The method of claim 1 , wherein the eluate provided from washing the protein A chromatography resin is introduced in the electrospray ionization mass spectrometer, wherein a flow rate of electrospray from the electrospray ionization is about 10 nL/min to about 50 nL/min.

6. The method of claim 1 , wherein the protein is a monoclonal antibody.

7. The method of claim 1 , wherein the protein is a product related impurity.

8. The method of claim 1 , wherein the protein is a bispecific antibody.

9. The method of claim 1 , wherein the protein is an impurity.

10. The method of claim 1 , wherein the protein is a monoclonal antibody variant.

11. The method of claim 1 , wherein the sample comprises at least two proteins.

12. The method of claim 1 , wherein the sample is subjected to condition selected from the group consisting of deglycosylation, oxidation, heat, ultraviolet light, cool-white light, or combinations thereof.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Apr 6, 2021
From: WANG, SHUNHAI
To: REGENERON PHARMACEUTICALS, INC.
Reel/Frame 055840/0757 →
Continuity (2)
Provisional Application 62796820 · Jan 25, 2019
Related Publication 20200240999A1 · Jul 30, 2020
References Cited (10)
Karas, M. et al. Nano-electrospray ionization mass spectrometry: addressing analytical problems beyond routine, Fresenius J Anal Chem (2000) 366 :669-676 (Year: 2000). [cited by examiner]
Canarelli S. et al., “On-line microdialysis of proteins with high-salt buffers for direct coupling of electrospray ionization mass spectrometry and liquid chromatography,” Journal of Chromatography A., Elsevier, Amsterd… [cited by applicant]
Rabah Gahoual et al., “Detailed Characterization of Monoclonal Antibody Receptor Interaction Using Affinity Liquid Chromatography Hyphenated to Native Mass Spectrometry,” Analytical Chemistry, vol. 89, No. 10, May 16, 2… [cited by applicant]
Markus Haberger et al., “Rapid characterization of biotherapeutic proteins by size-exclusion chromatography coupled to native mass spectrometry,” MABS, vol. 8, No. 2,Dec. 10, 2015, pp. 331-339. [cited by applicant]
Dhanashri Bagal et al., “Resolving Disulfide Structural Isoforms of IgG2 Monoclonal Antibodies by Ion Mobility Mass Spectrometry,” Analytical Chemistry, vol. 82, No. 16, Aug. 15, 2010, pp. 6751-6755. [cited by applicant]
International Search Report PCT Application No. PCT/US2020/014961, International Filing Date Jan. 24, 2020, Date of Mailing May 11, 2020. [cited by applicant]
Kukrer B. et al. Mass spectrometric analysis of intact human monoclonal antibody aggregates fractionated by size-exclusion chromatography, Pharmaceutical research, 2010, V. 27, p. 2197-2204 (p. 2199). [cited by applicant]
Dumont E. et al. Cell Clone Selection Using the Agilent Bio-Monolith Protein A Column and LC/MS, Agilent Technologies Application Note, publication No. 5991-5124EN, 2014; p. 1-10. [cited by applicant]
Cuykens F., Clayes M. Optimization of a liquid chromatography method based on simultaneous electrospray ionization mass spectrometric and ultraviolet photodiode array detection for analysis of flavonoid glycosides. Rapi… [cited by applicant]
Chen W.H. et al. High performance liquid chromatography-tandem mass spectrometry method for ex vivo metabolic studies of a rhenium-labeled radiopharmaceutical for liver cancer. European Journal of Mass Spectrometry, 201… [cited by applicant]