Granted Patent B2

US 12,693,274 · App. 18/104,669 · Granted Jul 28, 2026

Inventors: Shunhai Wang (Scarsdale, NY); Yuetian Yan (Chappaqua, NY)

Assignee: Regeneron Pharmaceuticals, Inc.

G01N30/7266G01N30/38G01N30/7273G01N2030/027

Abstract

Claims (28)

1 . A native liquid chromatography-mass spectrometry system, comprising:

a liquid chromatography system capable of separating a sample; and

an electrospray ionization mass spectrometry (ESI-MS) system in fluid communication with the liquid chromatography system, wherein the ESI-MS system comprises a multi-nozzle electrospray ionization emitter, a system for modifying a desolvation gas, wherein the desolvation gas is modified with about 5% ammonia in acetonitrile, and a mass spectrometer, wherein the mass spectrometer is configured to receive ions and characterize mass to charge ratio of ions.

2 . The native liquid chromatography-mass spectrometry system of claim 1 , wherein the system for modifying a desolvation gas comprises a container having a cap and comprising an organic solvent and an additional chemical component comprising an acid or a base, wherein the cap has an inlet line port and an outlet line port; a sheath gas inlet line for providing a sheath gas to the inlet line port; and a modified desolvation gas outlet line capable of connecting the outlet line port to the multi-nozzle electrospray ionization emitter.

3 . The native liquid chromatography-mass spectrometry system of claim 2 , wherein the acid is trifluoroacetic acid and wherein the base is triethylamine.

4 . The native liquid chromatography-mass spectrometry system of claim 2 , wherein the sheath gas is nitrogen.

5 . The native liquid chromatography-mass spectrometry system of claim 2 , wherein the sheath gas inlet line is partially inserted into to the inlet line port;

wherein the modified desolvation gas outlet line is partially inserted into to the outlet line port; and

wherein the sheath gas flows from the sheath gas inlet line through the container containing the organic solvent into the modified desolvation gas outlet line.

6 . The native liquid chromatography-mass spectrometry system of claim 1 , wherein the multi-nozzle electrospray ionization emitter includes eight nozzles.

7 . The native liquid chromatography-mass spectrometry system of claim 1 , wherein the liquid chromatography system comprises a size exclusion chromatography (SEC) column or an ion exchange chromatography (IEX) column.

8 . The native liquid chromatography-mass spectrometry system of claim 1 , wherein the native liquid chromatography-mass spectrometry system further comprises an analytical flow splitter for adjusting flow rate from the liquid chromatography system to the mass spectrometer, wherein the analytical flow splitter is capable of providing an electrospray with a solvent flow rate of about 1 to 5 μL/min.

9 . The native liquid chromatography-mass spectrometry system of claim 1 , wherein the mass spectrometer comprises an ion trap mass analyzer.

10 . A method of characterizing a non-reduced intact protein in a sample, comprising:

supplying the sample to a native liquid chromatography system capable of sample separation and fragmentation to obtain a fragment sample, the liquid chromatography system comprising a size exclusion chromatography system (SEC) column or an ion exchange chromatography (IEX) column;

analyzing the fragmented sample by use of an electrospray ionization mass spectrometry (ESI-MS) system in fluid communication with the liquid chromatography system, wherein the ESI-MS system comprises a multi-nozzle electrospray ionization emitter, a system for modifying a desolvation gas, wherein the desolvation gas is modified with about 5% ammonia in acetonitrile, and a mass spectrometer, wherein the mass spectrometer is configured to receive ions and characterize mass to charge ratio of ions to identify components of the protein to characterize the protein; and

wherein the method does not require deglycosylation of the protein in the sample prior to supplying the sample to the liquid chromatography system.

11 . The method of claim 10 , wherein the protein is an antibody, a fusion protein, recombinant protein, or a combination thereof.

12 . The method of claim 11 , wherein the antibody is a monoclonal antibody of isotype IgG1, IgG2, IgG3, IgG4, or mixed isotype.

13 . The method of claim 10 , wherein the system for modifying a desolvation gas comprises a container having a cap, the container comprising an organic solvent and an additional chemical component comprising an acid or a base, wherein the cap has an inlet line port and an outlet line port; a sheath gas inlet line for providing a sheath gas to the inlet line port; and a modified desolvation gas outlet line capable of connecting the outlet line port to the multi-nozzle electrospray ionization emitter.

14 . The method of claim 13 , wherein the acid is trifluoroacetic acid and wherein the base is triethylamine.

15 . The method of claim 13 , wherein the organic solvent is acetonitrile and/or wherein the sheath gas is nitrogen.

16 . The method of claim 13 , wherein the sheath gas inlet line is partially inserted into to the inlet line port;

wherein the modified desolvation gas outlet line is partially inserted into to the outlet line port; and

wherein the sheath gas flows from the sheath gas inlet line through the container containing the organic solvent into the modified desolvation gas outlet line.

17 . The method of claim 10 , wherein the multi-nozzle electrospray ionization emitter includes eight nozzles.

18 . The method of claim 10 , further comprising an analytical flow splitter for adjusting flow rate from the liquid chromatograph system to the mass spectrometer, wherein the analytical flow splitter is capable of providing an electrospray with a solvent flow rate of about 1 to 5 μL/min.

19 . The method of claim 10 , wherein the mass spectrometer comprises an ion trap mass analyzer.

Assignments (1)

Continuity (4)

References Cited (36)