# US 12,000,811 B2 · Method and system of identifying and quantifying antibody fragmentation > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,000,811 B2 - Document ID: US12000811B2 - Status: Granted - Kind: B2 - Application number: 17/951,614 - Filed: 2022-09-23 - Published: 2023-10-05 - Granted: 2024-06-04 - Earliest claimed: 2019-01-16 - Assignees: Regeneron Pharmaceuticals, Inc. - Applicants: Regeneron Pharmaceuticals, Inc. - Inventors: Yuetian Yan; Shunhai Wang - Web page: https://patentgrep.com/patent/US12000811B2 ## CPC - G01N 30/7266: {by electric field, e.g. electrospray} - G01N 30/88: Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 - G01N 33/563: involving antibody fragments - G01N 33/6848: {Methods of protein analysis involving mass spectrometry} - G01N 33/6857: {Antibody fragments} - G01N 30/34: of fluid composition, e.g. gradient (G01N30/36 takes precedence) - G01N 2030/8831: {involving peptides or proteins} - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract Methods and system for identifying and quantifying antibody fragments and identifying the site of fragmentation on an antibody are provided herein. ## Claims ### Claim 1 A method for quantification of a fragment of an antibody in a sample, said method comprising: contacting the sample to a chromatographic system having a mixed-mode size-exclusion chromatography resin with a charge-charge interaction functionality; washing the mixed-mode size-exclusion chromatography resin using a mobile phase to provide an eluent with the fragment; and quantifying the amount of the fragment of the antibody. ### Claim 2 (depends on 1) The method of claim 1, wherein the mobile phase can be selected from ammonium acetate, ammonium bicarbonate, or ammonium formate, or combinations thereof. ### Claim 3 (depends on 1) The method of claim 1, wherein the fragment is a degradation product of the antibody. ### Claim 4 (depends on 1) The method of claim 1, wherein the antibody is a monoclonal antibody. ### Claim 5 (depends on 1) The method of claim 1, wherein the antibody is a therapeutic antibody. ### Claim 6 (depends on 1) The method of claim 1, wherein the antibody is a bispecific antibody. ### Claim 7 (depends on 1) The method of claim 1, wherein the fragment can be a digestion product of the antibody. ### Claim 8 (depends on 1) The method of claim 1, wherein the mass spectrometer can be a tandem mass spectrometer. ### Claim 9 A method for identifying a fragment of an antibody in a sample, said method comprising; contacting the sample to a chromatographic system having a mixed-mode size-exclusion chromatography resin with a charge-charge interaction functionality; washing the mixed-mode size-exclusion chromatography resin using a mobile phase to provide an eluent with the fragment; determining the molecular weight of the fragment in the eluent using a mass spectrometer, wherein the mass spectrometer is coupled to the chromatographic system; and correlating the molecular weight data of the fragment to data obtained from at least one known protein standard to identify said fragment of said antibody; wherein the fragment is a degradation product of the antibody or the fragment is a digestion product of the antibody. ### Claim 10 (depends on 9) The method of claim 9, wherein the mobile phase can be selected from ammonium acetate, ammonium bicarbonate, or ammonium formate, or combinations thereof. ### Claim 11 (depends on 9) The method of claim 9, wherein the antibody is a monoclonal antibody. ### Claim 12 (depends on 9) The method of claim 9, wherein the antibody is a therapeutic antibody. ### Claim 13 (depends on 9) The method of claim 9, wherein the antibody is a bispecific antibody. ### Claim 14 (depends on 9) The method of claim 9, wherein the mass spectrometer can be a tandem mass spectrometer. ### Claim 15 A method for identifying a fragment of an antibody in a sample, said method comprising; contacting the sample to a chromatographic system having a mixed-mode size-exclusion chromatography resin with a charge-charge interaction functionality; washing the mixed-mode size-exclusion chromatography resin using a mobile phase to provide an eluent with the fragment; determining the molecular weight of the fragment in the eluent using a mass spectrometer, wherein the mass spectrometer is coupled to the chromatographic system; and correlating the molecular weight data of the fragment to data obtained from at least one known protein standard to identify said fragment of said antibody; wherein the mass spectrometer is a tandem mass spectrometer. ### Claim 16 (depends on 15) The method of claim 15, wherein the mobile phase can be selected from ammonium acetate, ammonium bicarbonate, or ammonium formate, or combinations thereof. ### Claim 17 (depends on 15) The method of claim 15, wherein the antibody is a monoclonal antibody. ### Claim 18 (depends on 15) The method of claim 15, wherein the antibody is a therapeutic antibody. ### Claim 19 (depends on 15) The method of claim 15, wherein the antibody is a bispecific antibody. ### Claim 20 (depends on 15) The method of claim 15, wherein the fragment is a degradation product of the antibody. ### Claim 21 (depends on 15) The method of claim 15, wherein the fragment is a digestion product of the antibody.