# US 2026/0086071 A1 · Monitoring polysorbate hydrolysis in pharmaceutical formulations using an ultrasensitive extraction-free fatty acid quantitation method > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2026/0086071 A1 - Document ID: US20260086071A1 - Status: Published application - Kind: A1 - Application number: 19/404,959 - Filed: 2025-12-01 - Published: 2026-03-26 - Earliest claimed: 2021-06-14 - Assignees: Regeneron Pharmaceuticals, Inc. - Applicants: Regeneron Pharmaceuticals, Inc. - Inventors: Sisi ZHANG; Hui Xiao; Ning Li - Web page: https://patentgrep.com/patent/US20260086071A1 ## CPC - 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)} - G01N 30/06: Preparation - G01N 33/6848: {Methods of protein analysis involving mass spectrometry} - G01N 33/6851: {Methods of protein analysis involving laser desorption ionisation mass spectrometry} - G01N 33/92: involving lipids, e.g. cholesterol {, lipoproteins, or their receptors (steroid hormones G01N33/743)} - 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 The present invention generally pertains to methods of quantifying free fatty acids in a pharmaceutical formulation. In particular, the present invention pertains to a method of quantifying free fatty acids released from polysorbate hydrolysis in a pharmaceutical formulation comprising a pharmaceutical product, polysorbate and free fatty acids, using liquid chromatography-mass spectrometry, without the use of an extraction step. ## Claims ### Claim 1 A method for quantifying free fatty acids in a pharmaceutical formulation comprising an antibody, polysorbate, and free fatty acids, the method comprising: (a) incubating said pharmaceutical formulation; (b) subjecting said formulation to liquid chromatography to separate said free fatty acids; and (c) using a mass spectrometer to quantify said free fatty acids, wherein said free fatty acids are not extracted from said formulation prior to subjecting said formulation to liquid chromatography. ### Claim 2 (depends on 1) The method of claim 1, further comprising adding internal free fatty acid standards to said formulation prior to incubating the formulation. ### Claim 3 (depends on 2) The method of claim 2, further comprising correcting said quantified free fatty acid concentrations to said internal standards. ### Claim 4 (depends on 1) The method of claim 1, further comprising generating a calibration curve using spiked-in fatty acids of known concentration, wherein said quantified free fatty acid concentrations are corrected to said calibration curve. ### Claim 5 (depends on 1) The method of claim 1, further comprising repeating the steps of claim 1 using at least one additional incubation time and comparing the quantification of free fatty acids obtained in each repetition to determine a rate of change of free fatty acid concentration over time. ### Claim 6 (canceled) ### Claim 7 (depends on 1) The method of claim 1, wherein said antibody is a monoclonal antibody. ### Claim 8 (depends on 1) The method of claim 1, wherein the concentration of said pharmaceutical product is between about 1 mg/mL and about 200 mg/mL. ### Claim 9 (depends on 1) The method of claim 1, wherein said polysorbate is polysorbate 20 or polysorbate 80, or wherein the concentration of said polysorbate is between about 0.001% and about 1%. ### Claim 10 (canceled) ### Claim 11 (depends on 1) The method of claim 1, wherein said free fatty acids are selected from a group comprising caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, oleic acid, stearic acid, linoleic acid, or a combination thereof, or wherein the concentration of said free fatty acids is between about 10 ng/ml and about 100 μg/mL. ### Claim 12 (canceled) ### Claim 13 (depends on 1) The method of claim 1, wherein said incubation is performed within a plastic tube or within a glass vial. ### Claim 14 (depends on 1) The method of claim 1, wherein said incubation is performed for between about 0 hours and about 36 months, or wherein said incubation is performed at between about 5° C. and about 37° C. ### Claim 15 (canceled) ### Claim 16 (depends on 2) The method of claim 2, wherein said internal standards are labeled with heavy isotopes, or wherein said internal standards are selected from a group comprising lauric-d23 acid, myristic-d27 acid, oleic acid-13C18, or a combination thereof. ### Claim 17 (canceled) ### Claim 18 (depends on 1) The method of claim 1, wherein said liquid chromatography system is reversed-phase chromatography and is coupled to said mass spectrometer. ### Claim 19 (canceled) ### Claim 20 (depends on 1) The method of claim 1, wherein said mass spectrometer is an electrospray ionization mass spectrometer. ### Claim 21 (depends on 1) The method of claim 1, wherein said mass spectrometer is a triple quadrupole mass spectrometer. ### Claim 22 (depends on 1) The method of claim 1, wherein said mass spectrometer is capable of performing LC-MS or LC-MRM-MS analyses. ### Claim 23 (depends on 1) The method of claim 1, wherein said formulation additionally comprises one or more excipients. ### Claim 24 (depends on 1) The method of claim 1, wherein said formulation additionally comprises histidine. ### Claim 25 (depends on 18) The method of claim 18, wherein a column used for said reversed-phase chromatography is an Acquity UPLC BEH C4 column. ### Claim 26 (depends on 9) The method of claim 9, wherein the concentration of said polysorbate is about 1%.