# US 8,541,178 B2 · Analysis of antibody drug conjugates by bead-based affinity capture and mass spectrometry > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,541,178 B2 - Document ID: US8541178B2 - Status: Granted - Kind: B2 - Application number: 12/464,250 - Filed: 2009-05-12 - Published: 2009-11-19 - Granted: 2013-09-24 - Earliest claimed: 2008-05-13 - Assignees: Genentech, Inc. - Applicants: Surinder Kaur; Ola Saad; Keyang Xu - Inventors: Surinder Kaur; Ola Saad; Keyang Xu - Web page: https://patentgrep.com/patent/US8541178B2 ## CPC - G01N 33/54326: {Magnetic particles} - A61P 35/00: Antineoplastic agents - A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 - G01N 33/531: Production of immunochemical test materials - G01N 33/6848: {Methods of protein analysis involving mass spectrometry} - G01N 33/6854: {Immunoglobulins} - G01N 33/94: involving narcotics {or drugs or pharmaceuticals, neurotransmitters or associated receptors} - 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) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS ## Abstract Methods to detect, characterize, and quantitate biological samples after administration of antibody conjugates, antibody-drug conjugates of Formula I, antibodies, and fragments and metabolites thereof, by immunoaffinity bead separation, chromatography, and mass spectrometry are disclosed; Ab-(L-D)p I wherein Ab is an antibody; D is a drug moiety; L is a linker covalently attached to Ab, and covalently attached to D; and p is 1, 2, 3, 4, 5, 6, 7, or 8. ## Claims ### Claim 1 A method for detecting antibody-drug conjugate compounds comprising: (i) providing an antibody-drug conjugate compound having Formula I: Ab-(L-D)p I wherein Ab is an antibody, wherein the antibody is specific against an extracellular domain (ECD) of a target receptor protein; D is a maytansinoid or monomethylauristatin drug moiety; L is a linker covalently attached to Ab, and covalently attached to D; and p is 1, 2, 3, 4, 5, 6, 7, or 8; (ii) contacting the antibody-drug conjugate compound with a biological source selected from a mammal, tissue, cell culture, plasma or serum; (iii) collecting a biological sample that has been contacted by the antibody-drug conjugate from the biological source; (iv) processing the biological sample to form an analysis sample by formulating, immobilizing, centrifuging, isolating, digesting, inducing or preventing blood cell clotting, hydrolyzing, or purifying to form a processed analysis sample; (v) capturing the processed analysis sample on immunoaffinity beads comprising an antigen specific for the antibody of the antibody-drug conjugate, wherein the antigen is the extracellular domain (ECD) of the target receptor protein; (vi) eluting the processed analysis sample; (vii) applying the processed eluted analysis sample to a separation media to effect separation of more than one sample constituent by capillary liquid chromatography wherein a separated sample constituent comprises the antibody-drug conjugate compound; (viii) establishing the mass to charge ratio of one or more separated sample constituents by mass spectrometry wherein the intact antibody-drug conjugate is detected. ### Claim 2 (depends on 1) The method of claim 1 further comprising repeating steps (iii) to (viii) one or more times. ### Claim 3 (depends on 1) The method of claim 1 wherein the biological sample is blood and the blood is processed to form plasma or serum. ### Claim 4 (depends on 1) The method of claim 1 wherein the analysis sample is denatured. ### Claim 5 (depends on 1) The method of claim 1 wherein the analysis sample is denatured by a denaturing reagent selected from formamide, dimethylformamide and acetonitrile. ### Claim 6 (depends on 1) The method of claim 1 wherein the analysis sample is treated with a reducing agent. ### Claim 7 (depends on 6) The method of claim 6 wherein the reducing agent is DTT or TCEP. ### Claim 8 (depends on 1) The method of claim 1 wherein the ECD is biotinylated. ### Claim 9 (depends on 8) The method of claim 8 wherein the biotinylated ECD binds to streptavidin coated paramagnetic immunoaffinity bead. ### Claim 10 (depends on 1) The method of claim 1 wherein the antigen is an anti-drug antibody. ### Claim 11 (depends on 1) The method of claim 1 wherein the immunoaffinity bead is a magnetic bead. ### Claim 12 (depends on 1) The method of claim 1 wherein the immunoaffinity bead comprises a porous polymer monolith. ### Claim 13 (depends on 1) The method of claim 1 wherein the immunoaffinity bead is configured in a flow-through channel in fluid communication with a collection reservoir. ### Claim 14 (depends on 13) The method of claim 13 wherein the immunoaffinity bead is configured in a flow-through vessel wherein the sample from the biological source is introduced at one end or orifice, and a sample is eluted from another end or orifice. ### Claim 15 (depends on 14) The method of claim 14 wherein the immunoaffinity bead is distributed in a plurality of flow-through vessels, each in communication with a separate collection reservoir. ### Claim 16 (depends on 15) The method of claim 15 wherein the vessels and reservoirs are configured in a 96 microtitre well format of 12×8 columns and rows, or a 384 microtitre well format of 24×16 columns and rows. ### Claim 17 (depends on 1) The method of claim 1 further comprising the step of treating the analysis sample with a degycosylating reagent. ### Claim 18 (depends on 1) The method of claim 1 wherein the deglycosylating reagent is PNGaseF. ### Claim 19 (depends on 1) The method of claim 1 wherein the separation media is a chromatography support. ### Claim 20 (depends on 19) The method of claim 19 wherein the chromatography support is a reverse-phase adsorbent. ### Claim 21 (depends on 20) The method of claim 20 wherein the reverse phase is polystyrene, or a graft or copolymer of polystyrene. ### Claim 22 (depends on 20) The method of claim 20 wherein an effluent from the chromatography support is intermittently analyzed by mass spectrometry to establish the mass to charge ratio of more than one of the separated cleared constituents. ### Claim 23 (depends on 1) The method of claim 1 wherein the antibody-drug conjugate compound is administered to a mammal at a dose of 0.1 to 10 mg/kg body weight. ### Claim 24 (depends on 1) The method of claim 1 wherein L is covalently attached to an amino, carboxyl or thiol of Ab. ### Claim 25 (depends on 1) The method of claim 1 wherein L is formed from a linker reagent selected from N-succinimidyl-4(2-pyridylthio)propanoate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP). ### Claim 26 (depends on 1) The method of claim 1 wherein L is selected from maleimidocaproyl (MC), maleimidopropanoyl (MP), and maleimidocaproyl-valine-citrulline -para-aminobenzyloxycarbonyl(MC-vc-PAB). ### Claim 27 (depends on 1) The method of claim 1 wherein D is DM1, having the structure: ### Claim 28 (depends on 1) The method of claim 1 wherein D is a monomethylauristatin. ### Claim 29 (depends on 28) The method of claim 28 wherein D is MMAE, having the structure: ### Claim 30 (depends on 28) The method of claim 28 wherein D is MMAF, having the structure: