Claims for Patent: 10,338,039

Summary for Patent: 10,338,039

Title:	Method for detecting monoclonal antibody using mass spectrometry
Abstract:	A method is provided for more easily detecting and quantifying a protein by regioselectively digesting a variable region of an Fab domain of a monoclonal antibody while suppressing proteolysis of an Fc domain. In the method, a porous body in which a monoclonal antibody is immobilized in pores and nanoparticles on which a protease is immobilized are brought into contact with each other in a liquid to perform selective proteolysis of the monoclonal antibody, and a resulting peptide fragment is detected using liquid chromatography-mass spectrometry (LC-MS), and a peptide having an amino acid sequence that includes an amino acid derived from a CDR2 region of a heavy chain or a light chain of the monoclonal antibody is detected.
Inventor(s):	Shimada; Takashi (Kyoto, JP), Iwamoto; Noriko (Kyoto, JP)
Assignee:	SHIMADZU CORPORATION (Kyoto-shi, JP)
Application Number:	15/556,018
Patent Claims:	1. A method of detecting a monoclonal antibody, comprising: bringing into contact in a liquid a porous body having pores in which a monoclonal antibody is immobilized and nanoparticles on which a protease is immobilized such that a selective proteolysis of the monoclonal antibody occurs; and detecting a peptide fragment by multiple reaction monitoring under conditions described below using liquid chromatography-mass spectrometry, TABLE-US-00009 Trastuzumab or trastuzumab-DM1 quantitation peptide and its mass spectrometry information Reten- Q1 Colli- Q3 Parent Frag- tion Pre sion Pre ion ment time Bias Energy Bias Peptide Sequence m/z ion m/z [minutes] [V] [V] [V] IYPTNGYTR 542.8 808.4 3.405 -40 -18 -30 (SEQ ID NO. 1) IYPTNGYTR 542.8 711.3 3.405 -40 -26 -38 (SEQ ID NO. 1) IYPTNGYTR 542.8 610.3 3.405 -40 -24 -22 (SEQ ID NO. 1) IYPTNGYTR 542.8 404.7 3.405 -20 -18 -15 (SEQ ID NO. 1) IYPTNGYTR 542.8 277.2 3.405 -20 -16 -30 (SEQ ID NO. 1) FTISADTSK 485.2 822.4 3.635 -18 -18 -32 (SEQ ID NO. 3) FTISADTSK 485.2 721.4 3.635 -11 -18 -26 (SEQ ID NO. 3) FTISADTSK 485.2 608.3 3.635 -18 -19 -32 (SEQ ID NO. 3) FTISADTSK 485.2 521.3 3.635 -18 -19 -38 (SEQ ID NO. 3) FTISADTSK 485.2 335.2 3.635 -18 -24 -24 (SEQ ID NO. 3) GLEWVAR 415.7 660.3 4.077 -30 -15 -24 (SEQ ID NO. 6) GLEWVAR 415.7 531.3 4.077 -30 -16 -40 (SEQ ID NO. 6) GLEWVAR 415.7 345.2 4.077 -16 -17 -24 (SEQ ID NO. 6) GLEWVAR 415.7 246.2 4.077 -30 -18 -17 (SEQ ID NO. 6) NTAYLQMNSLR 655.8 1095.6 4.144 -24 -23 -42 (SEQ ID NO. 7) NTAYLQMNSLR 655.8 1024.5 4.144 -24 -22 -40 (SEQ ID NO. 7) NTAYLQMNSLR 655.8 861.5 4.144 -24 -22 -32 (SEQ ID NO. 7) NTAYLQMNSLR 655.8 748.4 4.144 -24 -23 -28 (SEQ ID NO. 7) NTAYLQMNSLR 655.8 620.3 4.144 -24 -23 -32 (SEQ ID NO. 7) NTAYLQMNSLR 655.8 489.3 4.144 -24 -23 -25 (SEQ ID NO. 7) NTAYLQMNSLR 655.8 375.2 4.144 -24 -22 -19 (SEQ ID NO. 7) wherein the porous body has an average pore diameter in a range of 10 nm-200 nm, the nanoparticles have an average particle size in a range of 50 nm-500 nm, provided that the average particle size of the nanoparticles is larger than the average pore diameter of the porous body, the monoclonal antibody is trastuzumab or trastuzumab-DM1, and the peptide fragment includes the amino acid sequence of SEQ ID No: 1, 3, 6 and/or 7. 2. A method of detecting a monoclonal antibody, comprising: bringing into contact in a liquid a porous body having pores in which the monoclonal antibody is immobilized and nanoparticles on which a protease is immobilized such that a selective proteolysis of the monoclonal antibody occurs; and detecting a peptide fragment by multiple reaction monitoring under conditions described below using liquid chromatography-mass spectrometry, TABLE-US-00010 Bevacizumab quantitation peptide and its mass spectrometry information Parent Retention Q1 Pre Collision Q3 Pre ion Fragment time Bias Energy Bias Peptide Sequence m/z ion m/z [minutes] [V] [V] [V] STAYLQMNSLR 642.3 1024.5 4.147 -24 -25 -40 (SEQ ID NO. 9) STAYLQMNSLR 642.3 861.5 4.147 -24 -25 -20 (SEQ ID NO. 9) STAYLQMNSLR 642.3 748.4 4.147 -24 -22 -28 (SEQ ID NO. 9) STAYLQMNSLR 642.3 620.3 4.147 -24 -24 -32 (SEQ ID NO. 9) STAYLQMNSLR 642.3 489.3 4.147 -24 -22 -25 (SEQ ID NO. 9) STAYLQMNSLR 642.3 375.2 4.147 -24 -22 -27 (SEQ ID NO. 9) FTFSLDTSK 523.3 898.5 4.432 -38 -20 -34 (SEQ ID NO. 10) FTFSLDTSK 523.3 797.4 4.432 -38 -18 -30 (SEQ ID NO. 10) FTFSLDTSK 523.3 650.3 4.432 -38 -19 -34 (SEQ ID NO. 10) FTFSLDTSK 523.3 563.3 4.432 -38 -22 -40 (SEQ ID NO. 10) FTFSLDTSK 523.3 450.2 4.432 -20 -23 -30 (SEQ ID NO. 10) FTFSLDTSK 523.3 335.2 4.432 -20 -25 -24 (SEQ ID NO. 10) FTFSLDTSK 523.3 399.2 4.432 -38 -17 -11 (SEQ ID NO. 10) FTFSLDTSK 523.3 249.1 4.432 -38 -17 -17 (SEQ ID NO. 10) VLIYFTSSLHSGVPSR 588.3 832.4 4.515 -22 -19 -30 (SEQ ID NO. 11) VLIYFTSSLHSGVPSR 588.3 775.9 4.515 -22 -18 -28 (SEQ ID NO. 11) VLIYFTSSLHSGVPSR 588.3 719.4 4.515 -22 -18 -26 (SEQ ID NO. 11) VLIYFTSSLHSGVPSR 588.3 637.8 4.515 -22 -18 -24 (SEQ ID NO. 11) VLIYFTSSLHSGVPSR 588.3 564.3 4.515 -22 -22 -20 (SEQ ID NO. 11) VLIYFTSSLHSGVPSR 588.3 602.3 4.515 -22 -28 -22 (SEQ ID NO. 11) VLIYFTSSLHSGVPSR 588.3 359.2 4.515 -22 -26 -26 (SEQ ID NO. 11) VLIYFTSSLHSGVPSR 588.3 213.2 4.515 -22 -20 -23 (SEQ ID NO. 11) wherein the porous body has an average pore diameter in a range of 10 nm-200 nm, the nanoparticles have an average particle size in a range of 50 nm-500 nm, provided that the average particle size of the nanoparticles is larger than the average pore diameter of the porous body, the monoclonal antibody is bevacizumab, and the peptide fragment includes the amino acid sequence of at least one of SEQ ID No: 9-11. 3. A method of detecting a monoclonal antibody, comprising: bringing into contact in a liquid a porous body having pores in which a monoclonal antibody is immobilized and nanoparticles on which a protease is immobilized such that a selective proteolysis of the monoclonal antibody occurs; and detecting a peptide fragment by multiple reaction monitoring under conditions described below using liquid chromatography-mass spectrometry, TABLE-US-00011 Rituximab quantitation peptide and its mass spectrometry information Retention Parent ion Fragment time Q1 Pre Collision Q3 Pre Peptide Sequence m/z ion m/z [minutes] Bias [V] Energy [V] Bias [V] ATLTADK 360.2 648.4 2.809 -26 -14 -24 (SEQ ID NO. 13) ATLTADK 360.2 547.3 2.81 -13 -13 -20 (SEQ ID NO. 13) ATLTADK 360.2 434.2 2.81 -26 -16 -22 (SEQ ID NO. 13) ATLTADK 360.2 333.2 2.81 -26 -19 -24 (SEQ ID NO. 13) ATLTADK 360.2 262.1 2.81 -13 -21 -29 (SEQ ID NO. 13) LASGVPVR 399.7 685.4 3.426 -29 -17 -34 (SEQ ID NO. 15) LASGVPVR 399.7 614.4 3.426 -30 -16 -32 (SEQ ID NO. 15) LASGVPVR 399.7 527.3 3.426 -15 -16 -38 (SEQ ID NO. 15) LASGVPVR 399.7 371.2 3.426 -15 -17 -28 (SEQ ID NO. 15) ATSNLASGVPVR 586.3 912.5 3.728 -22 -25 -34 (SEQ ID NO. 16) ATSNLASGVPVR 586.3 685.4 3.728 -22 -23 -38 (SEQ ID NO. 16) ATSNLASGVPVR 586.3 614.4 3.728 -22 -25 -24 (SEQ ID NO. 16) ATSNLASGVPVR 586.3 527.3 3.728 -22 -25 -40 (SEQ ID NO. 16) ATSNLASGVPVR 586.3 470.3 3.728 -22 -19 -17 (SEQ ID NO. 16) ATSNLASGVPVR 586.3 371.2 3.728 -22 -17 -27 (SEQ ID NO. 16) wherein the porous body has an average pore diameter in a range of 10 nm-200 nm, the nanoparticles have an average particle size in a range of 50 nm-500 nm, provided that the average particle size of the nanoparticles is larger than the average pore diameter of the porous body, the monoclonal antibody is rituximab, and the peptide fragment includes the amino acid sequence of SEQ ID No: 13, 15 and/or 16. 4. The method according to claim 3, wherein the peptide fragment includes the amino acid sequence of SEQ ID No: 13. 5. The method according to claim 3, wherein the peptide fragment includes the amino acid sequence of SEQ ID No: 15. 6. The method according to claim 3, wherein the peptide fragment includes the amino acid sequence of SEQ ID No: 16. 7. The method according to claim 1, wherein the peptide fragment includes the amino acid sequence of SEQ ID No: 1. 8. The method according to claim 1, wherein the peptide fragment includes the amino acid sequence of SEQ ID No: 3. 9. The method according to claim 1, wherein the peptide fragment includes the amino acid sequence of SEQ ID No: 6. 10. The method according to claim 1, wherein the peptide fragment includes the amino acid sequence of SEQ ID No: 7. 11. The method according to claim 2, wherein the peptide fragment includes the amino acid sequence of SEQ ID No: 9. 12. The method according to claim 2, wherein the peptide fragment includes the amino acid sequence of SEQ ID No: 10. 13. The method according to claim 2, wherein the peptide fragment includes the amino acid sequence of SEQ ID No: 11.

Details for Patent 10,338,039

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Genentech, Inc.	RITUXAN	rituximab	Injection	103705	11/26/1997		2035-03-09
Genentech, Inc.	HERCEPTIN	trastuzumab	For Injection	103792	09/25/1998		2035-03-09
Genentech, Inc.	HERCEPTIN	trastuzumab	For Injection	103792	02/10/2017		2035-03-09
Genentech, Inc.	AVASTIN	bevacizumab	Injection	125085	02/26/2004		2035-03-09
Genentech, Inc.	RITUXAN HYCELA	rituximab and hyaluronidase human	Injection	761064	06/22/2017		2035-03-09
Genentech, Inc.	HERCEPTIN HYLECTA	trastuzumab and hyaluronidase-oysk	Injection	761106	02/28/2019		2035-03-09