Mass spectrometry of antibody conjugates

• US 7,662,936 B2
• Filed: 04/06/2005
• Issued: 02/16/2010
• Est. Priority Date: 04/07/2004
• Status: Active Grant

• Current Assignee: Genentech, Inc. (Roche Holding AG)
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: Mann, Emily, Kadkhodayan, Miryam
• Primary Examiner(s): Saunders; David A
• Application Number:
  US11/101,018
• Publication Number:
  US 20050232929A1
• Time in Patent Office: 1,777 Days
• Field of Search: 424/9.2, 424/178.1, 424/181.1, 424/183.1, 436/173, 530/391.7, 530/391.9
• US Class Current: 530/413
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: C07K 16/2866 against receptors for cytokines, lymphokines, interferons
• CPC Class Codes: C07K 16/32 against translation products of oncogenes
• CPC Class Codes: G01N 33/6848 Methods of protein analysis involving mass spectrometry
• CPC Class Codes: G01N 33/6854 Immunoglobulins
• CPC Class Codes: Y10S 436/825 Pretreatment for removal of interfering factors from sample
• CPC Class Codes: Y10T 436/24 Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry

Abstract

Methods to detect, screen, and quantitate biological samples after administration of antibody conjugates, antibody-drug conjugates of Formula I, antibodies, and fragments and metabolites thereof, by affinity 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

1. A method for detecting an antibody-drug conjugate comprising:

(i) separating the antibody-drug conjugate from a sample matrix by affinity separation wherein the antibody-drug conjugate comprises a monomethyl auristatin drug and a thio-ether linkage and wherein the thio-ether linkage is formed by reducing the interchain disulfide bonds of the antibody and conjugating the monomethyl auristatin drug to a cysteine thiol group of the reduced antibody, and wherein the antibody of the antibody-drug conjugate is an immunoreactive protein having an immunoglobulin structure;

(ii) eluting the separated antibody-drug conjugate; and

(iii) detecting the separated and eluted antibody-drug conjugate by electrospray, selective ion monitoring mass spectrometry.

2. The method of claim 1 wherein the antibody-drug conjugate is separated from the sample by immunoaffinity separation and reverse phase liquid chromatography.

3. The method of claim 1 wherein the sample matrix is plasma.