Analysis of antibody drug conjugates by bead-based affinity capture and mass spectrometry

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.