Development, Comparison of Quantitative Analytical Methods Evaluating Antibody-Drug-Conjugates in Plasma

Three LBA methods measuring total antibodies or conjugated antibody (Methods 1-3); hybrid LC-MS/MS for conjugated payload (method 4) and LC-MS/MS for free payload (method 5) were developed and their detection limits and assay ranges were determined. Plasma samples treated with Telisotuzumab Vedotin or Disitamab Vedotin were analyzed and compared. Although methods 2-4 are all for conjugated drug, they exhibit different aspects of the test article. Method 3 (anti-huIgG as capture and anti-MMAE as detection) has potential inaccuracy on conjugated antibody and payload since the aggregation and DAR uniformity issue could generate signal amplification unproportional to the molecule. Method 2 (reversed design of method 3) measures conjugated antibody regardless the payload number; while method 4 (hybrid LCMS) measures total number of the conjugated payload with no content of the antibody concentration. Combination of methods 2 and 4 provides better clarity and assessment of the test article species change.