10.1021/bc300491k.s001 Katherine R. Kozak Katherine R. Kozak Siao Ping Tsai Siao Ping Tsai Aimee Fourie-O’Donohue Aimee Fourie-O’Donohue Josefa dela Cruz Chuh Josefa dela Cruz Chuh Leslie Roth Leslie Roth Ryan Cook Ryan Cook Elton Chan Elton Chan Pamela Chan Pamela Chan Martine Darwish Martine Darwish Rachana Ohri Rachana Ohri Helga Raab Helga Raab Crystal Zhang Crystal Zhang Kedan Lin Kedan Lin Wai Lee T. Wong Wai Lee T. Wong Total Antibody Quantification for MMAE-Conjugated Antibody–Drug Conjugates: Impact of Assay Format and Reagents American Chemical Society 2013 assay reagent monomethyl auristatin E MMAE sandwich ELISA formats SHA cytotoxic drugs covalently measure ADC concentrations antibody species DAR fractions Total Antibody Quantification 2013-05-15 00:00:00 Journal contribution https://acs.figshare.com/articles/journal_contribution/Total_Antibody_Quantification_for_MMAE_Conjugated_Antibody_Drug_Conjugates_Impact_of_Assay_Format_and_Reagents/2414677 Antibody–drug conjugates (ADCs) are target-specific anticancer agents consisting of cytotoxic drugs covalently linked to a monoclonal antibody. The number of ADCs in the clinic is growing, and therefore thorough characterization of the quantitative assays used to measure ADC concentrations in support of pharmacokinetic, efficacy, and safety studies is of increasing importance. Cytotoxic drugs such as the tubulin polymerization inhibiting auristatin, monomethyl auristatin E, have been conjugated to antibodies via cleavable linkers (MC-vc-PAB) through internal cysteines. This results in a heterogeneous mixture of antibody species with drug-to-antibody ratios (DAR) ranging from 0 to 8. In order to characterize the assays used to quantitate total MC-vc-PAB-MMAE ADCs (conjugated and unconjugated antibody), we used purified fractions with defined DARs from 6 therapeutic antibodies to evaluate different assay formats and reagents. Our investigations revealed that for quantitation of total antibody, including all unconjugated and conjugated antibody species, sandwich ELISA formats did not always allow for recovery of all purified DAR fractions (DAR 0–8) to within ±20% of the expected values at the reagent concentrations tested. In evaluating alternative approaches, we found that the recovery of DAR fractions with semihomogeneous assay (SHA) formats, in which sample, capture, and detection reagents are preincubated in solution, were less affected by the antibody’s MMAE drug load as compared to traditional stepwise sandwich ELISAs. Thus, choosing the optimal assay format and reagents for total antibody assays is valuable for developing accurate quantitative assays.