Complex biotherapeutic modalities, such as antibody-drug conjugates (ADC), present significant challenges for the comprehensive bioanalytical characterization of their pharmacokinetics (PK) and catabolism in both preclinical and clinical settings. Thus, the bioanalytical strategy for ADCs must be designed to address the specific structural elements of the protein scaffold, linker, and warhead. A typical bioanalytical strategy for ADCs involves quantification of the Total ADC, Total IgG, and Free Warhead concentrations. Herein, we present bioanalytical characterization of the PK and catabolism of a novel ADC. MEDI3726 targets prostate-specific membrane antigen (PMSA) and is comprised of a humanized IgG1 antibody site-specifically conjugated to tesirine (SG3249). The MEDI3726 protein scaffold lacks interchain disulfide bonds and has an average drug to antibody ratio (DAR) of 2. Based on the structural characteristics of MEDI3726, an array of 4 bioanalytical assays detecting 6 different surrogate analyte classes representing at least 14 unique species was developed, validated, and employed in support of a first-in-human clinical trial (NCT02991911). MEDI3726 requires the combination of heavy-light chain structure and conjugated warhead to selectively deliver the warhead to the target cells. Therefore, both heavy-light chain dissociation and the deconjugation of the warhead will affect the activity of MEDI3726. The concentration− time profiles of subjects dosed with MEDI3726 revealed catabolism of the protein scaffold manifested by the more rapid clearance of the Active ADC, while exhibiting minimal deconjugation of the pyrrolobenzodiazepine (PBD) warhead (SG3199).

Robust and efficient LC-MS/MS assays were established and were effective in providing antipsychotic drug matrix comparator results in the intended clinical studies.

Bioanalysis of complex biotherapeutics, such as antibody-drug conjugates (ADCs), is challenging and requires multiple assays to describe their pharmacokinetic (PK) profiles. To enable exposure-safety and exposure-efficacy analyses, as well as to understand the metabolism of ADC therapeutics, three bioanalytical methods are typically employed: Total Antibody, Antibody Conjugated Toxin or Total ADC and Unconjugated Toxin. MEDI4276 is an ADC comprised of biparatopic humanized antibody attached via a protease-cleavable peptide-based maleimidocaproyl linker to a tubulysin toxin (AZ13599185) with an approximate average drug-antibody ratio of 4. The conjugated payload of MEDI4276 can undergo ester hydrolysis to produce the conjugated payload AZ13687308, leading to the formation of MEDI1498 (de-acetylated MEDI4276). In this report, we describe the development, validation and application of three novel multiplex bioanalytical methods. The first ligand-binding liquid chromatography coupled with tandem mass spectrometry (LBA-LC-MS/MS) method was developed and validated for simultaneous measurement of total antibody and total ADC (antibody-conjugated AZ13599185) from MEDI4276. The second LBA-LC-MS/MS assay quantified total ADC (antibody-conjugated AZ13687308) from MEDI1498. The third multiplex LC-MS/MS assay was used for simultaneous quantification of unconjugated AZ13599185 and AZ13687308. Additional stability experiments confirmed that quantification of the released warhead in the presence of high concentrations of MEDI4276 was acceptable. Subsequently, the assays were employed in support of a first-in-human clinical trial (NCT02576548).