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Bioanalytical Challenge Kumar, King, Clark & Gorovits Key terms


Drug–antibody ratio: Amount of drug conjugated per antibody molecule.


Total antibody assay: Measures total antibody–drug conjugate (ADC) concentration (sum of conjugated and unconjugated forms) irrespective of whether small molecule drug is attached to the antibody moiety or not.


Conjugated antibody assay: Measures total ADC concentration with at least one small molecule drug attached to the antibody moiety.


DAR-sensitive assay: Measures ADC concentration based on the valences of small molecule drugs on the ADC.


DAR-insensitive assay: Measures ADC concentration irrespective of the valences of small molecule drugs on the ADC.


include the conjugated antibody (DAR greater than or equal to one), the total antibody (DAR greater than or equal to zero), the antibody-conjugated small mol- ecule drug, the released small molecule drug (result- ing from in vivo deconjugation), the DAR distribution over time and the antidrug antibodies (ADAs) against any component of multicomponent ADC molecule. However, the precise collection of analytes needed depends on the detailed physicochemical characteris- tics of the ADC molecule and the endpoints required for understanding of the ADME properties and PK characteristics of the ADC molecule. Depending on the PK information sought, bioana-


lytical methods such as ligand-binding assays (LBA), LC separation coupled with MS detection (LC–MS), and combination of both methods are employed for analyzing the diversity of ADC analytes. LBA-based methods are often employed for monitoring the total antibody, conjugated antibody and antidrug anti- body analytes [8]. LC–MS-based methods are mainly utilized for the detection and quantitation of the antibody-conjugated small molecule drug, released unconjugated small molecule drug and its metabolites, and DAR distribution over time [9–11]. However, the


exact set of methods applied for understanding PK and immunogenicity of ADCs may evolve as more infor- mation is gained on ADC pharmacology from clini- cal trials and postmarketing data. This review focuses on challenges associated with ADC bioanalysis using LBA-based methods and the bioanalytical approaches and strategies that might be adopted for PK character- ization of diverse ADC analytes from early discovery to late nonclinical stages of drug development. The LBA-based ADA bioanalysis is outside the scope of this review article.


ADC bioanalysis at different stages of drug development The early to late stages of drug development may have different PK questions (such as ADC stability, expo- sure and safety) that need to be answered and thus may require different endpoints to address these ques- tions [9,12,13]. During early drug discovery stage that involves candidate selection, optimization and early nonclinical assessments, the goal of ADC bioanalysis is to provide insights into the overall stability, safety and efficacy characteristics of the drug candidates. Such information allows comparison of ADC candidates with various linker–small molecule drug combinations and facilitates selection and ranking of optimal can- didates that can progress to lead candidates with the best safety and efficacy profile. The commonly mea- sured analytes at early discovery stage are tabulated in Table 1 and may include the total antibody, conjugated antibody and/or antibody-conjugated small molecule drug,


released unconjugated small molecule drug


and its metabolites, and in vitro and in vivo changes in DAR distribution over time [9]. In addition, ADA might also be evaluated during early drug discovery stage, particularly if abnormal PK profile due to ADA- mediated clearance is suspected in initial nonclinical assessments [14]. During late nonclinical stage of drug development, the objective of ADC bioanalysis is


to gain better


Table 1. Analytes for antibody–drug conjugate bioanalysis from early drug discovery to late nonclinical stages of antibody–drug conjugate development.


ADC analytes Total antibody


Released/free small molecule drug and its metabolites In vitro and in vivo DAR distribution ADA


drug enabling studies. ‡


Drug discovery Nonclinical† ✓


Conjugated antibody and/or antibody-conjugated small molecule drug ✓ ✓ ✓


✓‡


✓ ✓ ✓





†Nonclinical herein refers to the post-lead candidate selection stage involving regulated TK and toxicity studies such as investigational new ADA evaluations in early discovery are done if ADA related changes in PK profile are observed.


ADA: Antidrug antibodies; ADC: Antibody–drug conjugate; DAR: Drug–antibody ratio.


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Bioanalysis (2015) 7(13)


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