Antibody–drug conjugates nonclinical support Bioanalytical Challenge
understanding of the nature of pharmacological and toxicological properties of the lead drug candidate. The observed toxicity and TK data provides insights about the exposure–response relationship, aids in predicting human PK, and facilitates translation of nonclinical data to clinical outcomes. The additional analyte measured at this stage typically includes ADAs along with the other multiple analytes measured in early drug discovery stage (Table 1) [9,12,13].
ADC bioanalysis using ligand-binding assays & associated challenges Ligand-binding assays offer unique advantages for quantitation of large molecule component of ADCs including high assay sensitivity and throughput, the broad range of quantitation, the requirement of mini- mal sample volume and their ability to measure the analyte of interest in the biological matrix without additional sample extraction steps. But the complex multicomponent structure, inherently heterogeneous and dynamically evolving in vivo behavior of ADCs presents unique challenges in ADC bioanalysis using LBAs:
• While single LBA is typically utilized for quan- titation of large molecule therapeutics, multiple LBAs such as the total antibody assay and the conjugated antibody assay are needed for the quantitation of diverse ADC-related analytes;
• Due to their heterogeneous nature and dynami- cally evolving in vivo behavior, the starting ADC reference material may not accurately represent the ADC composition present in the incurred samples, particularly at later time points [8,9]. Thus the selection of correct reference standard for quantitative LBAs for ADC bioanalysis pres- ents a major challenge. Current bioanalytical validation guidance does not address this ADC specific challenge;
• Depending on the question to be answered, DAR- sensitive or DAR-insensitive LBAs may be needed for ADC bioanalysis (discussed in detail in the later section). A DAR-sensitive assay attempts to measure ADC analyte concentration based on the number of small molecule drugs attached to the ADC, but may not be able to measure small molecule drug for all DAR species stoichiomet- rically. Ideally a DAR-sensitive LBA would be equivalent to the conjugated small molecule assay. The reverse is true for DAR-insensitive assays that attempt to measure various DAR components of the ADC equally, and hence are not biased toward the varying DAR values of the ADC.
future science group Since DAR sensitivity of an assay could be
governed by the critical (capture and detection) reagents and assay formats used [1,9,15,16], the design and development of DAR-sensitive and DAR- insensitive assays may require screening multiple assay formats, assay conditions and critical reagents. In addition, for a given drug candidate, due to dif- ferences in critical reagents and assay formats used, the DAR-sensitive and DAR-insensitive assays may deliver significantly different observed PK profiles and associated critical PK parameters such as clear- ance and drug exposure [15,17], which in turn may present significant challenges when early discovery PK data for the same drug candidate is compared with its late stage regulatory TK data. The vari- able sensitivity of the assays to the DAR values may also complicate direct comparison of the exposure results between various ADC candidates;
• Interference from unconjugated antibody (Ab) may reduce the specificity of bioanalytical assay designed for exclusive quantitation of intact ADC (fully con- jugated and partially conjugated). The specificity of an assay format exploiting target tumor anti- gens or antibodies against antibody components of ADCs as capture reagents to detect intact ADC can be affected by competition from the Ab result- ing from complete deconjugation in vivo. Theoreti- cally, this competitive binding of Ab may impact the observed concentration of intact ADC, par- ticularly at later time points when owing to in vivo deconjugation, the concentration of Ab in the sys- tem may rise relative to the ADC concentration. The consideration for Ab interference in the intact ADC quantitation could be taken in the context of target interference in the LBA assay. Similar to the target interference, the interference information may not be known upfront but evaluating it early would eliminate the risk of developing assays that may exhibit interference.
Table 2 shows an example for the impact of
Ab interference on the performance of an assay designed for exclusive quantitation of intact ADC. In the assay format, the intact ADC was captured using target protein and was detected using anti- bodies against small molecule drug. The accuracy of the assay was determined by spiking known concentrations of antibody component of ADC (i.e., Ab) into QC samples prepared with ADC dos- ing material. The QC samples quantitated against the ADC reference standard exhibited significantly reduced recovery at higher concentrations of Ab. Even a five-fold increase in concentration of cap- ture reagent showed no substantial improvement
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