Antibody–drug conjugates nonclinical support Bioanalytical Challenge For some ADCs, irrespective of whether the assay


format employs small molecule drug specific detec- tion or the small molecule drug specific capture, the DAR sensitivity of the assay may not be alleviated. Thus, like total antibody assay, the DAR sensitivtity of the conjugated antibody assay may also be gov- erned by the physicochemical characteristics of ADC. In addition, due to the high cytotoxic potency of the small molecule drug, generation of high quality anti- body reagents with high binding affinity and avidity against the small molecule drug poses a challenge. Table 6 displays DAR sensitivity of a conjugated antibody assay for an ADC containing lysine-based conventional conjugation chemistry. In this case, irre- spective of whether the assay format employed either small molecule drug specific detection or the small molecule drug specific capture, the conjugated anti- body assay performance for the ADC was affected by


A Detection reagent


(Biotin-anti-small molecule mAb or pAb)


ADC (DAR ≥ 1) Capture reagent


(antigen, anti-ld/anti-CDR mAb, antihuman mAb or pAb)


DAR values based on the DAR reference standards used. Thus in this situation, it is important to under- stand that irrespective of the assay format chosen, the critical reagents employed will deliver DAR-sensitive assay, so the observed results should be interpreted accordingly. Other considerations that may aid in overcoming


challenges associated with development and validation of LBAs for ADC bioanalysis include:


• Using the ADC dosing material as a reference stan- dard in both the total antibody and conjugated antibody assays. Development of multiple assays, with each assay utilizing reference standard with a specified DAR to determine impact of ADC hetero- geneity on the accuracy of assay is not practical. In addition, since clinical and nonclinical end points are measured following administration of the dos-


B Detection reagent


(Biotin-antigen, Biotin-anti-ld/anti-CDR mAb, Biotin-antihuman mAb or pAb)


ADC (DAR ≥ 1)


Capture reagent (anti-small molecule mAb or pAb)


Figure 2. Ligand-binding assay formats for the conjugated antibody analyte. (A) Capture and detection of the intact ADC using reagents specific for antibody framework and small molecule component of ADC, respectively. (B) Capture and detection of the intact ADC using anti-small molecule antibodies and antibodies against antibody component of ADC, respectively. ADC: Antibody–drug conjugate; CDR: Complementarity determining region; DAR: Drug–antibody ratio; Id: Idiotype; mAb: Monoclonal antibody; pAb: Polyclonal antibody.


future science group www.future-science.com 1613


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