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Bioanalytical Challenge Kumar, King, Clark & Gorovits


Table 5. Impact of change in assay format on drug–antibody ratio sensitivity of the conjugated antibody assay for an antibody–drug conjugate containing conventional cysteine-based conjugation chemistry.


Purified DAR species/QC


DAR 2 DAR 6 DAR 8 DAR 4†


Accuracy for anti-small molecule antibodies-based detection (%RE)


-70


100 127 2.5


Accuracy for anti-small molecule antibodies-based capture (%RE)


-47 9.0 -24


-4.0


DAR sensitivity of the conjugated antibody assay was evaluated in nonhuman primate (NHP) serum. The assay format on an ELISA platform involved either capture of intact ADC using target capture reagent and detection using antibodies against small molecule drug, or vice versa. Samples prepared with individually purified DAR components (DAR 2, 6, 8) at 175 ng/ml concentration (equivalent to mid QC concentration) in 100% NHP matrix were quantitated against the ADC reference standard (average DAR of 4) curve. The range of quantitation for the assay format employing antibodies against small molecule drug as detection reagent was 18.6–500 ng/ml in 100% NHP matrix. The assay format


employing antibodies against small molecule drug as capture reagent had a range of quantitation of 30.0–500 ng/ml in 100% NHP matrix. †


DAR 4 sample at mid-QC level was prepared using the ADC reference standard material. ADC: Antibody–drug conjugate; DAR: Drug–antibody ratio.


ing material, the use of ADC dosing material as a reference standard is appropriate;


• Mitigating Ab interference in the conjugated anti- body assay by changing the assay format in such a manner that the reagents against the small mol- ecule drug are used as capture reagents. Theoreti- cally, since such reagents do not directly bind to the antibody component of ADC, they may increase the assay specificity of conjugated antibody assay against intact ADC;


• Compared with the conventional ELISA platform, novel MSD and Gyrolab-based immunoassay plat- forms that claim to offer higher sensitivity, broad dynamic range, low sample volume and/or reduced matrix interference [32] can be used for attaining higher assay sensitivity for LBAs employed for ADC bioanalysis;


• Endogenous protein interference in the assay can be alleviated either by the use of critical reagents that


are not impacted by the binding of such proteins or by optimizing assay buffer composition by intro- ducing appropriate reagents that can block such interference in the assay [24,25,33]. In cases where it may not be feasible to eliminate endogenous pro- tein interferences, an increase in the minimum required dilution may be required.


Conclusion The complex multicomponent structure and dynamic and heterogeneous nature of ADCs require bioanalysis of multiple analytes for PK assessment of ADCs. This review has highlighted unique challenges associated with bioanalytical characterization of ADCs and the strategies to mitigate them. Since the stage of ADC development dictates what PK questions needs to be answered, the bioanalytical challenges associated in addressing those questions may also be unique to each stage. The bioana- lytical approaches and strategies therefore would need to be adapted depending on the desired endpoint. The assay formats and assay reagents for ADC bioanal- ysis may evolve as ADC drug candidates progress from


Table 6. Lack of impact of change in assay format on drug–antibody ratio sensitivity of the conjugated antibody assay for an antibody–drug conjugate containing conventional lysine-based conjugation chemistry.


Enriched DAR species† DAR 4


DAR 4.4 DAR 5.1


Accuracy for anti-small molecule antibodies-based detection (%RE)


-21 -10 30


Accuracy for anti-small-molecule antibodies-based capture (%RE)


-25 -29 31


DAR sensitivity of the conjugated antibody assay was evaluated in rodent serum. The assay format on an Mesoscale Discovery platform involved either capture of intact antibody–drug conjugate (ADC) via its antibody framework using target protein as the capture reagent and detection using antibodies against small molecule drug, or vice versa. Samples prepared with enriched DAR fractions (with average reported DAR of 4, 4.4 and 5.1) at 1 μg/ml concentration (equivalent to high quality control concentration) in 100% matrix were quantitated against


the ADC reference standard (average DAR of four). The range of quantitation for the standard curve was 10.0–1280 ng/ml in 100% matrix. †


The DAR heterogeneity in ADC was too complex to chromatographically isolate individual DAR species. Thus, enriched DAR species were


prepared by crude hydrophobic interaction chromatography fractionation of the ADC reference standard. DAR: Drug–antibody ratio.


1614


Bioanalysis (2015) 7(13)


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