Antibody–drug conjugates nonclinical support Bioanalytical Challenge
detection using antibodies against the small mol- ecule drug. The accuracy of the assay was deter- mined by quantitating ADC QC samples (ULOQ: 1300 ng/ml, High QC: 1000 ng/ml, Mid QC: 500 ng/ml, Low QC: 25 ng/ml, LLOQ: 10 ng/ml) spanning the range of quantitation against the ADC reference standard in both species. The ROQ for standard curve was 10–1300 ng/ml in 100% matrix.
Strategies & considerations to mitigate challenges associated with LBA-based ADC bioanalysis How PK parameters are used may be different at dif- ferent stages of drug development, and so are the asso- ciated bioanalytical challenges. The strategies and bio- analytical approaches employed for assay development may thus need to be adapted at each stage. This may require exploring multiple bioanalytical platforms, assay formats, critical reagents and biological matrix (plasma vs serum) to ensure that the most appropriate bioanalytical tools are used. In addition, the informa- tion about the biology (such as mechanism of action, targeted tumor antigen) and physicochemical charac- teristics (such as conjugation chemistry, type of linker, average DAR, small molecule drug properties) of ADC can aid in the design and development of robust and reliable LBAs for analyzing diverse ADC analytes. In general, during the early drug discovery stage,
employed [14,25]. In contrast, the late nonclinical drug development stage involving investigational new drug (IND) enabling studies typically require validated assays that follow regulatory guidance on criteria used to define the ROQ, precision, accuracy, specificity, selectivity, robustness and ruggedness [26]. In order to meet these expectations, specific reagents such as
where research teams are working with large number of similar compounds for a given target, the avail- ability of critical reagents, time and resources may be limited. As a result, flexible ‘fit-for-purpose’ assay development approaches and generic reagents against human IgG, or Fc region or (Fab’)2
region are often
target antigen proteins, monoclonal anti-idiotype (Id) antibodies, anti-complementary determining regions (anti-CDR) antibodies, etc. are frequently used in the validated assays. The usage of specific reagents may also raise questions whether the assay measures ‘free’ versus ‘total’ analyte concentration [27]. In the con- text of ADC bioanalysis, the ‘free’ analyte concentra- tion reflects concentration of ADC analytes that have at least one unoccupied target binding site (i.e., both antigen-free and -partially free analytes). Whereas the ‘total’ analyte concentration provides all forms of target antigen bound and unbound concentration of ADC analytes. The differences in critical reagents and assay for-
mats adopted for large molecule bioanalysis at differ- ent stages of drug development may have an impact on the observed analyte concentration and the associated PK profile and calculation of critical PK parameters. Though there are no issued regulatory guidelines or industry-wide best practices for assays validations spe- cific for ADCs, currently the assay validation guidelines for large and small molecules are applied for ADC bio- analysis. Thus, the challenges associated with large mol- ecule bioanalysis at various stages of drug development are applicable for ADC bioanalysis as well. In addition, during early non-regulated discovery
stage, when PK data is needed for design and selection of optimal ADC candidates that can advance to lead candidates, DAR-sensitive LBAs may be useful to bet- ter describe the changes in conjugated small molecule over time and associated PK parameters. At this stage in drug development, toxicology and efficacy studies conducted with multiple antibody, small molecule, linker and conjugation chemistry (and conjugation site) combinations often aid in selection and rank- ing of multiple ADC candidates. The need to obtain some measure of conjugated small molecule by either LC–MS or through the use of a DAR-sensitive con- jugated antibody assays is based on the mechanism of action based hypothesis that conjugated small mol- ecule drug is the main driver of efficacy at the site of action [28,29], and that the potency is directly propor-
Table 3. Matrix component interference in ligand-binding assay-based quantitation of intact antibody–drug conjugates.
QC samples ULOQ HQC MQC LQC
LLOQ
Accuracy in rodents (%RE) 8.6 -1.8 -15 -18 -14
HQC: High QC; LQC: Low QC; MQC: Mid QC; NHP: Nonhuman primates.
Accuracy in NHP (%RE) -2.0 -1.0 -16 -63 -59
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