Bioanalytical Challenge Kumar, King, Clark & Gorovits
Table 4. Impact of change in critical reagents on the drug–antibody ratio sensitivity of the total antibody assay.
QC samples HQC - ADC MQC - ADC LQC - ADC HQC - Ab MQC - Ab LQC - Ab
Accuracy for Fc specific detection (%RE) Accuracy for LC specific detection (%RE) 13
8.0
7.0 8.0 130 70 17
jugated, fully conjugated and partially conjugated) antibody using target protein as a capture reagent, and either Fc- or LC-specific anti-human IgG as detection reagents. The accuracy of the assay was evaluated by quantitating QC samples prepared with ADC or pre- pared with Ab against the ADC reference standard. HQC, MQC and LQC represent the high, mid and low concentration range of the standard curve, respectively with HQC at 750 ng/ml, mid QC at 500 ng/ml and low QC at 100 ng/ml concentrations in 100% matrix.
Conjugated antibody assay Conjugated antibody assay is used for monitoring anti- body concentration bearing at least one small molecule drug (i.e., DAR is greater than or equal to 1). Because the intact ADC is the active therapeutic analyte, the conjugated antibody assay is used to measure active ADC concentration and to determine ADC PK param- eters. In systemic circulation, the conjugated antibody concentration may change owing to the elimination of intact ADC and due to the complete deconjugation of ADC to the Ab. In order to detect intact ADC, the conjugated anti-
body assay typically employs critical reagents that bind both the small molecule drug component as well as the antibody component of ADC (Figure 2). Similar to the total antibody assay, conjugated antibody assays also exhibit sensitivity to the site of conjugation and the DAR values of the ADC. The binding of anti-small molecule drug antibodies to the small molecule drug component of ADC might be hindered by the solvent accessibility of the conjugation site. In addition, pro- portional binding may not be possible due to the steric hindrance from multiple adjacently conjugated small molecule drugs. Thus, the conjugated antibody assay may provide inaccurate measurement of higher and lower DAR species in systemic circulation [9,16]. The assay format and critical reagents chosen for
the conjugated antibody assay may also influence DAR sensitivity of
the assay, which in turn may impact observed intact ADC concentration in vivo. Theoreti- 1612 Bioanalysis (2015) 7(13)
-2.0 2.0
-5.0 -10 0.5
Ab: Unconjugated antibody; ADC: Antibody–drug conjugate; HQC: High QC; LC: Light chain; LQC: Low QC; MQC: Mid QC.
cally, an assay format employing anti-small molecule drug antibodies as the detection reagent might exhibit more DAR sensitivity because the observed assay sig- nal might be proportional to the total number of small molecule drug conjugated to the ADC (Figure 2). In other words, higher DAR species may exhibit higher assay signal compared with the lower DAR species. Whereas, an assay format utilizing anti-small molecule drug antibodies as capture reagent might not be as DAR-sensitive because such antibodies might capture intact ADC through its small molecule drug compo- nent irrespective of the number of small molecule drug conjugated to the ADC [15]. An objective criterion to determine if the assay for-
mat selected for the conjugated antibody assay has rendered the assay performance DAR-sensitive, is to evaluate recovery of the samples prepared with the enriched or individually purified DAR species against the ADC reference standard with average reported DAR [8]. If all samples exhibit percentage recovery within the acceptable range of the assay (e.g., ±20% of nominal), it reflects that the chosen assay format is the optimal choice for DAR-insensitive assay. How- ever, if the recovery of samples prepared using various DAR species falls outside of the acceptable range for the assay, further evaluation of assay format and/or reagents is warranted. The impact of assay format on the DAR sensitiv-
ity of a conjugated antibody assay for an ADC with conventional cysteine-based conjugation chemistry is illustrated in Table 5. In this case, when the assay format involving target protein as capture reagent and the anti-small molecule drug antibodies as detection reagent was employed for intact ADC quantitation, it yielded an overestimation of individually purified high DAR species and underestimation of low DAR spe- cies relative to the average DAR standard. However, changing the assay format to using anti-small molecule drug antibodies as capture reagent and target protein as detection reagent, significantly improved percentage recovery of both low and high DAR species.
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