Bioanalytical Challenge Kumar, King, Clark & Gorovits
Table 2. Unconjugated antibody interference in intact antibody–drug conjugate assay. Spiked unconjugated antibody conc. (μg/ml) Accuracy for LQC (%RE)
0
2.5 5.0 25
HQC: High QC; LQC: Low QC.
in the recovery of ADC QC samples when spiked with Ab (data not shown). Accuracy is defined as the closeness of agree-
ment between the observed concentration (E) and the nominal concentration (T) and is expressed as percent relative error (%RE).
%RE `j T
= ET 100# -
The assay performance was evaluated in rodent
serum for an ADC containing lysine-based con- ventional conjugation chemistry. The assay for- mat on a Mesoscale Discovery (MSD) platform involved capture of intact ADC via its antibody framework using target protein as the capture reagent and detection using antibodies against the small molecule drug. High (1000 ng/ml) and low (25 ng/ml) QC ADC samples spiked with varying concentrations of Ab (0–25 μg/ml) were quan- titated against the ADC reference standard. The range of quantitation (ROQ) for standard curve was 10–1300 ng/ml in 100% matrix;
• Due to highly potent cytotoxic activity of small molecule component of ADCs, the potential tox- icity concerns are usually higher for ADCs. As a result, compared with large molecule therapeutics, relatively lower dose ranges may be selected for nonclinical and clinical studies for ADCs. LBAs for ADC bioanalysis thus may require high assay sensitivities (in the low ng/ml range) than typically expected for large molecule therapeutics [18–21];
• The selection of an appropriate biological matrix also presents unique challenge during ADC bio- analysis. The preferred matrix for bioanalysis of small molecule therapeutics using LC–MS plat- form and large molecule therapeutics using LBA platform are typically plasma and serum, respec- tively. However, since ADCs contain both small and large molecule therapeutics, the selection of optimal matrix for ADC bioanalysis becomes debatable.
1608 Bioanalysis (2015) 7(13) Depending on the linker lability and ADC sta-
bility, the processing of serum may cause deconju- gation and release of cytotoxic small molecule drug that may interfere in quantitation of small molecule analyte of some ADCs. The plasma matrix is thus preferred for LC–MS-based quantitation of the antibody-conjugated small molecule drug and/or the released unconjugated small molecule drug and its metabolites [9]. But the plasma processing is also associated with its own set of challenges such as the selection of appropriate anticoagulant from the list of available anticoagulants such as EDTA, heparin or sodium citrate; the appropriate volume of blood collected in collection tubes to ensure optimum blood/anticoagulant
ratio; and the freeze–thaw stability of plasma [22,23];
• Though not commonly observed, LBA perfor- mance may be impacted by the binding of known matrix components to either
large molecule or
small molecule component of multicomponent ADC [24]. Such interferences may reduce desired sensitivity of the assay.
Table 3 shows an example for the impact of
known matrix component interference on the assay performance in serum samples from rodents and nonhuman primates (NHP). Though similar assay format and similar experimental conditions were employed for quantitation of intact ADC in both species, the assay performance reflected by signifi- cantly reduced recovery of QC samples at the lower end of
the ROQ was impacted in NHP serum
samples. Further analysis revealed that the spe- cific binding of a known matrix protein to one of the component of multicomponent ADC in NHP serum caused the observed decrease in recovery of QC samples (data not shown).
The assay performance was evaluated in two indi-
vidual species, rodents and NHP, for an ADC with lysine-based conventional conjugation chemistry. The assay format on an MSD platform involved capture of intact ADC via its antibody framework using target protein as the capture reagent and
future science group
-19 2.0 -11 -65
Accuracy for HQC (%RE) -22 -18 -27 -79
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