Statistical and bioanalytical considerations for establishing a depletion criterion for specificity testing during immunogenicity assessment of a biotherapeutic

Abstract

Immunogenicity assessment of fully human monoclonal antibody-based biotherapeutics requires sensitive and specific ligand binding assays. One of the components of specificity is the depletion of signal by a relevant biotherapeutic that is commonly based on an arbitrary depletion criterion of inhibition of the original response or reduction of the signal below the screening assay cut point (ACP). Hence, there is a need to develop a statistically derived physiologically relevant specificity criterion. We illustrate an optimization approach to determine the concentration of biotherapeutic required for the specificity evaluation. Naïve donor sample sets with and without circulating drug and antitherapeutic/drug antibody (ADA) were prepared. Next, a depletion cut point (DCP) using naïve and ADA-containing donor sets with the optimized biotherapeutic concentration was evaluated. A statistically derived design of experiment was used to establish a validated DCP. A reliable DCP requires naïve (no ADA) donors treated only with an optimized concentration of biotherapeutic. The additional DCPs generated using two distinct concentrations of ADA-spiked sample sets led to a physiologically irrelevant criterion that was not necessarily representative of real-time samples. This increased the risk of false positives or negatives. In this study, well-defined bioanalytical and statistical methods were employed to validate a DCP to confirm the presence of biotherapeutic specific ADA in human serum samples. A physiologically relevant and effective strategy to confirm specificity in immune reactive samples, especially those that are close to the ACP, is proposed through this study.

Fig. 1

Evaluation of biotherapeutic concentrations in depletion buffer at various ADA levels. Five dose–response curves were created consisting of increasing levels of ADA (0.01, 0.04, 0.16, 0.50, 1.0, 20, 80, 160 μg/mL). Each curve was depleted with one out of five biotherapeutic concentrations (1, 7.5, 25, 100, 400 μg/mL). The lowest concentration of biotherapeutic (1 μg/mL) was not able to reduce the signal at the highest concentration of ADA (last point on the curve) which indicates the possibility of reporting false negatives in a study where high amounts of ADA could be present. Biotherapeutic performance was comparable at 25, 100, and 400 μg/mL in depletion buffers. Note: the ADA concentration was transformed prior to plotting on the X-axis

Fig. 2

a–c Optimization of excess soluble biotherapeutic concentration for specificity assay in the presence of circulating biotherapeutic and ADAs. Three ADA panels were prepared (100, 0.5, and 0.05 μg/mL) consisting of four serum curves each with increasing concentrations of biotherapeutic (mimicking circulating drug in serum). Each curve per panel was depleted using one of four soluble biotherapeutic concentrations (7.5, 25, 100, and 400 μg/mL), giving a final number of 12 curves with 12 different combinations. Panels a–c illustrate that buffer A (7.5 μg/mL of biotherapeutic) does not deplete as robustly as the other three concentrations. Buffers B, C, and D perform comparably in panels a and b, but panel c demonstrates high variability due to low level of ADA (50 ng/mL). This phenomenon may lead to false positives or negatives in response to the already circulating drug. Buffer C offers the most consistent depletion across ADA and circulating drug. Note: the circulating drug concentration was transformed prior to plotting on the X-axis

Fig. 3

Comparing clinical study specific predoses with the naïve donor validation data set. S/N percent depletion was calculated for 16 predose samples from a phase 1 clinical study and for 96 donor samples (48 healthy and 48 disease specific). The plot indicates that although baseline samples depleted a bit higher, they are still within the distribution of naïve donors from the DCP validation experiment

Fig. 4

Comparison of arbitrary 50% depletion threshold vs. DCP established using clinical study specific predoses. Study samples (N = 22) were tested in the confirmatory assay and analyzed using both available depletion options (net ECL and S/N% depletion). When using net ECL depletion (left half of the plot), 18 out of 22 samples confirm as positive because their depletion is above the 50% threshold (Note: the remaining four samples [denoted in red] were considered positive even though they were under the 50% threshold because their treated S/N value fell below the untreated S/N ACP). Eight samples out of 22 would confirm as positive if S/N% depletion and the DCP of 23% were employed (right half of the plot). When the DCP or criterion II produced by spiking donors with 20 ng/mL of ADA was applied, all samples would be positive due to the unrealistic DCP of −7%. But on the other hand, if the cut point generated by adding 160 ng/mL of ADA to the donor set would be used, none of the samples would confirm positive due to the artificially elevated DCP (43%)