An electrochemiluminescence assay was developed for ADA detection based on a homogeneous bridging format. Assay performance followed a multitered approach in accordance with the FDA guidance, which includes screening, confirmation, and determination of antibody titer.
25 Briefly, duplicate unknown and control samples were incubated with acid dissociation buffer to disrupt potential ADA‐drug complexes. A mix of capture/detection reagents (avelumab conjugated with biotin or SULFO‐Tag [Microcoat Biotechnologie GmbH, Germany]) was diluted in neutralization buffer, allowing binding between ADA and labeled drugs. Samples were transferred into streptavidin precoated/preblocked MSD GOLD SECTOR microplates (Meso Scale Discovery) to capture formed complexes on a solid phase. After a wash step, MSD Read Buffer (Meso Scale Discovery) was added, and signals were acquired. Assay results were based on comparisons between the sample readout and a plate‐specific screening cut point. Screening results were reported as “putative positive” (equal or above the cut point) or “negative” (below the cut point). Putative positive samples were tested for specificity by undergoing competition testing in the presence and absence of avelumab; if the percentage decrease in signal in the presence of drug was greater than or equal to the confirmatory cut point, the sample was considered ADA‐positive. To evaluate assay tolerance, positive controls (BioGenes GmbH, Germany) were spiked with increasing concentrations of avelumab (the healthcare business of Merck KGaA, Darmstadt, Germany) until responses in the assay fell below the assay cut point. The 20 ng/mL positive control tolerated up to 25 μg/mL of avelumab, the 250 ng/mL positive control tolerated up to 100 μg/mL, and the 500 ng/mL positive control tolerated up to 100 μg/mL. The assay had a sensitivity of 10 ng/mL in the absence of avelumab. Assays were validated according to US and European regulatory guidelines.
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25 Briefly, duplicate unknown and control samples were incubated with acid dissociation buffer to disrupt potential ADA‐drug complexes. A mix of capture/detection reagents (avelumab conjugated with biotin or SULFO‐Tag [Microcoat Biotechnologie GmbH, Germany]) was diluted in neutralization buffer, allowing binding between ADA and labeled drugs. Samples were transferred into streptavidin precoated/preblocked MSD GOLD SECTOR microplates (Meso Scale Discovery) to capture formed complexes on a solid phase. After a wash step, MSD Read Buffer (Meso Scale Discovery) was added, and signals were acquired. Assay results were based on comparisons between the sample readout and a plate‐specific screening cut point. Screening results were reported as “putative positive” (equal or above the cut point) or “negative” (below the cut point). Putative positive samples were tested for specificity by undergoing competition testing in the presence and absence of avelumab; if the percentage decrease in signal in the presence of drug was greater than or equal to the confirmatory cut point, the sample was considered ADA‐positive. To evaluate assay tolerance, positive controls (BioGenes GmbH, Germany) were spiked with increasing concentrations of avelumab (the healthcare business of Merck KGaA, Darmstadt, Germany) until responses in the assay fell below the assay cut point. The 20 ng/mL positive control tolerated up to 25 μg/mL of avelumab, the 250 ng/mL positive control tolerated up to 100 μg/mL, and the 500 ng/mL positive control tolerated up to 100 μg/mL. The assay had a sensitivity of 10 ng/mL in the absence of avelumab. Assays were validated according to US and European regulatory guidelines.
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