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Application Note


A Simoa™ pharmacokinetic bridging assay


T


herapeutic drugs based on biologi- cal materials (antibodies, hor- mones, cytokines, enzymes, fusion


proteins) may have the ability to elicit an immune


response in the host.


Immunogenicity of a therapeutic is defined as the unwanted elicitation of an immune response by the host to the therapeutic. The measurement of this immune response is typically assessed by quantifying the host’s production of antibodies against the therapeutic molecule. Immunogenicity of a therapeutic can be a major roadblock in the development of a product. Here we describe a proof of concept


assay to demonstrate use of the Simoa plat- form as a tool to measure levels of anti-drug antibodies (ADA) against Adalimumab (Humira®). To compare performance, equivalent assays were run with the Simoa platform and a standard plate-based ELISA.


Reagents and methods In both assays, a fully-human monoclonal


(IgG1/kappa) antibody drug, Adalimumab (Creative Biolabs), was used as the capture reagent. The detection reagent was the same drug labelled with biotin. As a target, the


plate, detection was performed with biotinylated Adalimumab at 1µg/ml in the same buffer. Following detection, the plate was washed and SbG enzyme was added at 500pM in Quanterix SbG diluent. After enzyme labelling, the plate was washed again then incubated with RGP substrate. Resorufin fluorescence was measured as an assay readout. Adalimumab was conjugated to magnet-


fully-human monoclonal anti-


Adalimumab IgG1 (HCA204, clone AbD18655, Biorad), was spiked into sam- ples at varying concentrations.


ELISA assay Adalimumab was coated overnight at 1µg/ml on a high-binding ELISA plate. The plate was washed and blocked with 5% BSA in PBST. For the calibration curve, anti-Adalimumab antibody, HCA204, was titrated between 1-1,000ng/mL in a 2% BSA + PBST + 10% human serum buffer. After capturing HCA204 and washing the


ic microparticles using EDC+SNHS chem- istry. For the calibration curve, anti- Adalimumab antibody HCA204 was titrat- ed between 1-100ng/mL in a 2% BSA + PBST + 10% human serum buffer. The same detection antibody used for the ELISA, 1µg/mL biotinylated Adalimumab, was used for Simoa. Labelling was per- formed with 50pM SBG. The assay was run on the Simoa HD-1 platform as a three-step assay with incubation times of 60, 15, and 15 minutes for each of the steps.


Results The Simoa assay and ELISA assay both showed a dose dependent response to HCA204 in a calibration curve, indicating that either assay could be used to measure the concentration of auto-antibodies against Adali-mumab in patient samples. The Simoa assay demonstrated about 8x higher signal to background ratios across the calibration curve (<100ng/mL). The Simoa assay and plate ELISA both show fairly linear dose responses up to 100ng/mL. Upper limit of quantification (ULOQ) for the Simoa and plate ELISA assays are 200ng/mL and 215ng/mL, respectively. The lower limit of quantifica- tion (LLOQ) for the Simoa and plate ELISA assays are 0.14ng/mL and 0.98ng/mL, respectively. Limit of detection (LOD), determined as three standard devi- ations above the zero calibrator, was 0.05ng/mL for Simoa, while the plate ELISA showed a LOD of 0.5ng/mL.


Drug Discovery World Spring 2019 21


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