Application Note
A Simoa™ pharmacokinetic bridging assay P
harmacokinetics (PK) refers to the movement of a drug through the body. The distribution, absorption,
metabolism and excretion of a drug are all important pharmacokinetic factors to be considered in the development of therapeu- tics. The ability to detect and quantify the concentration of a therapeutic molecule with high sensitivity may be necessary for effective pharmacokinetic studies, especially if the drug is administered at a low dose. Here we describe a proof of concept assay to demonstrate the Simoa platform as a tool for measuring concentration of the biother- apeutic IgG Adalimumab (Humira®). A sandwich ELISA was created using antiAdalimumab antibodies as capture and detection. To compare performance, equiv- alent assays were run with the Simoa plat- form and a standard plate-based ELISA.
Reagents and materials In both assays, a monovalent Fab (HCA202, clone AbD18654, Biorad) was used as the capture reagent. The detection reagent was a fully-human monoclonal IgG1 (HCA204, clone AbD18655, Biorad). As a target, the
IgG1/kappa drug (Adalimumab, Creative Biolabs) was spiked into samples at varying concentrations.
ELISA assay HCA202 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,
Adalimumab was titrated between 1- 1,000ng/mL in a 2% BSA + PBST + 10% human serum buffer and added to the plate for a two-hour incubation. After capturing Adalimumab and washing the plate, detec- tion was performed with biotinylated HCA204 at 1µg/ml in the same buffer with a one- hour incubation. After detection, the plate was washed and Streptavidin beta- galactosidase (SbG) enzyme was added at 500pM in Quanterix SbG diluent for a 30min incubation. Following enzyme labelling, the plate was washed again then incubated with RGP substrate. Resorufin fluorescence was measured after one hour as an assay readout.
Drug Discovery World Fall 2019 35
biotinylated HCA204, was used for Simoa. Labelling was performed with 50pM SbG. The assay was run on the Simoa HD-1 platform as a two-step assay with incuba- tion times of 60 minutes for capture/detect and 15 minutes for SBG labelling.
Figure 1: Schematic image of PK Bridging Simoa ELISA. Anti-idiotypic capture antibody, Fab format (purple, HCA202), monoclonal antibody drug (gold, Adalimumab), anti-idiotypic detection antibody, Ig format (blue, HCA204), labelled with Biotin. Image: Bio-rad
Simoa assay HCA202 was conjugated to magnetic microparticles using EDC+SNHS chem- istry. For
the calibration curve,
Adalimumab was titrated between 0.1- 100ng/mL in a 2% BSA + PBST + 10% human serum buffer. The same detection antibody used for the ELISA, 1ug/mL
Results The Simoa assay and ELISA assay both showed a dose dependent response to Adalimumab in a calibration curve, indicat- ing that either assay could be used to mea- sure the concentration of Adalimumab in samples. The Simoa assay demonstrated about 10x 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 quantification (LLOQ) for the Simoa and plate ELISA assays are 0.06ng/mL and 0.8ng/mL, respectively. Limit of detection (LOD), determined as three standard devia- tions above the zero calibrator, was 0.029ng/mL for Simoa, while the plate ELISA showed a LOD of 0.46ng/mL. DDW
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