Working with Pharmacokinetic Bridging Assays

Pharmacokinetics (PK) is defined as the movement of a drug through the body. The absorption, distribution, metabolism, and excretion of a drug are all key pharmacokinetic factors to be taken into account while developing therapeutics.

The potential to detect and measure the concentration of a therapeutic molecule with high sensitivity may be essential for effective pharmacokinetic studies, particularly if the drug is dispensed at a low dose.

This article describes a proof-of-concept assay to establish the Simoa platform as a tool for quantifying the concentration of biotherapeutic IgG adalimumab. Anti-adalimumab antibodies were used as capture and detection to develop a sandwich ELISA. The performance was compared by running equivalent assays with the Simoa platform and a standard plate-based ELISA.

Reagents and Materials

Both assays employed a monovalent Fab as the capture reagent. A fully human monoclonal IgG1 was used as the detection reagent. As a target, the IgG1/kappa drug was spiked into samples at changing concentrations.

ELISA Assay

The monovalent Fab was coated on a high-binding ELISA plate overnight at 1 µg/mL. The plate was cleansed and blocked using 5% BSA in PBST. To obtain the calibration curve, adalimumab was titrated between 1 and 1000 ng/mL in a 2% BSA + PBST + 10% human serum buffer and added to the plate for a 2-hour incubation.

After trapping adalimumab and cleansing the plate, the detection was carried out with the biotinylated human monoclonal IgG at 1 µg/mL in the same buffer with 1-hour incubation. Once the detection was completed, the plate was cleansed and Streptavidin beta-galactosidase (SbG) enzyme was added at 500 pM in Quanterix SbG diluent for a 30-minute incubation. After enzyme labeling, the plate was cleansed again and subsequently incubated with RGP substrate. Resorufin fluorescence was quantified after 1 hour as an assay readout.

Simoa Assay

EDC+SNHS chemistry was used to couple monovalent Fab to magnetic microparticles. The calibration curve was obtained by titrating adalimumab between 0.1 and 100 ng/mL in a 2% BSA + PBST + 10% human serum buffer.

For Simoa, the same detection antibody which was employed for the ELISA—1 μg/mL biotinylated monoclonal IgG—was used. Labeling was carried out with 50 pM SbG. The assay was carried out on the Simoa HD-1 platform as a two-step assay with incubation times of 60 minutes for capture/detect and 15 minutes for SBG labeling.

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), labeled with Biotin. Image: Bio-rad.

Figure 1. Schematic image of PK Bridging Simoa ELISA. Anti-idiotypic capture antibody, Fab format (purple), monoclonal antibody drug (gold, adalimumab), anti-idiotypic detection antibody, Ig format (blue), labeled with Biotin. Image: Bio-rad.

Results

Both the Simoa assay and the ELISA assay exhibited a dose-dependent response to adalimumab in a calibration curve, denoting that either of them could be employed to determine the concentration of adalimumab in samples. The Simoa assay exhibited nearly 10x higher signal-to-background ratios across the calibration curve (<100 ng/mL).

The Simoa assay and plate ELISA exhibit quite linear dose responses up to 100 ng/mL. The upper limit of quantification (ULOQ) for the Simoa assay is 200 ng/mL, and for plate ELISA assay, it is 215 ng/mL. The lower limit of quantification (LLOQ) for the Simoa is 0.06 ng/mL, and for plate ELISA assay, it is 0.8 ng/mL. Limit of detection (LOD), established as three standard deviations above the zero calibrator, for Simoa and the plate ELISA was 0.029 ng/mL and 0.46 ng/mL, respectively.

Data

Signal-to-background ratio of the Simoa assay versus plate ELISA for calibrators under 111 ng/mL.

Figure 2. The signal-to-background ratio of the Simoa assay versus plate ELISA for calibrators under 111 ng/mL.

Calibration curve for Simoa assay (0.137–100 ng/mL)

Figure 3. Calibration curve for Simoa assay (0.137–100 ng/mL)

Calibration curve for plate ELISA assay (1.37–1000 ng/mL)

Figure 4. Calibration curve for plate ELISA assay (1.37–1000 ng/mL)

Simoa versus plate ELISA curves (relative units)

Figure 5. Simoa versus plate ELISA curves (relative units)

About Quanterix

Quanterix is on a mission to change the way in which healthcare is provided by giving researchers the ability to closely examine the continuum from health to disease. In order to make this vision a reality, we brought together the most experienced management team, renowned scientists, industry leading investors and expert advisors, to form a collaborative ecosystem, united through the common goal of advancing the science of precision health.


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Last updated: Aug 13, 2019 at 4:01 AM

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