Using Octet QK for the Quantitation of a Humanized IgG2 Therapeutic in Cynomolgus Monkey Plasma

Enabling Ligand Binding Assay Reagent Development

Mark Dysinger^a, Lindsay E. King^a, and Islay Campbell^b

Highlights

• Label-free reagents
• Compatible with complex matrices
• Wider dynamic range
• Minimal dilutions
• Fast assay development
• No wash steps, as required in ELISA
• Fewer full-time employees needed

The following data were presented by Mark Dysinger and Lindsay King as a poster at the 2009 American Association of Pharmaceutical Scientists (AAPS) annual meeting¹. The purpose of this work was to determine the quantitative capabilities of the Octet QK system that utilizes BioLayer Interferometry (BLI) technology in comparison to a validated chemiluminescent ELISA for potential use for regulated TK sample analysis.

Quantitation of analytes using ligand binding assays is a well established approach with multiple detection modalities (colorimetric ELISA, electrochemiluminescence, fluorescence, etc). On the other hand, label-free technologies such as Surface Plasmon Resonance (SPR) on the Biacore platform are well established for the evaluation of binding kinetics. Recently, these technologies are also emerging as approaches for the quantitation of analytes.

ForteBio’s Octet system utilizes BioLayer Interferometry (BLI) technology to directly measure the binding of unlabeled analyte in real-time to a ligand immobilized on a biosensor tip surface. This platform can be used for both kinetic and quantitative analyses of analytes.

The gold standard for large molecule analyte quantitation is ELISA. For the purposes of this investigation reagents used in a previously validated sandwich ELISA assay were used to construct an Octet BLI assay that was analytically qualified to determine the technology’s potential use in TK sample analysis. The analytical qualification parameters used to define the acceptable limits in which the data are deemed reliable are as follows:

• Regression Model - Fifteen standard concentrations ranging from 100 µg/mL to 0.25 µg/mL analyzed on each of five days (single replicate each).
• Accuracy and Precision - Seven sample concentrations (ULOQ, HQC, MHQC, MQC, MLQC, LQC, LLOQ) analyzed in triplicate on each of five days.
• Matrix Evaluation - Ten individual lots of matrix (no humanized IgG2 added) analyzed for potential assay interference/background.
• Selectivity Evaluation - Same ten individual lots of matrix spiked with humanized IgG2 at HQC and LQC levels and analyzed for potential assay interference.

Qualification Notes

• Each sample, 1 replicate = 2 biosensors/well
• Standards and qualification samples were generated on a Hamilton Star automated system

A five-day qualification of the Octet QK yielded accuracy, precision, and selectivity results that were comparable to those of the chemiluminescent ELISA validation. Although the Octet platform appears very different from ELISA, the actual experiments for analytical validation/qualification were essentially identical. Accuracy and precision results were generated from seven QC sample levels (ULOQ, HQC, MHQC, MQC, MLQC, LQC, LLOQ), and selectivity results were generated from high and low concentrations of therapeutic spiked into ten individual lots of monkey plasma. The dynamic range for the assay was established at 0.4–50 μg/mL (compared to 0.1–10 μg/mL in the ELISA validation). The minimum required dilution was 1:2 versus 1:10 used in the ELISA. For assay selectivity, 90% of lots tested within 100±20% recovery.

Although the Octet BLI assay did not have as low an LLOQ value as the ELISA assay (0.4 µg/mL vs. 0.1 µg/mL), the dynamic range was greater. The Octet assay has a 1:2 required minimum dilution whereas the ELISA requires a 1:10 minimum dilution which suggests that there was less matrix interference in the Octet BLI assay.

Label-free technologies are becoming established in Ligand Binding Assay (LBA) reagent development. Instruments such as Biacore, dotLab, and Octet are used to determine reagent specificities and relative affinities, which aid in the characterization and subsequent selection of these reagents for assay development and optimization. Based on our observations during this investigation, the Octet system is simple to use and requires minimal hands-on training. Data analysis is executed via Octet software, allowing for linear, 4PL, or 5PL regression. The minimal assay prep and absence of wash steps result in less direct FTE time compared to ELISA. Assay development was fast, results are generated in real time, label-free reagents can be used, and the technology is compatible with complex samples as demonstrated by the 1:2 minimum required dilution. With the addition of an available CFR Part 11 compliant software package, this Octet BLI assay could be applied for the quantitation of biotherapeutics in a regulated environment.

The ability to quantify an analyte of interest in complex matrices while using label free reagents gives Octet a unique niche. The ability to see results in real time confirms assay functionality. The data presented support the use of the Octet QK system for the quantitation of a humanized IgG2 therapeutic in cynomolgus plasma. Furthermore, the extended dynamic range (as compared to the validated ELISA method) of the qualified assay suggests that it would be of particular utility in TK studies where animals are typically dosed with high amounts of drug. 

References

¹ Dysinger, M. and King, L.E. Use of Bio-Layer Interferometry (Octet) for the Quantitation of a Humanized IgG2 Therapeutic in Cynomolgus Monkey Plasma; The AAPS Journal. 2009, 11(S2); 2329.

ForteBio Notes

ForteBio’s Octet platform consists of the Octet QK, Octet QK^e, Octet QK384, Octet RED and Octet RED384 systems, each with distinctive capabilities. The data described in this note were generated on the Octet QK system, which is a first-generation instrument. The other Octet systems have been outfitted with higher performance spectrometers that provide higher signal to noise resolution and thus, greater sensitivity. The Octet RED and RED384 provide the broadest dynamic range for quantitation and kinetic analysis, greatest sensitivity, ability to measure small molecule - protein binding and ability to measure fast binding interactions.

^a Pfizer Global Research and Development, Groton/New London Laboratories, Pfizer, Inc, Groton, CT 06340
^b ForteBio, Inc, Menlo Park, CA 94025

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