Purpose: In today’s modern bioanalytical laboratory, various platforms may be used to develop ligand binding assays (LBAs) during drug development. Each platform has pros and cons based on the requirements of the assay being developed. One of the key attributes of an assay platform is the run time. Using the biolayer interferometry capabilities of the Octet Red 384 instrument, our goal was to develop a generic Ig dip and read rapid assay format that could be used to quantitate IgG therapeutics, significantly reduce assay run time, and save on reagent requirements. An assay format utilizing a two-step assay process on the Octet Red 384 via streptavidin biosensor tips was successful, however, it was determined that with a one-step direct assay format via anti-human IgG Fc biosensor tips, the results were more precise and accurate, and the overall run time was significantly reduced. This one-step assay was qualified in 20% mouse plasma with assay sensitivity in the low g/mL range. While the sensitivity was not as low as other assay platforms, the run time was reduced by more than 50% compared to other standard LBA platforms (30 minutes for 96 data points). No additional assay reagents beyond anti-human IgG Fc biosensor tips were required using the one-step assay configuration.
Methods: Two assay formats were tested on the Octet Red 384. The two-step assay format utilized streptavidin biosensor tips and a biotinylated goat anti-human IgG capture antibody to detect the human IgG therapeutic. This two-step assay format had approximately a 1 hour run time for 96 data points. The one-step, or direct, assay format utilized pre loaded anti-human IgG Fc quantitative biosensor tips and required very little optimization beyond ensuring the capture reagent did not cross react with mouse IgG. This direct method had an approximate 30 minute run time for 96 data points. For both assay formats, the capture reagent concentration and buffer conditions were optimized, and the range of quantitation assessed using drug-spiked serum quality control samples. For the one-step assay, additional assay performance parameters such as selectivity, specificity, and accuracy and precision were evaluated.
Results: It was determined that a one-step assay method on the Octet Red 384 was much more robust and also faster than the two-step assay. After only a few basic experiments, the one-step dip and read Octet Red 384 assay was optimized and qualified for sample analysis. Recovery ranges for all QCs were within 20% (84% to 115%) of nominal concentration in the one-step assay format. Although the assay was not as sensitive (lower limit of quantitation at 2.5 µg/mL) as other contemporary platforms such as Mesoscale Discovery (MSD) and Gyrolab, the Octet Red 384 could be beneficial for a fast assay turn around in early drug discovery studies that typically use higher dosing concentrations and sensitivity is fit for purpose. Additionally, the sample volume requirement for this generic Ig dip and read assay was optimized to be comparable to other standard LBA platforms by using a 384 well sample loading plate, which utilized only 40 µL of diluted sample per well.
Conclusion: Feasibility of a rapid, precise and accurate one-step generic human IgG dip and read assay on the Octet Red 384 was assessed and demonstrated success. The platform has the flexibility to use anti-human IgG, streptavidin or other user-defined protein as a capture reagent, for example, target or anti-idiotype antibody. There were various benefits compared to other LBA assay platforms, most significantly the reduction in assay run time, real-time, label-free detection, and the need for only one reagent. This platform would be suitable for studies that have a higher dosing regimen and could reduce the sample analysis turn-around time by more than 50% when compared to other standard LBA assay platforms.