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Ligand binding assays (LBAs) play a critical role in quantifying serum therapeutic protein (TP) levels in pharmacokinetic and pharmacodynamic assessments. Described herein is the impact of the bioanalytical platform, reagent-antibody affinity, and incubation time on obtaining free or total TP levels. An ELISA-based LBA that quantifies humanized anti-sclerostin monoclonal antibody (TPx) was used as the model system. All kinetic experiments were performed by Bio-Layer Interferometry (BLI) on a Pall ForteBio Octet RED384 system equipped with Streptavidin (SA) Biosensor probes. The sensor tips were loaded with biotinylated-Anti-Fc mAb, which were subsequently captured with TPx. Sensor probes were then dipped in wells containing Sclerostin (Scl) protein. Kinetic parameters (kon, koff, and KD values) were determined. The importance of having a complete picture of the binding interaction from a BLI kinetic assay as compared to the use of end-point data from ELISA is higlighted. Overall results of this investigation provide a better understanding of how the choice of reagents and the platform for LBAs can affect accurate quantitation of either the free or total TP concentrations.

PubMed

This is a comparability study of a biosimilar trastuzumab with respect to its reference product to evaluate the physicochemical and biological aspects. Trastuzumab is a monoclonal antibody that interferes with the human epidermal growth factor receptor (HER2), which overexpresses in certain invasive forms of breast cancers. A Pall ForteBio Octet QK384 instrument equipped with Streptavidin Biosensor probes captured with Biotinylated FcRn investigated the binding of trastuzumab to FcRn. Overall results of this investigation suggest that there is a high degree of similarity in the physicochemical properties of the biosimilar trastuzumab with respect to the reference product as evident from comparable biological activity.

PubMed

The authors present rationale and strategies for the development and analytical qualification of a BLI assay for the quantitation of a humanized antibody therapeutic in cynomolgus monkey plasma. They used an Octet QK system, a first-generation instrument based on BLI technology. Reference is made at several points in the publication to the Octet RED-type systems that are capable of higher sensitivity detection in comparison to the Octet QK system. Results of the qualification were compared to those of a validated ELISA used to quantitate the same therapeutic. Selectivity, matrix effect, and precision and accuracy were found to be similar between the two methods. The main difference between the two assays was in the dynamic range (0.1-10 μg/mL for ELISA vs. 0.4-50 μg/mL for BLI). A direct quantitation comparison of sample results for the two methods shows a high degree of agreement (r2=0.979, slope=1.017).

PubMed

In this article scientists at Genentech review the different bioanalytical strategies that have been implemented to characterize various antibody-drug conjugates (ADCs) and discuss the challenges and issues associated with these approaches. The Octet platform is discussed as a convenient alternative to fluidics-based systems for the analysis of ADCs.

PubMed

The authors compare performance of the Octet system to ELISA and MSD techniques for detection of anti-drug antibodies (ADA) against an investigational therapeutic human IgG1 mAb called CNTO X. They found that the Octet platform assay was the most sensitive and ELISA the least sensitive for detection of low-affinity ADAs. The assay also tolerated the presence of free drug to 10X greater levels than the MSD assay, and 100X greater levels than the ELISA assay. The MSD and Octet platform assays were applied to the bioanalysis of cynomolgus monkey sera from a pre-clinical multiple dose study of CNTO X. The Octet system indicated 3 positive animals developed ADA as early as day 15 of the dosing phase while drug was present at nearly 1 mg/mL. ECLIA detected only one of these, and only in a day 57 recovery sample after drug had cleared from circulation. The authors conclude that the Octet instrument is a promising platform for detection of lower affinity ADAs and is particularly suitable for ADA detection when drug persists at levels that negatively impact bridging immunoassays.

PubMed

Judy Shih, Ph.D., Pharmacokinetics and Drug Metabolism, Amgen, Octet System User Workshop - NBC, 2012 Quality assay reagent is the key for ligand binding assays, particularly for assay specificity and sensitivity. Assay reagent screening, characterization and selection process has been well established. ELISA has been widely and routinely used. Complementary tools include Biocore and cell based assays. This presentation demonstrates that the Octet platform is another complementary tool with high throughput and increased efficiency. Two case studies address assay reagent screening, paring, and bridging assessment. The third case study describes how the Octet platform facilitated assay troubleshooting. Furthermore, the platform has been implemented for reagent biotinylation assessment. In summary, the Octet platform is an efficient tool for ligand binding assays.