A newly published paper in Biotechniques from the laboratory of Dr Darren Tomlinson at the University of Leeds, UK, demonstrates key characteristics exhibited by Affimer reagents in capturing target proteins from complex matrices and shows how they outperform commercially available Fab fragments as critical reagents in clinical PK assays.
Four inhibitory anti-idiotypic Affimer reagents to the therapeutic antibodies trastuzumab, rituximab, adalimumab and ipilimumab showed excellent performance in this study in terms of target specificity, sensitivity and assay dynamic range in a standard sandwich ELISA format for PK assays. Furthermore, the calibration curve metrics for these assays and the batch-to-batch reproducibility of anti-ID Affimer reagents were shown to meet regulatory guidelines, demonstrating the significant advantages offered by the use of anti-ID Affimer reagents as antibody alternatives in the development of clinical PK assays.
A simple negative selection protocol successfully drove binding towards the target therapeutic antibody, generating highly specific Affimer binders that exhibit minimal matrix effects in 10% human serum and no cross-reactivity with non-specific mAbs, without any need for affinity maturation. Calibration curve metrics for all four Affimer anti-ID reagents and the lot-to-lot reproducibility over four separate lots of anti-trastuzumab Affimer reagent met FDA criteria for critical reagents for bioanalytical assays, highlighting the PK assay performance potential of Affimer anti-ID reagents.
Assay dynamic range is important to allow a wider range of sample concentrations to be analysed in a single assay. Compared to commercially available assay kits for trastuzumab, rituximab and adalimumab, the developed Affimer ELISAs all showed superior dynamic range, while the anti-ipilimumab Affimer ELISA was comparable with commercially available assays.
All of the Affimer ELISAs were established with a universal detection system, where the specific Affimer reagent for the antibody was used for capture and teamed with a generic anti-human Fc Affimer reagent for detection. In comparison, most antibody-based PK assays require a bridging assay format, using two different target specific affinity reagents, to ensure the required level of specificity in the end assay.
The universal detection possible with Affimer ELISAs allows for faster assay design and development, requiring only one specific affinity reagent to establish a specific ELISA, and overcomes issues of low sensitivity and inconsistency associated with bridging assays.
Anti-ID Fab fragments can be selected in vitro from phage libraries, in a similar way as Affimer reagents. The team at Leeds University compared commercially available Fab fragments to Affimer reagents for the four therapeutic antibodies examined in this study, with each of the ELISAs carried out to perform optimally according to the affinity reagent used. For each antibody target analysed, the Fab-based ELISA showed a reduced dynamic range compared to the Affimer ELISA, and also an increased matrix effect for both the rituximab and adalimumab assays. This comparison illustrates the high specificity of Affimer reagents and their suitability for use as critical reagents in clinical PK assays for drug development and monitoring.
This study clearly demonstrates that the Affimer platform can be reliably used to develop anti-ID reagents for immunoassays, to measure clinically relevant concentrations of therapeutic antibodies with accuracy and precision metrics that meet regulatory criteria. The performance improvements offered by Affimer reagents in terms of high specificity, low matrix effects, broad dynamic ranges, flexible universal detection formats and low lot-to-lot variation allow ease of assay development and assurance of supply, preventing delays in an arena where speed to market is essential. As the development of antibody therapeutics continues to expand, Affimer anti-ID reagents may represent key reagents for use in critical PK assays.
Avacta Life Sciences Principal Assay Scientist Dr. Amanda Nicholl will present data from the paper in a webinar on September 18th. Register to attend here. (link is external)