Validation of a flow cytometry-based assay to assess C5aR receptor occupancy on neutrophils and monocytes for use in drug development.

The C5a/C5a receptor (C5aR) pathway, a key component in the proinflammatory immune response, is an attractive therapeutic target since its dysregulation is implicated in a variety of autoimmune and inflammatory disorders. The objective of the present study was to validate a receptor occupancy (RO) assay for a human anti-C5aR monoclonal antibody drug candidate, NNC0215-0384 (NN0384). This flow cytometry-based assay measures the percentage (%), median fluorescence intensity (MFI), and molecules of equivalent soluble fluorochrome (MESF) of NN0384 binding to its target cells, neutrophils and monocytes, in whole blood from normal healthy donors and rheumatoid arthritis (RA) patients with clinically active disease. The validation parameters assessed included postcollection and postprocessing sample stability, intra- and interassay precision, an analyst-to-analyst comparison, a comparison of normal healthy donor and RA patient sample postcollection stability, and a laboratory-to-laboratory comparison and assay transfer. The cumulative results indicate that the assay was reproducible, met the clearly defined acceptance criteria for the validation parameters tested, and was transferable to another laboratory. In conclusion, this RO assay is suitable for use to accrue pharmacodynamic biomarker data in a multicenter, global clinical trial.

Recommendations for the development and validation of flow cytometry-based receptor occupancy assays.

Receptor occupancy measurements demonstrate the binding of a biotherapeutic agent to its extra-cellular target and represent an integral component of the pharmacodynamic (PD) portfolio utilized to advance the development and commercialization of a therapeutic agent. Coupled with traditional pharmacokinetic (PK) assessments derived from serum drug concentration, receptor occupancy data can be used to model PK/PD relationships and validate dose selection decisions throughout the drug development lifecycle. Receptor occupancy assays can be even more challenging to develop than other flow cytometric methods (e.g. surface immunophenotyping). In addition to typical considerations regarding stability of the cell type of interest, stability of the target-bound therapeutic agent and stability of the target receptor must be taken into account. Reagent selection is also challenging as reagents need to be evaluated for the potential to compete with the therapeutic agent and bind with comparable affinity. This article provides technical guidance for the development and validation of cytometry-based receptor occupancy assays.

Recommendations for the validation of flow cytometric testing during drug development: I instrumentation.

Flow cytometry is a powerful and flexible analytical tool used during all stages of drug development. While substantial effort is invested in development and validation of analytical methods, instrument validation is often neglected. Flow cytometers are evolving at a pace that surpasses the protracted timeline of drug discovery and development. Therefore, it becomes fundamentally important to the success of the study to document the validated state of the flow cytometer and verify data integrity at the time of study conduct. It is important to bear in mind that validation strategies are critical components of the entire process involved in bringing new therapeutic options to the medical community; drugs which eventually manifest as successful new treatments for those individuals afflicted with disease. Formal industry guidance is provided through Good Laboratory Practices [GLP], which require validation of all computerized systems and equipment used to support pre-clinical studies for regulatory submissions. Key elements of instrument validation processes have been delineated through guidance documents published by regulatory agencies and industry working groups to support the rigorous compliance needs of GLP. However, most testing to support drug development is conducted in less strict regulatory environments. Such comprehensive validation efforts may not be appropriate for laboratories supporting early discovery or basic research, however, laboratories involved in regulated stages of development, such as pre-clinical and clinical phases, should consider these recommendations. This paper presents a consensus methodological approach that the authors have used successfully to ensure data integrity in flow cytometric studies conducted during drug development.