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.

Role of receptor occupancy assays by flow cytometry in drug development.

The measurement of the binding of a biotherapeutic to its cellular target, receptor occupancy (RO), is increasingly important in development of biologically-based therapeutic agents. Receptor occupancy (RO) assays by flow cytometry describe the qualitative and/or quantitative assessment of the binding of a therapeutic agent to its cell surface target. Such RO assays can be as simple as measuring the number of cell surface receptors bound by an antireceptor therapeutic agent or can be designed to address more complicated scenarios such as internalization or shedding events once a receptor engages the administered therapeutic agent. Data generated from RO assays can also be used to model whether given doses of an experimental therapeutic agent and their administration schedules lead to predicted levels of receptor occupancy and whether the receptor is modulated (up or down) on cells engaged by the therapeutic agent. There are a variety of approaches that can be used when undertaking RO assays and with the ability to measure distinct subsets in heterogeneous populations, flow cytometry is ideally suited to RO measurements. This article highlights the importance of RO assays on the flow cytometric platform in the development of biotherapeutic agents.

Recommendations for the evaluation of specimen stability for flow cytometric testing during drug development.

The objective of this manuscript is to present an approach for evaluating specimen stability for flow cytometric methods used during drug development. While this approach specifically addresses stability assessment for assays to be used in clinical trials with centralized testing facilities, the concepts can be applied to any stability assessment for flow cytometric methods. The proposed approach is implemented during assay development and optimization, and includes suggestions for designing a stability assessment plan, data evaluation and acceptance criteria. Given that no single solution will be applicable in all scenarios, this manuscript offers the reader a roadmap for stability assessment and is intended to guide the investigator during both the method development phase and in the experimental design of the validation plan.

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.