(M0930-09-50) Robustness Testing of an Assay for Flow Cytometric CAR Persistence Determination

Purpose: Flow cytometric determination of CAR persistence by specific fluorescent labeling and detection of circulating CAR T cells after application of the cellular drug product is used in clinical trials as part of immune monitoring and may be defined as a secondary endpoint (e.g. PK parameter analysis) in the clinical trial protocols. Due to the nature of rare cell analysis, a robust and reliable assay with well-selected reagents is the basis for longitudinal measurements on patient samples from multi-center, global clinical trial.

Methods: According to the guidelines of the International Council for Harmonization (ICH) the robustness of an assay is defined as the assay’s capacity to remain unaffected by small, but deliberate variations in assay parameters and provides an indication of its reliability during normal usage. The individual practical steps of the assay (bulk lysis of whole blood, fluorescence staining procedure, and flow cytometric measurement) were evaluated individually using a low level risk assessment approach. This method was used to identify the critical main parameters (factors, X) and parameter interactions to be investigated in the robustness study. The statistical Design of Experiments (DOE) approach was then used to define the group of parameters to be tested. Different responses (Y) were defined as readouts, which were then examined with regard to repeatability and accuracy. Due to the size of the study (USA and Europe with two central measurement centers), the consistency of two sets of reagents were investigated as a further part of the robustness testing to ensure that comparable results can be expected.

Results: Here we present results obtained during the robustness testing indicating a robust assay with suitable parameter optima and edge of failure ranges (design space). All in all 15 different parameters were tested to achieve the most robust method possible. Here we focus on four parameters (incubation time of the CD19 CAR detection reagent, hold time of the stained samples before measurement, staining time of the secondary staining, and age of the secondary staining cocktail) affecting the staining steps as representative data of the robustness testing. The lot-to-lot consistency testing was embedded in an assay variance test (2 reagent sets, 4 operators, 2 devices, 6 donors), revealed two highly comparable reagent sets suitable for the realization of the analyses of the clinical trial.

Conclusion: With the addition of on-site method validation in the clinical research organization's (CRO’s) laboratories (data not shown here), the assay is fit for purpose for immune monitoring of CAR persistence within clinical trials.

References: ICH Topic Q 2 (R1), Validation of Analytical Procedures:Text and Methodology, June 1995 CPMP/ICH/381/95
Little TA., Design of experiments for analytical method development and validation, Bio. Pharm. Int. 27(3) (2014).
Coffy T, Grevenkamp M, Wilson A et al. Bioassay qualification using design of experiment. Bioprocess Int. 11(6), 42–49
(2013).

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Parameter optimum and edge of failure analysis of "age of staining cocktail" and "inubation time of second staining".


Results of assay variance experiments.