Recommendations for the validation of flow cytometric testing during drug development: II assays

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(Table 1). Definitive quantitative assays include calibrators fit to a regression model to calculate absolute values and reference standards that are well characterized and fully representative of the endogenous measurand. Definitive quantitative assays can be both accurate and precise. Relative quantitative assays utilize responseconcentration calibration, however in this scenario the reference standards are not fully characterized or truly representative of the endogenous measurand. As such, imprecision can be demonstrated for a relative quantitative method, but accuracy can only be estimated. With quasi-quantitative assays there is a relationship between the response and the measurand but calibration standards are not used. Thus, quasi-quantitative methods can be validated for imprecision, but not accuracy. Qualitative methods generate categorical data. Flow cytometric methods largely fall in the two latter categories and are essentially therefore quasi-quantitative or qualitative.Multi-color flow cytometry is a unique technology, which enables the analysis of heterogeneous cellular systems and provides multiparametric information at a cellby-cell level. The strength of flow cytometry lies not only in the ability to simultaneously measure multiple parameters, but also in the flexibility to report them in different ways. The appropriate data output depends on the biology of the system being investigated, the analytical or scientific question being asked, and the intended use of the results. A wide variety of data outputs can be reported usually expressed in terms of several characteristics of cells, or cell subsets, in the sample tested for example, percentage of positive events, absolute counts, median fluorescence intensity, quantitative antigen expression levels, ratiometric indices, markers coexpression, or relative nucleic acid content.

Section: Validation Samplesmentioning

confidence: 99%

Section: Assay Of Standard or Reference Materialsmentioning

confidence: 99%

Validation of cell‐based fluorescence assays: Practice guidelines from the ICSH and ICCS – part V – assay performance criteria

Wood

Jevremović

Béné

et al. 2013

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236

“…(Fig. 6) The fit‐for‐purpose, iterative approach was applied to the validation strategy in that, as the intended use of the data and the associated regulatory requirements changed, the assay was re‐optimized and re‐validated appropriately 24, 25, 26. The prototype assay was developed during the drug discovery phase and initially used to detect binding of anti‐SEMA4D candidates to cellular SEMA4D.…”

Section: Discussionmentioning

confidence: 99%

Saturation monitoring of VX15/2503, a novel semaphorin 4D‐specific antibody, in clinical trials

Fisher

Seils

Reilly

et al. 2015

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BackgroundReceptor occupancy, or saturation, assays are often utilized in preclinical and clinical development programs to evaluate the binding of a biologic to a cellular target. These assays provide critical information regarding the dose of drug required to “saturate” the target as well as important pharmacodymamic (PD) data. A flow cytometric method was developed to measure the degree of Semaphorin 4D (SEMA4D; CD100) saturation by VX15/2303, an investigational monoclonal antibody specific for SEMA4D.MethodsThe assay detects VX15/2503, a human IgG4 specific for SEMA4D, with an IgG4‐specific monoclonal antibody.ResultsData generated allowed assessment of two related SEMA4D‐specific pharmacodynamic (PD) markers: (1) The measurement of cellular SEMA4D (cSEMA4D) saturation by VX15/2503, and (2) the cell membrane expression levels of cSEMA4D.ConclusionsThis assay specifically and reproducibly measured cSEMA4D saturation and expression levels. Evaluation of the SEMA4D‐specific PD markers were critical in determining the clinical saturation threshold of cSEMA4D by VX15/2503. © 2015 he Authors Cytometry Part B: Clinical Cytometry Published by Wiley Periodicals, Inc.

“…Typically, flow cytometry‐based assay validation procedures are “fit‐for‐purpose” in that the goal is to assess assay performance primarily in conditions applicable to the study 50. However, there is also a standard set of parameters characterized for most flow cytometry‐based RO assays including assessments of intra‐ and inter‐assay precision and sample stability 51.…”

Section: Considerations When Developing Flow Cytometry‐based Ro Assaysmentioning

confidence: 99%

Receptor occupancy assessment by flow cytometry as a pharmacodynamic biomarker in biopharmaceutical development

Liang¹,

Schwickart²,

Schneider³

et al. 2015

Receptor occupancy (RO) assays are designed to quantify the binding of therapeutics to their targets on the cell surface and are frequently used to generate pharmacodynamic (PD) biomarker data in nonclinical and clinical studies of biopharmaceuticals. When combined with the pharmacokinetic (PK) profile, RO data can establish PKPD relationships, which are crucial for informing dose decisions. RO is commonly measured by flow cytometry on fresh blood specimens and is subject to numerous technical and logistical challenges. To ensure that reliable and high quality results are generated from RO assays, careful assay design, key reagent characterization, data normalization/reporting, and thorough planning for implementation are of critical importance during development. In this article, the authors share their experiences and perspectives in these areas and discuss challenges and potential solutions when developing and implementing a flow cytometry‐based RO method in support of biopharmaceutical drug development. © 2015 The Authors Cytometry Part B: Clinical Cytometry Published by Wiley Periodicals, Inc.