Biomedical Engineering Reference
In-Depth Information
Similar principles could be applied to the use of target molecule-conjugated beads.
The Ag, antidrug Ab, receptor, or ligand, could be conjugated to beads and the use of a
fluorescent dye-conjugated secondary detection reagent allows drug quantification.
The focus of this chapter is to define key parameters for flow cytometric PK assay
development and validation using cell-based analysis. Examples of flow cytometric
PK analysis provided in this chapter are two antibody drugs that bind to cells
expressing surface target antigen. Detection is accomplished by using a second Ab
(FITC or PE conjugated) recognizing heavy chain of the Ab drug. Emphasis will be on
assay development that is unique for flow cytometric PK assay. Validation of flow
cytometric PK assays should follow the same guidance from the FDA and ICH on
analytical method validation as described later in this chapter.
11.2 ASSAY DEVELOPMENT
11.2.1 Flow Cytometer
Most of the flow cytometers meet the instrument requirement for PK analysis.
Typically, PK analysis can be done using a single color. Adding a cell viability dye,
PI (propidium iodide) or 7-AAD (7-aminoactinomycin), in the analysis requires a
second color. Cytometers with a 488 nm (blue) laser are sufficient for most analysis
using fluorochrome such as FITC (fluorescein isothiocyanate), PE (phycoerythrin),
and so on. A second 633 nm (red) laser will allow the use of additional fluorochrome
such as APC (allophycocyanin). Selection of a stable fluorochrome that can retain
signal over time is important to reduce assay variability and improve overall assay
performance.
Cytometers that collect analogue data or digital data are appropriate for PK
analysis. The use of a sensitive instrument will allow quantification of drug at low
levels. Although some newer cytometers can generate a much higher florescence
digital signal in comparison to the analogue signal, both types of instruments are
adequate for PK analysis (shown in examples described later). The instruments can
also provide a degree of automation. Depending on the blood volume of the sample, an
instrument with a tube loader or 96-well high-throughput system has added advan-
tages for sample acquisition in comparison to single-tube acquisition. Most of the
analytical software for flow cytometric data analysis can be used. Software that allows
batch analysis has added advantages.
11.2.2 Cell Characterization to Select Cells with Homogeneous, High, and
Stable Surface Target Ag Expression
During assay development, cell characterization is important and the use of well-
characterized cells is themost critical component in the development of a high-quality
flow cytometric PK assay.
11.2.2.1 Homogeneous and High Target Antigen Expression
Primary cells or
cell lines that naturally express the target Ag can be used. Alternatively, appropriately
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