IMMUNOGENICITY TESTING USING FLOW CYTOMETRY - Flow Cytometry in Drug Discovery and Development - page 200

the need for lengthy cell separation methods. However, use of the flow cytometer as a
platform for developing immunogenicity assays does not always take advantage of
these capabilities. Immunogenicity method development may employ cells expres-
sing target proteins for biotherapeutic binding and the detection of the binding event
or the resulting signal is recorded. Neutralizing antibodies would interfere with the
drug target binding and result in lower recorded values. Applying bioassays in this
manner for immunogenicity testing more closely mimics physiological conditions.
The use of flow cytometry for immunogenicity testing also offers the advantage of cell
viability assessment and cell gating options that are not typically available in nonflow
cytometry-based bioassays. Changes in cell population growth characteristics are
readily detected from the scatter profile and are used with the concurrent cell viability
measurements. The acceptable ranges of thesemeasurements can be incorporated into
the overall assay run acceptance criteriawhenmonitoring assay performance. The use
of flow cytometry also offers the advantage of investigating both cell surface binding
and intracellular signaling using permeabilized cells that are able to maintain their
capacity to respond to cell surface receptor stimulation. Early signal events such as
tyrosine and serine phosphorylation of cellular proteins [3], generation of cAMP, or
inositol-phosphate hydrolysis can be measured in response to drug stimulation of
permeabilized cells.
The advantages of flow cytometry assays are rapid and quantitative measurements.
Depending on the mode of action of the drug and the selected readout, flow cytometry
can offer an alternative assay format without using harsh denaturation steps and
lengthy sample manipulation, as in the case of a fluorescent thymidine analogue that
can be used to measure newly synthesized DNA in situ [4]. The use of flow cytometry
as an assay platform for evaluating neutralizing activity requires a functional binding
or signal transduction event involving a fluorescent reagent. The biological event to be
measured must occur with sufficient signal in the presence of the intended biological
matrix, so that a change in the signal due to neutralization can be detected [1]. The
challenges of applying flow cytometry to immunogenicity testing lie in the under-
standing of the technical aspects of flow cytometry, readout selected, and internal
variability of the assay and applying an assay cut point or threshold to define a sample
as positive or negative. This chapter will describe the use of flow cytometry for the
measurement of immunogenicity using as examples a biotherapeutic that binds a cell
surface receptor and a biotherapeutic enzyme that is actively internalized.
10.2 SELECTION OF ASSAY REAGENTS
Numerous well-characterized fluorescent dyes are available for the purpose of
preparing dye-reagent conjugates for use in assay development, validation, and
sample testing [5]. Assay reagents can be either labeled directly with a fluorescent
dye or used unlabeled with a secondary labeled reagent for detection. Common
conjugation chemistries via functional groups on the protein as well as considerations
for characterization of functional binding and specificity are necessary to be able to
select optimum assay reagents. Conjugation of a protein may result in heterogeneity