IMMUNOGENICITY TESTING USING FLOW CYTOMETRY - Flow Cytometry in Drug Discovery and Development - page 213

packages are available for free downloading at the Purdue CytometryWeb site: www.
cyto.purdue.edu/flowcyt/software.htm. Some of these applications have good report-
generating functions as well, which may be important in some laboratory settings.
Both FlowJo and FCS Express applications offer 21 CFR Part 11 compliant solutions
that are necessary when testing samples in a GLP-regulated lab using validated flow
cytometers.
10.3.4 Technical Considerations
10.3.4.1 Biological Matrices Matrix interference may be described as the effect
of a substance present in the sample that causes a deviation of themeasured value from
the true value. The interferences may be endogenous or exogenous. Endogenous
interferences include such substances as lipids, heterophilic antibodies, rheumatoid
factors (RF), complement, binding proteins soluble biotherapeutic target, ligands, and
receptors [24-27]. Exogenous interferences include anticoagulants, concomitant
medications, and sometimes inappropriate sample storage conditions that change
the biological components of the sample [27].
In neutralizing antibody assays, interferences present in samples, whether
endogenous or exogenous, may mimic the binding of the drug or affect the detection
of neutralizing activity in the sample. Depending on the nature of the interaction, a
sample result may be falsely positive or falsely negative.
During immunogenicity testing using flow cytometry, assay interferences caused
by matrix substances may present as a nonspecific signal when cells bind molecules
that cross react with drug or when cells are responsive to these nonspecific compo-
nents. This falsely elevated assay signal could potentially result in a reduced ability to
detect neutralization activity by artificially increasing the apparent concentration of
drug used in the assay. When matrix substances interfere with the binding of
neutralizing antibodies present in the sample, again the potential exists to report
erroneous sample results. Sample pretreatment may be necessary to remove or reduce
the level of interfering substances, or a different assay format considered that is less
susceptible to such interferences.
Furthermore, a specificity assay can be used to distinguish the pharmaceutical
drug target binding event or signaling from that signal produced by other structu-
rally related molecules. Similarly, studies can be designed to determine whether
there is nonspecific interaction occurring between matrix components and neu-
tralizing antibody. An assessment of specificity should be conducted using appro-
priate disease-state matrices so that the structurally related molecules are present in
the amounts anticipated in the test sample. An example of a specificity assay for a
cell surface NAb binding assay involves the use of an irrelevant cell surface marker.
For enzyme uptake assays, an irrelevant enzyme conjugate whose cell-mediated
uptake is unaffected by the presence of specific neutralizing antibodies may be used.
In either assay, detector reagent and enzyme conjugate titrations are conducted to
optimize conditions for the specificity assay and the validation parameters outlined
in Ref. [1] are applied.