Biomedical Engineering Reference
In-Depth Information
10.3.4.2 Drug Interferences In addition to the interfering substances mentioned
previously, biotherapeutics with relatively long half-lives may still be in circulation at
the time the sample is taken. This circulating biotherapeutic may interfere in the NAb
assay by binding and sequestering drug-specific antibody or by binding to the target
and inappropriately increasing the neutralizing signal. Where the biotherapeutic has a
relatively short half-life, such as an enzyme, there is no apparent assay interference.
In both the situations, timing of sample collection should be thoughtfully considered
to reduce the impact of biotherapeutic on the detection of neutralizing activity in a
study sample.
To minimize the potential for drug being present in the sample, it is recommended,
when possible, that samples be collected prior to initiation of each treatment. For
biotherapeutic molecules with short half-lives, this practice will ensure a drug-free
sample. When the biotherapeutic has a long half-life, obtaining a drug-free sample is
more challenging. It may be necessary to collect samples over the span of the clinical
trial and evaluate them for circulating drug prior to testing for NAb. Alternatively, the
study may be designed to continue collecting samples from the patient until drug is no
longer detectable in circulation.
10.3.4.3 Interference by Circulating Target Similar to the challenges described
with circulating drug, modulation of signal may occur when soluble target is present
in circulation. The biological mechanisms of therapeutic molecules must be taken
under consideration and, if possible, samples collected from treated and untreated
patient populations should be evaluated for the presence of soluble target. If soluble
target is confirmed to be in circulation, their impact can be minimized by removal or
use of competitive assay formats.
10.3.4.4 Staining Patterns Attention should also be given to unexpected patterns
of staining that may present as secondary peaks on either side of the main histogramor
as a trailing, diminishing extension of the main histogram peak. Some causes of
abnormal staining patterns to consider are uneven staining due to decreased receptor
exposure resulting from cell clumping or overcrowding of treated cells in culture.
Variability in staining patterns may also be explained by heterogeneity of the target
cell population that can result from excessive cell passaging or from culture
conditions favoring a given subpopulation. Since these artifacts can interfere with
assay cut point assignment and determination of neutralizing activity in a sample, it is
therefore important to determine the reasons for these types of anomalous staining
patterns and incorporate assay strategies to minimize them.
10.4 CLINICAL APPLICATION
The safety and efficacy of administered biotherapeutics drive immunogenicity
concerns and appropriate testing of immune responses. Figure 10.7 illustrates three
patients with varying responses to antibody-mediated neutralizing activity over a
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