However, this is not the case for bioassays and ELISAs, as
there are many other factors required for an assay to work
correctly, and any one can go awry. In contrast to a physicochemical assay, it is not acceptable simply to run a system
suitability sample and then assume every subsequent run of
samples and controls will be fine. Each run is different and
does not relate to a prior run – or even a different plate in the
same set of runs. Problems that could be missed if system
suitability tests were relied on include incorrectly prepared
buffers, mechanical issues, or problems with the instruments.
Everything must be self-contained within each individual
assay, and it is not known until after the assay has been run
whether the acceptance criteria have been met.
As bioassays involve complex biological systems, and
often living systems such as cells, there is a lot of scope for
variability from one run to another. It is common for them
to include more than one cell type, and ligands that can
induce cell responses, whether positive or negative. And
there is a whole host of variables that must be optimised to
achieve reliable and accurate results. The carbon dioxide
levels, humidity and temperature in the incubator must be
correct. The cell density can change between assay plates
and prior cell passage flasks. The cell health and percentage
viability can change in terms of log phase growth and lag
phase. The medium contains many different components,
and the levels of each nutrient, including L-glutamine,
should be optimal. The timing of the addition of assay
components can have an effect, whether for the addition
of the cells, the active or the neutralising material. Other
important variables include the induction time, which can
run from minutes to days, and the read-out reagent, which
might be a RedOx dye, a luminescent or fluorescent reagent,
or an ELISA read-out.
However, not every assay response must be identical
from one assay to the next. The potency of a test sample is
a relative measurement compared to a standard and, in the
majority of cases, a parameter that impacts response to a
test sample will have an equivalent response to a standard.
Thus the absolute values of the response curve may not be
critical. Rather, the dose–response curve must be deemed
adequate, with the assay acceptance criteria laying down
objectively what represents ‘adequate’.
There are two levels at which an assay can be judged.
Multiwell plates have become the standard plat- form for carrying out the majority of potency assays, as they facilitate serial dilutions and allow multiple kits to be run simultaneously.
This significantly reduces the amount of operator time
required compared to running individual samples. The 96
well plate is more commonly used for enzyme-linked immu-nosorbant assays (ELISAs) and bioassays, with 384 well
plates largely being used for high-throughput screening, as
most of the automated washing systems bioassays rely on
are designed exclusively for 96 well plates.
If a physicochemical assay is being run, it is reasonable to
assume that the system will work as expected, with all the
controls and assay parameters being reproducible from one assay to the next. Once the system suitability criteria have been
passed, it is safe to go straight ahead and run both samples
and controls, in the confidence that all is working as it should.
■ By Dr. Michael Sadick, Senior Manager, Large Molecular Analysis and Characterization,
Catalent Pharma Solutions
The need to track key indicators when performing a potency assay to determine
whether the assay is accurate, or indicative of a biological component going bad.
Monitoring Potency Assays