eBook: Analytical Method and QC Testing Strategies for Biologics Production - 29

(2) Sensitivity: Again, a sample at LOD was compared
to the NTC. 100% of the samples showed a higher Ct
value for the NTC than the LOD (30 fg) sample. This
clean separation between the LOD sample and the
NTC provides confidence that a sample as low as 30 fg
can be detected accurately. (Figure 7)
(3) Specificity: The specificity experiment was also very
similar to the one on the 7500 Fast, where a variety of
cross-reactive DNA types were spiked into a sample
at 3 ng. The data show that for every cross-reactant
(e.g. CHO DNA, HEK293 DNA, E. coli DNA) the signal
was much higher than the 30 fg sample. Thus, both
the Sf9 and Baculovirus assays are highly specific in
the presence of genomic DNA from other sources.
(Figure 7)
(4) Singleplex vs multiplex performance: When the assay
is run with a singleplex Baculovirus standard versus a
duplex Baculovirus and Sf9 combination standard, the
standard curves overlap and no significant difference
was observed between the two. This is also the case for
singleplex Sf9 and the combination standard. As such,
the duplex assay is reliable, even when measuring only
one of the analytes. (Figure 8)
(5) Spike recovery: For spike recovery, Sf9 and Baculovirus
standards were spiked into PBS (sample control) and a
Figure 6. Standard curve performance across multiple sites, days, and operators on the QuantStudio 5 instrument. No significant difference
was found across all points on the standard curve. High precision, accuracy, linearity, and efficiency were achieved to enable quantitative
results across a broad range of DNA concentrations. (NTC = no-template control, IPC = internal positive control)
29

eBook: Analytical Method and QC Testing Strategies for Biologics Production

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