eBook: Analytical Method and QC Testing Strategies for Biologics Production - 14

Results: R2 of >0.98 was achieved for all four standard
curves.
Range Assessment
The range of an analytical procedure is the interval
between the upper and lower concentrations of
analyte in the sample (including these concentrations)
for which it has been demonstrated that the analytical
procedure has a suitable level of precision, accuracy,
and linearity.
Why this is necessary: To verify the range of the assay
across the DNA concentrations analyzed to generate
the standard curve.
Study design: Four independent curves, prepared from
a 10-fold dilution series of CHO standard DNA with a
concentration range of 3 ng to 30 fg, were analyzed.
Acceptance criteria: Assay quantitative range should be
consistent in a minimum of four independent standard
curves.
Results: The assay quantitative range was consistent
in the four independent standard curves and it was
determined to be 0.03-3000 pg for CHO DNA.
Accuracy Assessment
The accuracy of an analytical procedure expresses the
closeness of agreement between the value which is
accepted, either as a conventional true value or an
accepted reference standard value, and the value found.
Why this is necessary: It determines how close the
test results are for a specific analyte compared to the
true measure, which is calculated by the amount of
standard DNA spiked into test samples.
Study design: Three samples were tested at four
different DNA spike levels: 100, 10, 1, and 0.1 pg.
Acceptance criteria: Accuracy is acceptable when the
quantity of spike DNA levels are found within a range
of ± 2 to 4-fold of the comparative standard value.
Alternatively, it could be 50-150% of the comparative
standard value with a %CV less than 25%.
Results: The assay was able to quantitate 100 pg, 10 pg,
1 pg, and 0.1 pg of DNA from the sample test matrix
within the acceptance range of ± 2 to 4-fold of the
comparative standard value.
Additionally, an internal validation study of the
KingFisher Flex automated sample preparation system
using the CHO, E. coli, and Vero assays, performed
with two different instruments, demonstrated highly
efficient recovery with accurate and precise quantitation
(very low %CV) of DNA recovered from multiple test
sample matrices. " This shows the robustness of both
the resDNASEQ assays and the PrepSEQ sample prep
process using the KingFisher Flex automated magnetic
bead handler, " remarks Brewer.
Quantitation Limit Assessment
The quantitation limit of an individual analytical
procedure is the lowest amount of analyte in a
sample which can be quantitatively determined
with suitable precision and accuracy. Quantitation
limit is a parameter of quantitative tests for low
level compounds and sample matrices and it is used
particularly for the determination of impurities and/or
degradation products.
Why this is necessary: To verify accurate quantitation at
the Limit of Quantitation (LOQ) of the assay.
Study design: Six samples of the surrogate bulk drug
substance, each spiked with 30 fg of DNA, were tested.
(Note: The overall method LOQ with spiking at this
level is 1.5 pg DNA/mL test sample) The reason for
using the bulk drug substance surrogate matrix for
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