eBook: Analytical Method and QC Testing Strategies for Biologics Production - 12

(2) High specificity, sensitivity, and efficiency
researchers to develop their own method to purify the
standard DNA.
According to Brewer, resDNASEQ qPCR primer/probe
sets were developed with the help of advanced
informatics to design an assay that is both specific to the
species and highly sensitive. " The use of sophisticated
informatics allowed us to select a high-copy, repetitive
element dispersed across the genomes of the species
that we're targeting, so the assay is designed to be
highly specific and as sensitive as possible, " he remarks.
The sensitivity of resDNASEQ is reflected in its limit of
quantitation (LOQ): 1.5 pg DNA/mL test sample for
most mammalian species and 15 pg DNA/mL test
sample for E. coli.
(3) AccuSEQ software for data analysis
The inclusion of purpose-built software by Applied
Biosystems marks another differentiator of the
resDNASEQ system. Instead of manually carrying out
calculations, researchers can rely on the software
to perform the calculations required for accurately
reporting test results, including the mean quantity
of DNA in the sample tested, the standard deviation,
and the percent coefficient of variation for the sample
replicates typically tested with a quantitative method.
Also, the AccuSEQ software enables compliance with
the guidance provided in the US Food and Drug
Administration's (FDA) 21 CFR Part 11 - Electronic
Records. These regulations are set around data security
and data integrity related to electronic documentation
and electronic signatures.
(4) Highly-characterized DNA reference standards
Quantitation of the DNA present in the test samples
are achieved by comparing the amount of DNA in the
unknown sample to the standard curve generated
from qPCR analysis of known amounts of host-cell
standard DNA. But some commercially available qPCR
assays do not include DNA reference standards, leaving
The resDNASEQ assay kit comes with a highlycharacterized
DNA reference standard. Brewer notes
that this component is particularly important because
the regulatory expectation for host cell DNA testing
is to use a quantitative assay, not a detection assay
(a positive-negative readout).
" Detection assays
are validated differently than a quantitative test for
impurities, " he clarifies.
Establishing acceptance criteria
Once the analytical method is developed, to establish
acceptance criteria, a round of pre-validation tests
should be conducted. This type of qualification involves
running multiple samples through experiments that
would be typical of the actual validation.
" The point of the pre-validation is to get a feeling for the
performance of the method - you can look at various
metrics like percent recovery, coefficient of variation,
etc. to figure out what you might assign for the assay
acceptance criteria, " says Brewer. " Analyzing the results
from the pre-validation, perhaps with the help of a
statistician, will help you confidently set appropriate
acceptance criteria for the actual validation. "
The criteria can be set in an acceptable range to allow
for variation, but they must comply with regulatory
guidance. For example, USP Chapter 509 states that
percent recovery should be between 50-150% and
percent coefficient of variation (%CV) should be 30%
or less.
Method Validation Case Study
Method validation is the process used to confirm that
the analytical procedure employed for a specific test
is suitable for its intended use. As mentioned above,
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