eBook: Analytical Method and QC Testing Strategies for Biologics Production - 25

In the PrepSEQ sample prep kit, is a lysis buffer to
disrupt the sample and separate out nucleic acids from
protein complexes. It also contains magnetic beads
to bind and extract the nucleic acids. During sample
preparation, the beads (with bound DNA/RNA) are
collected, supernatants removed, and a series of wash
steps carried out to remove any inhibitors. Ultimately,
the DNA or RNA can be eluted off of the beads into
a PCR-compatible nucleic acid solution. This workflow
can be managed manually or via an automated
instrument called the KingFisher Flex, which increases
throughput to 96 samples at a time. The PrepSEQ
kit has been tested with success on many different
sample types (residual host cell DNA, plasmid DNA,
mycoplasma, and a variety of viruses) and challenging
matrices (low pH, high salt, high protein, etc.)
" Because it's so easy to set up and quick to use, it
allows users to test a wide variety of samples in a more
consistent way with less chance of contamination and
fewer chances of errors, " says Norman.
Once the nucleic acid is extracted, it enters the PCR
instrument which uses a TaqMan assay to measure
the amount of residual DNA with high sensitivity,
specificity, reproducibility, and amplification
efficiency (within 10% of 100%). The AccuSEQ
software records all data, and any changes to the
data, to ensure full traceability and compliance in the
GMP environment according to the FDA's 21 CFR Part
11 - Electronic Records.
(2) Highly sensitive duplex assay
The duplex nature of the assay was designed to
further streamline residual DNA testing, allowing drug
developers to cover two steps in the testing menu by
using one assay. The performance of the duplex assay
is no different to singleplex assays, as shown in the
case studies that follow. In fact, the duplex assay is
able to detect both Sf9 and baculovirus residual DNA
with very high sensitivity (limit of quantitation is 0.3
pg for both).
(3) Robustness and reliability
As the validation studies below demonstrate,
the resDNASEQ duplex assay is extremely robust,
performing consistently well across many different
scenarios, including multiple instruments, operators,
and sites with different levels of experience.
Validation of the resDNASEQ duplex
assay on two instrument systems
The validation study was done on two instruments:
1) Applied BiosystemsTM
Instrument, and 2) Applied BiosystemsTM
5 Real-Time PCR Instrument. Data was collected by
eight operators using two sample prep methods,
across three continents and four days.
Case study on 7500 Fast:
(1) Precision, efficiency, and linearity: The standard curve
performance was assessed by running 18 plates across
3 continents (locations in US, Singapore, and the UK).
No significant difference was found across all points on
the standard curve. Results demonstrate high overall
precision down to the limit of quantitation (LOQ) of
0.3 pg. The PCR efficiency was found to be very close
to 100% (within ± 10%), with high accuracy (R2
> 0.99)
and linearity (slope: -3.1 to -3.6) across the entire range
of DNA concentrations tested.
Furthermore, three different measures of precision
were evaluated: run-to-run, instrument-to-instrument,
and operator-to-operator. Results show high precision
across all three measures for the entire standard
curve. As such, the data are consistent across multiple
runs, instruments, operators, and sites, even when
quantitating low levels of DNA. (Figure 2)
7500 Fast Real-Time PCR
QuantStudioTM
25
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