eBook: Analytical Method and QC Testing Strategies for Biologics Production - 11

How to design validation studies
with a comprehensive commercial
testing kit
When it comes to validating an assay developed
in-house, the process is very similar to validating a
commercially available assay, says Brewer, but generally,
the development and qualification process prior to
validation of an internally developed method is very time
and labor intensive. " There is a lot of experimentation
and documentation that goes into developing a method
in-house, including selection of critical reagents, assay
optimization, and development of SOPs, etc., " says
Brewer. " With a commercially available assay, you're
buying something that's essentially ready to use, so the
process is much more streamlined. "
The user guide that comes with a commercially
available assay helps researchers develop their own
SOPs and quickly qualify the performance of the
method for use with customer-specific samples. Such
guided qualification can significantly shorten the
timeline from initial evaluation to a validated analytical
method, adds Brewer.
resDNASEQ System for Residual DNA Testing
Thermo Fisher's resDNASEQ is a fully integrated realtime
qPCR-based system for measuring residual host
cell DNA. It combines a sample preparation method
that enables quantitative DNA recovery with high
precision from a variety of sample matrices, and
specific qPCR-based assays for the host cell line used in
the process being tested. This includes CHO (hamster),
Human, E. coli (bacterial), Vero (monkey), NS0 (mouse
tumor), Pichia pastoris (fungal), MDCK (canine kidney),
HEK293 (human embryonic kidney) and Sf9 (insect)/
Baculovirus host cell lines.
The following list highlights some of the key
differentiators of the resDNASEQ system:
(1) Sample preparation
resDNASEQ employs Applied Biosystems' PrepSEQ
- a system that uses magnetic bead-based sample
preparation chemistry. First the proteins in the sample,
product and host cell, are enzymatically digested, a
solution is added to drive association of the DNA with
the magnetic beads, and then a DNA binding step is
performed. Following binding, impurities or potential
qPCR inhibitors are removed via two wash steps, and
then the DNA is eluted with a buffer that's compatible
with qPCR.
" One of the unique things about this chemistry is that
you get quantitative or near-quantitative recovery of
the DNA from the test samples, " Brewer says, claiming
that minute quantities of DNA (think picogram or
femtogram levels) can be accurately recovered from
challenging sample matrices with high levels of protein
(10-100 mg/mL of protein), low pH, and significant
levels of salt and buffers typical of biopharmaceutical
purification processes.
While a manual protocol is available for lower
throughput applications, sample preparation can be
automated with the KingFisher Flex instruments that
allow high-throughput purification of DNA from 24-96
samples per run. Brewer notes that, although there are
other methods that give a similar level of performance,
most cannot be adapted to an automated platform.
Additionally, methods that rely on alcohol precipitation
to concentrate the DNA sometimes have issues with
qPCR
inhibitors co-precipitating and preventing
accurate quantitation of the DNA.
11
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