Biomedical Engineering Reference
In-Depth Information
Electron microscopy (EM) suffers from issues of specificity. First, species identifi-
cation of a virus through EM is not reliable. Second, it is commonly not possible to
determine if a virus particle has been inactivated, as the visual difference between active
and inactive virus may not be always apparent.
Nucleic acid technology (NAT) assays include variations on polymerase chain
reaction (PCR) or reverse transcriptase-polymerase chain reaction (RT-PCR). The
benefits of these assays are their exceptional speed and low cost when compared to
cell culture-based assays. PCR techniques employ virus-specific probes to ensure signal
specificity, which allows study designs in which multiple viruses are spiked at one time
[6]. Quantitative PCR (Q-PCR) allows one to quantify virus clearance with sensitivity
approximately 100 times that of a conventional cell-based infectivity assay. Real-time
Q-PCR allows one to quantify clearance during the time course of the experiment, not
simply at the beginning and the end of the experiment. Still, PCR experimental design
must account for issues related to endogenous viral DNA in preinactivation samples
(background signal) and the possibility that a virus inactivation mechanism does not lead
to a change in PCR signal.
8.5 DEVELOPING A VIRUS CLEARANCE DESIGN SPACE
The technical issues outlined above must be acknowledged. Nevertheless, the industry
should not wait for their complete resolution. With the premise that a good design space
exists within every robust process, design spaces can be approximated with today's
technology and understanding of biology, chemistry, and engineering principles. One
may segment the construction of a design space into six key elements (Fig. 8.1), some of
which may be executed in parallel and some of which find a parallel to current practices
and regulatory guidance.
8.5.1 Define Product Virus Safety-Related CQAs
A manufacturing process has the overarching aim of reliably meeting product CQAs.
Thus, the process design space is predicated on the definition of the product design
space. It has been acknowledged that “the design space approach to process definition
is frequently hampered by inadequate information regarding product quality needs,
that is, the deliverables (product specification)” [7]. With this dynamic as a backdrop,
some biopharmaceutical manufacturers seek to document the maximum possible viral
clearance level from the process. However, regulatory guidance does not stipulate an
absolute log reduction value (LRV) requirement. To this point, Appendix 5 of the ICH
Q5A guideline is sometimes interpreted as a mandate to achieve a 6 log 10 safety
margin per dose for endogenous retroviral particles, when in fact this appendix is
intended to illustrate how to perform the calculation of estimated virus particles per
dose.
To design in an appropriate level of viral clearance, one must establish target LRVs
for the various models or relevant viruses. Risk assessment tools can aid in this
determination [8]. The more common questions to consider are the following:
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