ChasRiver_Aug2022_Perspectives-Toward-a-Treatment - 8

PERSPECTIVES
Going directly into the brain mitigates some of the
toxicity concerns as well, which is a real benefit. Many
studies have shown that with systemic administration higher
doses can elicit an immune response to the virus, which
can be quite serious. Going directly into brain tissue greatly
reduces this risk.
Also, much smaller amounts of virus are needed when
you go directly into brain tissue, at least an order of
magnitude difference between what would be needed to go
systemically. So, in addition to the safety benefits, there are
manufacturing and scaling benefits to direct brain injection.
BEN MOORE:
We have done a significant number of surgical approaches
or administrations to optimize delivery. We have looked
through several specific structures in mice, and now in
nonhuman primates, to try to understand the most effective
approach and locus of delivery.
Anjali Sarkar: Did you face any challenges in
scaling the AAV manufacturing process? Did you
need to change the construct or the production
process to overcome these challenges?
RENO FRANCHETTI:
Being a CDMO, and having manufacturing experience,
you know there are challenges with any construct. In this
case, we dealt with a challenge in the downstream processing,
where we wanted to ensure we had a good full-capsid
ratio. A constant discussion industrywide revolves around
methods to increase full-capsid ratio to make sure you have
pure material. The original plan was to go with two-column
purification because typically that is much more scalable, and
we want to be able to scale this process when going to the full
GMP (good manufacturing practices) or clinical route.
But at this stage and under the current contract to deliver
this material, we did change our process to utilize one column
followed by iodixanol gradient ultracentrifugation. We tested
this at the small scale and saw favorable results using our
8 | GENengnews.com
internal DLS (Dynamic Light Scattering) Stunner, which gives
you quick results for both titer and full-capsid ratio. That
was the chosen path forward. We scaled up to 10 L and 50 L,
which we then analyzed using analytical ultracentrifugation.
We ended up seeing 84% or higher full-capsid ratios.
We do see the possibility of having to scale this up to at
least 200 L in the future. That is something we have done
with ultracentrifugation but given more time and process
development capabilities in the near future, we hope to still
include that double column process, so that it's scalable for
Paros Bio when taking this to a much larger scale.
Anjali Sarkar: With the complex structure of
the inverted terminal repeat (ITR)-based plasmid
for gene delivery, were there any scaling
challenges that had to be overcome in the
manufacturing process and, what role did
the CDMO play in this effort?
BEN MOORE:
One key problem concerned the stability of our plasmid
when we were producing it at larger quantities. Our plasmid
is flanked by ITRs, which are regions of DNA that tend to be
hard to replicate with high accuracy. Working with Charles
River to manufacture our plasmid, we found out that our
plasmid carrying our gene of interest PSEN1 had alterations
in the ITR region.
There are two major consequences to having changes in
ITRs. First, it can lower production efficiency. The second is
uncertainty. We have a good understanding of how normal
ITRs behave and how they lead to durable expression of
the gene. It's important for us to stand on the mountain of
research that has previously been done, and to use those
identical sequences. So, it was helpful to be able to ensure
that the ITR sequences remained intact in the plasmid
production process.
We were happy that Reno's excellent scientific team at
Charles River was not only able to catch this instability
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ChasRiver_Aug2022_Perspectives-Toward-a-Treatment

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