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Key Insights Into Overcoming mRNA Process Challenges
which seem very promising for reducing process
timelines and improving product quality, " he
explains. " There has been a lot of pressure on
plasmid manufacture, particularly to GMP, to
support gene therapy and mRNA applications.
Hence with these supply chain issues
alternative approaches such as rolling circle
amplification may see their use accelerated. "
In Vitro Transcription
After pDNA is manufactured in an E.colibased
fermentation process, it is harvested,
linearized, purified, and used as templates
for the enzymatic in vitro transcription (IVT)
process, yielding the desired mRNA molecule.
IVT is currently the cost-driving step in mRNA
process development. " IVT is what generates
the product, but it is a highly complex step. It
requires the careful addition of a number of
diverse components in addition to the DNA
template, such as enzymes and nucleotides to
synthesize the mRNA, " explains Dr. Liddell. " You
also need a capping reagent that will be added
to the five-prime end of the mRNA. Design of
Experiments approaches are typically used for
process optimization of these different components
to optimize yield and quality. Currently,
reactions are batch based, but alternate reactor
designs could be devised to reduce inventory
of expensive raw materials, eventually even
moving to a continuous reaction scheme.
This may be harder to develop, but it could
make a big difference in overall productivity. "
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Dr. Zhu says the need for additional optimization
has led to issues and costs in GeneLeap
Biotech's work with mRNA. " We have found
that each vendor's T7 RNA polymerase has a
different 'flavor,' which means we will optimize
one IVT system using the T7 from one vendor
but then have to optimize again if we switch
to a different one. That is why sustainable
supply from a reliable vendor is critical for us. "
Equipment fit for the unique characteristics
of mRNA is also essential. " For the upstream,
we need equipment that is designed for
cell-free expression platforms, " adds Dr. Zhu.
" We can use some of the functionality on
the bioreactors available now, but they are
missing some important functions we need,
like certain inline monitoring, which will help
with scale-up by monitoring the different
parameters that vary with mRNA models. "
Compounding raw material variability challenges
is the impurity profile of mRNA molecules that
can vary with each project, requiring different
purification steps from case to case. " mRNA
is a very large molecule-30 to 50 nanometers-which
is way beyond the size of proteins
and comparable to viral vectors. That means
they do not interact well with conventional
chromatography resins, where you will likely get
only surface adsorption, " explains Dr. Liddell.
The varying impurity profiles of mRNA from
the IVT step calls on options in purification


Table of Contents for the Digital Edition of Cytiva_Jan2022_mRNAVaccinesANewEra

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