GEN-Thermo_Oct22_MakingThePromiseOf - 16

Scaling Up IVT for mRNA Production
high-temperature inactivation step that is part of the
R&D stage is easy to do in a microtube, but single-use
plastics used in medium-scale production cannot
tolerate high temperatures, and this step cannot be
transferred to large-scale production. It is therefore
crucial to consider the full process and include necessary
filtration and purification steps in your workflow
early. Ensuring a consistent supply of high-quality
raw materials will help you optimize concentration,
quality, yield, and cost, and enable your output to be
more representative as you increase mRNA production
scale.
3. Optimize temperature and reaction time
Increasing IVT reaction time (Figure 2) and optimizing
temperature can help to maximize yield and product
quality, and there is much interest in testing different
IVT temperatures. Temperatures both lower and
higher than 37°C have been shown to have an impact
on improving mRNA quality. This will need to be optimized
for the mRNA in question. For example, when
working with longer transcripts such as self-amplifying
RNAs, a lower temperature is more favorable.
Lower temperatures also mean longer reaction times,
which can result in unwanted by-products, such as
double-stranded RNA (dsRNA). These will necessitate
an additional polishing step to be incorporated into
your workflow.
Whatever the optimal temperature for your reaction,
it is important to maintain a constant temperature
throughout the IVT reaction volume. For example, if
your process uses a temperature higher than 40-45°C,
you'll need to use an appropriate bioreactor, such as
the Thermo Scientific™ HyPerforma™ single-use series,
that allows you to control both temperature and
mixing, to avoid generating truncated transcripts.
4. Strike a balance between reaction
concentration and yield
Effect of reaction time on RNA yield
200
180
160
140
120
100
80
60
40
20
30
60
Reaction time, min
Figure 2. Yield of RNA of varying lengths at different reaction
times.
16 | GENengnews.com
90
120
The concentrations of enzymes and NTPs should
be optimized for improved yield, but optimization
is not linear and can be process- and product-dependent.
You may even see yields decrease as you
increase scale. Starting with single components
rather than premixed solutions will allow you to
test a range of conditions to optimize yield. Buffer
composition can also affect yield, so don't forget
to factor in buffer formulation, especially Mg2+
Na+ concentrations, in your optimization. Yield can
and
100 b
500 b
1 kb
2 kb
be improved by increasing the concentration of
RNA polymerase, but this will come at increased
cost as reaction volumes increase. It's also worth
noting that RNA polymerases from different
suppliers will require additional optimization,
which may also increase costs. Some single-use
bioreactor materials may also have limitations in
temperature compatibility.
RNA yield, µg
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