GEN-Thermo_Oct22_MakingThePromiseOf - 20

Scaling Up IVT for mRNA Production
Question 2: What is most important when scaling an IVT reaction?
" Maintaining the IVT reaction temperature constant
through the liquid solution with sufficient mixing
and with an abundance of raw materials, to avoid
truncated transcripts. " -Pirkko
" Selecting appropriate GMP-grade raw material
and ensuring a steady supply from your vendors as
well as protecting your mRNA from degradation,
from either pH or temperature changes, or with
RNase inhibitors. " -Vaida
" Identifying the most suitable equipment for scale-up
and having a representative scale-down model. It's a
good idea to have a few intermediate scales to assess
the linearity of your scale-up. " -Sirat
" It is crucial to pre-warm the reaction components
before reaction setup (as using cold reagents may
decrease yield significantly). You should always
make sure you're doing reaction setup and incubation
in a DNase- and RNase-free environment to
prevent degradation and loss of RNA. " -Monika
Question 3: What advice would you have for anyone starting out?
" Using single raw material components
instead of premixed enzymes and nucleotides
will allow you to test a range of conditions.
Remember that the composition of buffers,
especially Mg2+
and Na+
, can impact the yield,
so the buffer formulation is important to know,
but try not to include too many variables in
the design of experiments (DOE). " -Pirkko
" There are a lot of variables that can be optimized
for IVT, such as temperature, pH, reaction
duration, batch or continuous production, co- vs.
post-transcriptional capping, the equipment
used, such as single-use, heating, or stirrer type,
as well as the amount of enzymes used. Always
consider your mRNA length and sequence,
perform risk assessment, and select the most
influential variables for your process optimization
strategy. " -Vaida
20 | GENengnews.com
" DOE is critical to help ensure you have a good
understanding of the impact of process prior to
scale-up, as changes are always easier to make at
small scale. Have at least 2-3 replicates for each
condition while designing a DOE, and consider
all process factors that impact yield and product
purity and quality, such as NTP concentration
and Mg2+
concentration. Then ensure all necessary
analytical methods have been developed
and are reliable to help you draw the right
conclusions from your DOE results. " -Sirat
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