Thermo_Apr2021_The_mRNA_TherapeuticsBoom - 4

* ds-mRNA
* DNA
* Enzymes

* Enzymes - by pass
500
0

20
The
mRNA10Theraptutics
Boom 30Washing
Sample load Equilibrate/Flush

50
Elution

60
Base clean

70
Equilibrate

0
80

Elution Volume, CV

Fig. 4 Chromatogram showing efficient separation of a 2000nt mRNA from an IVT mixture at a
load concentration of 2 mg/mL. Elution was performed using H2O and yielded in >95% recovery.

Below
detection
limit

Fig. 2: High recovery and purity independent of sample type used. Recovery of mRNA from pure mRNA and unpurified IVT mixture,
Fig. 5noHigh
recovery
and purity
independent
ofinsample
used.
Recovery
of (right).
mRNANofrom
pure
showed
differences
(left). Amount
of protein
was determined
load, flowtype
through
(FT) and
elution pools
proteins
were
mRNA
and
unpurified
IVT
mixture,
showed
no
differences
(left).
Amount
of
protein
was
determined
in
detected in the elution pool, indicating excellent impurity removal of IVT mixture products.
load, flow through (FT) and elution pools (right). No proteins were detected in the elution pool, indicating
excellent impurity removal of IVT mixture products.

We do not control the design, and different designs
impact recovery even with samples straight from an
The
POROS
Oligo
(dT)25 affinity
can
trigger
complexity
in the purification
process. " resin
IVTdemonstrates:
mixture. Low nonspecific binding and the affinity
approach
only allow polyA species to bind. Recovery
The
Oligo (dT)25
resin, asat
a purification
platform,
 POROS
Efficient
elution
different
load concentrations
rates are greater than 90% and, in most cases, greater
can be cross applied to different constructs, empha Flook.
Excellent
recovery
withchallenges
high puritythan
independent
ofcolumn
sample
type used
95%. Adjusting the
size according
to
sizes
The resin addresses
the current
need makes the process flexible and scalable.
involved with large-scale mRNA purification for poten-

Absorbance, 260nm (mAU)

tial clinical use by simplifying the downstream
process,
Fromby
a purity
perspective, evaluation of the proteinImpurity
analysis
HPLC
increasing purity and yield, and allowing for scale-up
aceous load showed that primarily enzymes from
Improved impurity
without
the IVT mixture
clearanceare seen in the flow-through but not
600 the use of toxic chemicals.
Unpurified
IVT
mixture
detected within the elution peak. " If we analyze the
400
mRNA
Efficient
15.7%
fractions
from the elutionFig.
peak6starting
withremoval
about
200
of impurities compared
Validating
the POROS Oligo (dT)25 Resin
0
17% product-related impurities, a reverse-phase spin
to the spin column
150
Standard
mRNA
contains between 1000-5000 nucleocolumn slightly reduces itmethod.
to 13%, " says
Flook.
Current spin column purification method
HPLC analysis of
100
tides, which is the size the resin was designed to optimixture,
" All of13.4%
the process-relatedunpurified
componentsIVT
are removed
50
mally operate in. Optimized conditions can maximize
spin
column
purified
with the POROS Oligo (dT)25 resin, and we see
a
0
the binding capacity, even for larger RNAs, to achieve
mRNA
and
mRNA
purified
significant reduction in product-related impurities, "
300 efficient purification process.
with our
POROS
a more
Purified with POROS Oligo (dT)25 resin
6.2%Flook. " This means
continues
affinityOligo
resin (dT)25
does a
200

100 a binding perspective, we have seen customers
" From
0
achieving
up to 5mg/ml of 4000 bp mRNA, " says Flook.
0.0
2.0
4.0
6.0
8.0
10.0
" This is significantly higher than what you would see
Time (min)
with some of the research products. "

resin.

much better job of removing non polyA species than
7.0
8.0
9.0
reverse
phase.
" All remaining
product-related impurities are polyadenylated, as expected.

Overall, the POROS Oligo (dT)25 resin demonstrated
Astandardized
experiment
looked
at
the
binding
efficient
elution
at different
load concentrations and
Purification with POROS Oligo (dT)25
resin
leads
to significant
capacity of three different sizes of mRNA without optiexcellent recovery with high purity regardless of
reduction of impurities
mizing conditions for each independently. Size did not
sample type.
4CONCLUSIONS
| GENengnews.com

The POROS Oligo (dT)25 resin addresses the current challenges involved

https://www.thermofisher.com/us/en/home.html https://www.genengnews.com

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