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30 Pharmaceutical Technology
®
The Real Message Behind Commercial mRNA Products April eBook 2023 PharmTech.com
Drug
Delivery
of degradation or process impurities. dsRNA is an
unwanted by-product of IVT because it activates the
innate immune system.
T he reduc t ion of dsR NA usi ng opt i m ized I V T
process yields material that is lower in dsRNA than
when standard IVT RNA is purified using cellulose
purification (one main way of removing dsRNA con-
taminants) (5). Furthermore, when RNA produced
using optimized IV T is injected in vivo, no signifi-
cant increase in interferon alpha is detected, indi-
cating that this material does not activate the innate
immune system (5). Capping efficiency is also high at
>95%, see Figure 5.
Optimized IVT processes
significantly improve mRNA quality
Because optimized IVT processes generate RNA that is
significantly low in dsRNA, cumbersome downstream
processes, such as reverse-phase high-performance liq-
uid chromatography (RP–HPLC), are not required. In-
stead, the optimized downstream process can strongly
focus on the removal of process-related impurities such
as proteins, DNA, and buffer components. This allows
one to achieve extremely low levels of residuals (near
complete removal of DNA and proteins) with high
Novel UTR combinations elicit altered expression levels
Ratio
(baseline:
ctr3)
2
1
0
1 2 3 4
5 6
7
8 9 10
11 12
13
14
15 16
17
18 19
20 21
22
23
24
25 26
27 28
29 30 31 32 33
34 35 36
37
38 39 40
Control
1
Control
2
Control
3
Control
4
FIGURE 4. Selection of UnTranslated Region (UTR) combinations impacts
expression. The graph represents an in vitro luminescence screen employing the
top 40 UTR combinations following transfection in immature dendritic cells.
RNA yield
3000
2000
1000
0
[RNA]
(ng/µl)
IVT1 IVT2 IVT3
5
3
2
0
dsRNA
content
(µg
dsRNA/mg
RNA)
4
1
IVT1 IVT2 IVT3
dsRNA content
TLR4
huCD40L
huCD70
A B
IVT1 IVT2 IVT3
Capping
efficiency
(%)
capping efficiency
pp1
pp2
pp3
pp4
Ratio NTP: CleanCap
100
90
85
95
FIGURE 5. Optimized IVT processes significantly improve mRNA quality.
TABLE I. Illustrates some of the key
critical quality attributes (CQAs) and
typical values associated with mRNA
production, which are lower than
the limit of quantification (LOQ).
CQA Typical value
Bioburden < 1 CFU/5ml
1
Endotoxin < 1 EC/ml
1
Residual pDNA < 25 pg/mg
Residual protein < 0.4 µg/mg
1
* Typical value is