APR May/June 2022 - 38

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DRUG DELIVERY
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the uridine analogue, 1-methyl uridine.3
This is easily achieved in
the process of making mRNA from a DNA template by including
the modified nucleotide rather than uridine. The architecture of
RNA sequences to achieve effective expression has been the major
development challenge for this type of product. Additionally, the
formulation methods, lipid nanoparticles (LNPs) necessary to achieve
in vivo delivery and expression mean that mRNA and its delivery
approach are difficult to separate.
Summary of Different Systems
The required components of a successful mRNA therapeutic are shown
in Table 1 and described diagrammatically in Figure 1.
Table 1. mRNA/saRNA components and function.
Structure Approach
5' cap
Co-capping with cap analogues or post
transcription enzymic capping. Authentic cap-1
structure essential to avoid activation of the innate
immune system and clearance of the mRNA.
5' UTR
Serves two key functions: to stabilize mRNA and
to facilitate scanning by small ribosome subunit to
localize the start codon.
Replicase
non-structural
proteins
Transgene
3' UTR
Self-amplifying mRNA has two open reading frames.
The first frame, like conventional mRNA, codes for
the antigen of interest. The second frame codes for
an RNA-dependent RNA polymerase (and its helper
proteins) which replicates the mRNA construct in
the cell.
Sequence encoding for the protein to be made from
the mRNA.
Role in gene expression by influencing the
localization, stability, export, and translation
efficiency of an mRNA.
Poly A tail
Contains binding sites for poly(A) binding proteins
(PABPs), affecting the export, stability, decay,
and translation of an mRNA. PABPs bound to the
poly(A) tail may also interact with proteins, such
as translation initiation factors, that are bound to
the 5' cap of the mRNA. This interaction causes
circularization of the transcript, which subsequently
promotes translation initiation.
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mRNA saRNA
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Manufacturing Considerations
for RNA
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The way that RNA is produced, through an enzyme catalyzed reaction
from a DNA template compared to molecules like antibodies expressed
by cell culture processes, are that the impurities present at the end
of an in-vitro transcription (IVT) reaction are all defined and present
at known amounts. This facilitates the rational design of purification
process for RNA post IVT, with Figure 2 demonstrating an example
workflow for RNA synthesis and purification. DNA plasmid template
is linearized, purified and used as template for IVT following which
the mRNA is purified. RNAs are large molecules being 1-5kb (ca 2MD)
for mRNA and at least 12kb (ca 6MD) for saRNA. As such they are in
the same size range as the smaller viral vectors. Hence manufacturing
needs to be designed for particles rather than molecules in solution.
An advantage of the large size of RNA molecules means that use
can be made of the size difference between RNA and IVT reaction
components (unreacted nucleotides, transcription enzymes, digested
DNA template components) so that significant purification can
be achieved by cross flow filtration. Subsequent chromatographic
purification is then required for polishing of impurities and product
related impurities (dsRNA specifically).
Some specific manufacturing considerations for mRNA include the
extreme sensitivity of the molecule to adventitious RNase activity
which needs to be controlled in manufacture. Failure to do this
results in rapid loss of RNA in processing and batch failures. Extreme
precautions are required to ensure that RNase activity is eliminated
through effective clean-in-place regimes, inclusion of RNase inhibitors
in the process and use of high-grade reagents which need to be
confirmed to be RNase activity free.
Another manufacturing consideration is the specialized nature of the
supply chain for RNA where many key reagents (including capping
reagents, IVT enzymes, encapsulation lipids) are only available from
single suppliers which means careful management of raw material
inventories to avoid manufacturing timelines being governed by
material supply.
Figure 1. A - mRNA architecture schematic.
B - saRNA architecture schematic.
Figure 2. Schematic of the RNA manufacturing process.
Post TFF2, RNA enters formulation.
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| May/June 2022
APR May/June 2022

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