3.4. Synthetic mRNA produced in E. coli can be
purified at large-scale and is functional in human cells
To exemplify the utility of our E. coli -based system for
large-scale mRNA synthesis we manufactured GFP-mRNA in 1 L production
processes. The first purification step required is total RNA extraction
from host cell factories. While this can be achieved with commercial
kits when production scales are < 1 ml, larger volumes require
a scalable cost-efficient procedure. To achieve this, we adapted the
RNASwift method previously described by Nwokeoji et. al for
extraction of dsRNA products from E. coli that utilises NaCL and
SDS to lyse cells and precipitate macromolecular contaminants (Nwokeoji
et al., 2016). To maximise both total RNA yield and RNA quality, we i)
introduced a lysozyme digestion step upstream of RNASwift, ii) lowered
the lysis incubation temperature from 90ºC to 65ºC, and iii) added an
ethanol precipitation step downstream of RNASwift. Using this modified
RNASwift unit operation we were able to routinely obtain large yields
(10 mg per 0.5 g wet cell mass) of high-quality total RNA (RNA Integrity
Numbers > 9.5, as determined by capillary gel
electrophoresis).
While small amounts of product-mRNA can be purified from total RNA using
oligo-dT magnetic beads (see Fig 3C), larger quantities require
chromatographic operations. To show that mRNA manufactured in E.
coli can be purified using a liquid chromatography separation step, we
utilised a 1 ml monolithic oligo-dT(18) column in combination with an
AKTA PCC system. Figure 5B shows a chromatogram representative of this
purification process, indicating conductivity as a measure of salt
concentration, and the UV trace of material eluted from the column.
Capillary gel electrophoresis analysis of pre- and post-purification
samples showed that both SelfCirc-GFP and TermtRNA-GFP molecules could
be efficiently purified by an affinity-capture chromatographic unit
operation (Fig. 5C-D). However, TermtRNA-GFP was isolated at much high
purity, 71% as compared to 38% for SelfCirc-GFP, where SelfCirc-GFP
samples showed a considerable wide peak of impurities representing
~30% of total RNA. This may be due to SelfCirc-GFP
molecules having considerably smaller polyA tails than TermtRNA-GFP
species, 50 nt and 120 nt respectively, preventing use of elution
conditions that deliver both high yield and high purity. Further
mRNA/DNA engineering to increase the encoded polyA tail length should
permit product isolation with reduced process/product related
impurities. Either way, for both molecule-formats, it is clear that for
most applications a second chromatographic unit operation would be
needed to achieve requisite purity profiles, such as a size-exclusion
chromatography step. The use of two chromatographic unit operations is
standard for purification of other high-value macromolecules, including
recombinant proteins and IVT-derived mRNA (Fan et al., 2023; Rosa et
al., 2021; Sripada et al., 2022). A simplified process flow diagram for
large-scale and small-scale in vivo mRNA production processes is
shown in Figure 5A.
Finally, to validate that mRNA products manufactured in E. coliwere functional in mammalian cells, we transfected purified SelfCirc-GFP
and TermtRNA-GFP into Human embryonic kidney cells (HEK). While
SelfCirc-GFP contains an internal ribosome binding site (IRES),
obviating the need for post-purification processing, TermtRNA-GFP
required the enzymatic addition of a Cap-0 structure to enable
translation initiation. As shown in Figure 5E-F, both synthetic mRNA
molecular formats were translatable in HEK cells, facilitating similar
levels of GFP protein expression. Translational efficiency of
SelfCirc-GFP molecules would likely be further enhanced via
determination and selection of optimal IRES elements (Wesselhoeft et
al., 2018). Indeed, this may provide a route to encode cell-type
specificity into mRNA gene therapeutics (Plank et al., 2013).