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).