IVT Workflow Overview
Manufacturing of mRNA for therapeutic applications is accomplished through either co-transcriptional or post-transcriptional capping (sometimes called enzymatic capping). In co-transcriptional capping, wild-type T7 (WT T7) RNA polymerase can achieve ~95% capping efficiency when optimized with trinucleotide cap analogs, and less than 80% capping efficiency with dinucleotide cap analogs (such as ARCA). Given the loss in capping efficiency, manufacturers prefer post-transcriptional capping with Vaccinia Capping Enzyme (VCE) when dinucleotide caps are used, as it can achieve >95% capping, but this also introduces additional process and purification steps.
Codexis enzymes are designed to address these trade-offs manufacturers face to improve overall process efficiency and quality. The Codex® HiCap RNA Polymerase is the first enzyme engineered to improve co-transcriptional capping efficiency using either trinucleotide or dinucleotide cap analogs, while achieving >95% capping efficiency with both cap analog formats.
Codex® HiCap RNA Polymerase
Improve capping efficiency
Improved co-transcriptional capping efficiency with the engineered Codex® HiCap RNA polymerase produces higher yields of the desired, capped mRNA product. Whereas use of WT T7 RNA polymerase requires a delicate balance between the cap analog and GTP concentrations due to indiscriminate selection by the wild-type polymerase, forcing manufacturers to trade-off yield for improved capping efficiency or undertake challenging GTP feeding strategies throughout the reaction.
Maximize yields
Codex® HiCap RNA Polymerase was engineered for selectivity of cap analogs over GTP at transcription initiation, enabling manufacturers to run IVT reactions with higher concentrations of GTP, maximizing overall yields of the desired capped mRNA, even with lower cap analog concentrations. In contrast, indiscriminate selection of GTP over cap analogs by WT T7 RNA polymerase requires manufacturers to restrict GTP concentrations, limiting overall mRNA yield.
Reduce dsRNA impurities
Engineered Codex® HiCap RNA Polymerase generates 50x less dsRNA impurities compared to WT T7 RNA polymerase creating higher quality mRNA. Impurities formed during IVT include short, abortive transcripts, uncapped transcripts, and double-stranded RNA (dsRNA). Each may elicit immunogenic responses in vivo, requiring extensive downstream purification prior to formulation.