Increasing yield of in vitro transcription reaction with at‐line high pressure liquid chromatography monitoring

Abstract

The COVID‐19 pandemic triggered an unprecedented rate of development of messenger ribonucleic acid (mRNA) vaccines, which are produced by in vitro transcription reactions. The latter has been the focus of intense development to increase productivity and decrease cost. Optimization of in vitro transcription (IVT) depends on understanding the impact of individual reagents on the kinetics of mRNA production and the consumption of building blocks, which is hampered by slow, low‐throughput, end‐point analytics. We implemented a workflow based on rapid at‐line high pressure liquid chromatography (HPLC) monitoring of consumption of nucleoside triphosphates (NTPs) with concomitant production of mRNA, with a sub‐3 min read‐out, allowing for adjustment of IVT reaction parameters with minimal time lag. IVT was converted to fed‐batch resulting in doubling the reaction yield compared to batch IVT protocol, reaching 10 mg/ml for multiple constructs. When coupled with exonuclease digestion, HPLC analytics for quantification of mRNA was extended to monitoring capping efficiency of produced mRNA. When HPLC monitoring was applied to production of an anti‐reverse cap analog (ARCA)‐capped mRNA construct, which requires an approximate 4:1 ARCA:guanidine triphosphate ratio, the optimized fed‐batch approach achieved productivity of 9 mg/ml with 79% capping. The study provides a methodological platform for optimization of factors influencing IVT reactions, converting the reaction from batch to fed‐batch mode, determining reaction kinetics, which are critical for optimization of continuous addition of reagents, thus in principle enabling continuous manufacturing of mRNA.

Scalable multimodal weak anion exchange chromatographic purification for stable mRNA drug substance

“…In a typical chromatographic experiment, a freshly prepared IVT reaction mixture (batch IVT reactions were performed as described elsewhere [15,16]) was diluted at least 10-fold with selected mobile phase A (MPA) to provide pH buffering and binding conditions. Chromatographic purification was performed on ÄKTA Purifier fast protein liquid chromatography system with two pumps and a multiwavelength UV-Vis detector (2 mm flow cell path length).…”

“…Yeast factories have recently been identified as an interesting alternative to in vitro transcription (IVT) reaction with potential to significantly decrease the cost of mRNA production [12], yet development efforts are still in its infancy. Productivity of IVT reaction, the unit operation with highest cost-of-goods (CoGs) profile in the process, has increased from 5 to 12 g/L in batch [13,14] or fed-batch modes [15,16]. Innovations in purification of mRNA DS have been significant, though veiled in a complex web of intellectual property, driving further need for freely available approaches to isolate highly pure mRNA DS with high yield.…”

“…Although initial reports of CIM PrimaS demonstrated very promising baseline separation of mRNA from pDNA, which enabled robust analytical methods for monitoring mRNA production [15,16,26], it had not been evaluated for fractionation of crude IVT mixture to isolate mRNA at preparative scale and no mitigation for potential hydrolysis of mRNA due to high elution pH has been proposed. We thus set out to optimize mRNA isolation from IVT mixture with CIM PrimaS with a threefold aim: retain high selectivity for mRNA, while simplifying the buffer system and mitigate the potential hydrolytic effect of high elution pH in order to develop a robust and scalable purification for mRNA DS with high binding capacity.…”

Effect of plasmid DNA isoforms on preparative anion exchange chromatography

“…Prior to analysis, the samples were diluted with double deionised water to achieve the loading of 55 ng of nucleic acids per well. AGE was conducted with 1% agarose gels at 100 V for 60 min or 100 V for 120 min and stained with Sybr Gold from Thermo Fisher Scientific (Waltham, MA, USA) [27].…”

Analytical separation of plasmid DNA isoforms using anion exchanging chromatographic monoliths with 6 µm channels

“…Purity and/or isoform composition requirements vary depending on the application of the pDNA. High purity of SC pDNA isoform is required in gene therapy platforms, whereas isoform composition is usually not defined for the preparation of IVT mRNA production, where linearization step follows the pDNA purification [5][6][7]. In any case, pDNA must be purified from all other contaminants present in the E. coli lysate, such as cell debris, RNA, residual proteins and genomic DNA.…”