Scalable mRNA Machine for Regulatory Approval of Variable Scale between 1000 Clinical Doses to 10 Million Manufacturing Scale Doses

Abstract

The production of messenger ribonucleic acid (mRNA) and other biologics is performed primarily in batch mode. This results in larger equipment, cleaning/sterilization volumes, and dead times compared to any continuous approach. Consequently, production throughput is lower and capital costs are relatively high. Switching to continuous production thus reduces the production footprint and also lowers the cost of goods (COG). During process development, from the provision of clinical trial samples to the production plant, different plant sizes are usually required, operating at different operating parameters. To speed up this step, it would be optimal if only one plant with the same equipment and piping could be used for all sizes. In this study, an efficient solution to this old challenge in biologics manufacturing is demonstrated, namely the qualification and validation of a plant setup for clinical trial doses of about 1000 doses and a production scale-up of about 10 million doses. Using the current example of the Comirnaty BNT162b2 mRNA vaccine, the cost-intensive in vitro transcription was first optimized in batch so that a yield of 12 g/L mRNA was achieved, and then successfully transferred to continuous production in the segmented plug flow reactor with subsequent purification using ultra- and diafiltration, which enables the recycling of costly reactants. To realize automated process control as well as real-time product release, the use of appropriate process analytical technology is essential. This will also be used to efficiently capture the product slug so that no product loss occurs and contamination from the fill-up phase is <1%. Further work will focus on real-time release testing during a continuous operating campaign under autonomous operational control. Such efforts will enable direct industrialization in collaboration with appropriate industry partners, their regulatory affairs, and quality assurance. A production scale-operation could be directly supported and managed by data-driven decisions.

Digital Twin Fundamentals of mRNA In Vitro Transcription in Variable Scale Toward Autonomous Operation

“…The reaction conditions have already been optimized for the batchwise production of mRNA. In addition, it has been shown that the continuous and scalable production of mRNA is feasible by generating a segmented flow and that comparable yields can be achieved as in the batchwise mode …”

“…The continuous production was performed by generating a segmented flow in a plug flow reactor with a 1/16 in. diameter and a length of approximately 18 m. The feasibility of the slug generation was already demonstrated . The reaction mixture formed the slug phase, and oleic acid was used as the slug generating phase.…”

“…Since supplementation of urea has already been shown in the literature to reduce dsRNA during in vitro transcription, urea was supplemented during the continuous in vitro transcription . However, this resulted in a decrease in yield and a significant increase in truncated mRNA in continuous production.…”

“…In many studies, nucleotide concentrations, magnesium concentrations, and their interactions with each other have already been reported to be the most significant factors in IVT. ,− Some of these will give specific concentrations of magnesium that vary over a wide range from 12 to 60 mM. ,,− Others also indicate optimal ratios of NTP to magnesium ions . Furthermore, the counterion to magnesium ions plays a role, as magnesium acetate can be used at higher concentrations, unlike magnesium chloride. , …”

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

“…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.…”