Ask the Expert: High-Yield mRNA Processing — From Plasmid to Highly Purified Product

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Interest in industrial-scale production of messenger RNA (mRNA) has surged amid rapid development of mRNA-based vaccines against SARS-CoV-2. During an
18 February 2021 Ask the Expert presentation, Aleš Štrancar (chief executive officer of BIA Separations, a Sartorius company) reminded attendees that no platform approach yet exists for mRNA production and that much remains to be learned about manufacturing such products at commercial scales. He described current production challenges and shared BIA’s efforts to devise flexible mRNA purification tools.

Štrancar’s Presentation
Understanding Reagents: Štrancar emphasized the need for impurity control during mRNA manufacturing. The process usually entails isolation of plasmid DNA (pDNA), plasmid linearization, mRNA production by in vitro transcription (IVT), and finally, mRNA purification. Poor understanding of interactions among reagents can diminish mRNA yield and purity. For instance, quality of raw materials varies across lots. Key enzymes can aggregate, fragment, and express alongside contaminants such as residual host-cell protein and DNA. Likewise, pDNA (often expressed using Escherichia coli) can come with impurities such as host-cell protein, DNA, RNA, and endotoxin. Unnecessary addition of bovine serum albumin (BSA) as an enzyme stabilizer compounds contamination concerns.

Such impurities not only jeopardize product safety and drive up purification costs, but also interact in ways that form stable aggregates and complexes with mRNA, diminishing product yield and purity. Minimizing reagent impurities and interactions among them could improve process economy and reduce costs significantly.

Early Optimization: Although drug sponsors often outsource plasmid expression, keeping that activity in house could increase impurity control. Doing so enables sponsors to know what contaminants must be addressed at later process stages. Considering long lead times for contract services instigated by the pandemic, in-house pDNA expression also could help ensure consistent plasmid supply.

Štrancar recommended minimizing contaminants before plasmid linearization. BIA Separations isolates pDNA — including open-circular (OC) plasmids and multimers — using alkaline lysis with an adjustment to 0.5–1.0 M CaCl2, followed by a two-step filtration process. Then partial purification of pDNA is performed using a CIMmultus diethylaminoethyl (DEAE, a weak anion exchanger) monolith. That step recovers 80–90% of pDNA. Partial purification, Štrancar explained, maximizes subsequent yield and purity of linear plasmids. He added that OC plasmids and multimers should not be removed at this stage because they also can be linearized. More rigorous purification can be performed after linearization using a strongly hydrophobic CIMmultus C4-HLD monolith, usually with ~80% recovery of highly purified linear pDNA.

IVT and Beyond: Štrancar recommended reassessing reagent purity after linearization. Contaminants will bind with mRNA produced during IVT, and sponsors do not want to lose product at the IVT stage because it requires expensive materials such as antireverse cap analog (ARCA). Štrancar pointed out that sponsors can optimize the IVT reaction using high-performance liquid chromatography (HPLC). CIMac PrimaS columns facilitate such analyses because they separate mRNA from building blocks and reagents, enabling calculation of requisite ARCA concentrations. Using that method, BIA Separations lowered its ARCA consumption from 8 mM to 4 mM per process, providing significant cost savings. Equally important, the CIMac PrimaS workflow enabled BIA scientists to increase the transcription factor to >100. Such an increase makes more mRNA product available for purification.

Of course, IVT generates its own impurities, including double-stranded (ds) mRNA. CIMmultus Oligo dT columns separate ds and single-stranded (ss) mRNA effectively, enabling ~95% target recovery. After a capping step, target mRNAs can undergo a final polishing step on a CIMmultus C4-HLD column, resulting in 70–80% recovery of highly purified mRNA. Štrancar added that using a PATfix HPLC system for these steps could enable in-process control, expedite process development, and augment process robustness.

Questions and Answers
What titers can BIA’s process yield? The process is fully scalable. During final polishing, BIA usually dilutes drug substance to 1 mg/mL, but further concentration is possible.

Why should an IVT factor exceed 100? A high transcription factor (>100) indicates that an IVT reaction can yield a given amount of mRNA using fewer plasmids than are used in a typical process. Ultimately, the goal is to generate the highest amount of mRNA from a given number of plasmids.

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