Standard and Practical Process to Fast-Track mRNA Drug Product Manufacturing
The central dogma of life has DNA on one side, protein on the other, and RNA sitting right in the centre. RNA, specifically mRNA, is the intermediary step in this dogma and is being used as the essential cargo in many therapies. Expertise in manufacturing high-quality DNA and enzymes used in the in vitro transcription (IVT), capping, and the tailing process is fundamental to mRNA production for several therapeutic modalities. Based on our 25 years of experience as a contract development and manufacturing organisation (CDMO), we present considerations and practical solutions at each step of production to streamline the workflow with the appropriate quality system to deliver mRNA therapeutically.
THE STANDARD WORKFLOW OF AN IVT REACTION
Plasmid Vector Design Serving as a Linear Template
Using a consciously designed plasmid backbone that has optimized untranslated regions (UTRs) and poly(A) tail with a well-placed restriction site for linearisation is critical in the first step of mRNA manufacturing as they may affect the downstream processes. The 5’ and 3’ UTRs regulate the transcription of the gene of interest, affect translation efficiency, and provide molecule stability. This is where experience in the design process provides the opportunity for optimisation. For example, customers who decide to encode a poly(A) tail within their linear DNA template design also must contend with the limitations of E. coli in propagating structural DNA elements like the poly(A) tail. Once the template is manufactured, linearisation with a blunt end restriction site is recommended as a best practice (Step 1 in Figure 1). Before proceeding into the IVT reaction (Step 2 in Figure 1), the digestion should be fully optimised to a 100% rate. Failure to achieve complete digestion prior to IVT can produce impurities with subsequent steps of the reactions. These considerations in plasmid vector design should be accounted for when choosing a manufacturing partner to get the optimal mRNA synthesis.
Cap it – By Either Enzymatic or Co-transcriptional Methods
Both enzymatic and co-transcriptional capping is accepted by industry standards for the manufacturing of mRNA. With co-transcriptional capping, the big advantage is that it is a one-pot reaction. However, enzymatic capping has been observed to be more efficient than co-transcriptional since it is the natural capping method using wild-type enzymes (Step 3 in Figure 1).
When establishing manufacturing standards, capping efficiency can be determined by state-of-the-art analytical methods. The challenge is that conditions must be optimised for every RNA construct. Best practices include using liquid chromatography followed by mass spectrometry to evaluate the final product. Therefore, when selecting your manufacturing partners, ask what methods will be utilised in determining capping efficiency. It should be clear how the capping efficiency is being measured, and the process should be tailored to the specific individual construct(s).