mRNA - Understanding What Fits

November 30, 2022 / by Nate Spangler, Ph.D.

Production paths to mRNA

My last post gave a short introduction to the manufacture of in vitro transcribed (IVT) messenger RNA (mRNA) for vaccines and therapies, outlining the steps from preclinical to clinical stages of production. In this post, I’ll touch on manufacturing considerations, beginning with the upstream process for IVT mRNA.

Taking the first steps
Each program entering clinical-stage development has unique requirements. Designing a tailored, scalable manufacturing process begins with questions to better understand what fits. For example:

• What is the application? Ex vivo or in vivo?
• Is activation of an innate immune response a concern?
• What will the manufacturing scales be from preclinical to commercial stages?
• What are the requirements for mRNA purity and activity?
• What are the tolerances for contaminants and impurities

The construct design also guides and limits manufacturing choices. For example:

• Does the DNA template have a T7 or SP6 promoter?
• Is the poly(A) tail template encoded?
• Is the construct designed for a GAG cap analog?
• What is the transcript length?

General questions lead to more specific until a detailed understanding of the program’s requirements are achieved and manufacturing choices have been narrowed to the best options. Small-scale scouting and process demonstrations will lead to data-informed decisions about what can work best for scale-up manufacturing in clinical to commercial stages.

Upstream process scouting and demonstration begin with standardized parameters generally tailored to mRNA or saRNA. If needed, molecule-specific process optimization can be performed because not all constructs behave similarly in the IVT and capping reactions.

Several process factors can affect the product yield, such as integrity, purity, and/or activity. For example: reaction temperature and time, enzyme and substrate concentrations, and the use of certain raw materials such as cap analogs and base-modified NTPs.

To keep program timelines to a minimum, process scouting, demonstration, and optimization work is performed in parallel with analytical methods and master cell bank generation, so that these major steps converge as soon as possible on the critical path to the IVT mRNA engineering run followed by GMP manufacturing.

To wrap up, early process scouting, demonstration, and optimization work for IVT mRNA production can pay dividends later without increasing the overall program timeline. In my next post, I’ll discuss downstream challenges in mRNA production.

• Read Nate's previous post, Four major steps involved in mRNA manufacturing
• Watch Nate's presentation, Self-amplifying and messenger RNA for Vaccines and Therapies
• Want to learn more? Contact Nate Spangler with your questions
• Visit the mRNA section of our website