A social enterprise established by the U.K. government is turning its attention to improving the development and scale-up of new RNA therapies and vaccines. The Center for Process Innovation (CPI), established by the U.K. government in 2004, announced last year the development of a new GMP manufacturing facility and training center dedicated to RNA therapeutics.
A CPI senior scientist, Daniel Myatt, PhD, presented at PEGS Europe in November on several new techniques for characterizing the properties of mRNA vaccines that use self-amplifying RNA (saRNA).
“As mRNA is a relatively new biologic, there are a variety of questions about the biophysics of the RNA molecules,” says Myatt. And “only by understanding the size, shape, and structure of the RNA molecule can we build a robust production process.”
According to Myatt, mRNA and saRNA are a distinct class of biologics that need different protocols and analytical techniques to characterize physical properties, such as size and shape. These, he writes, help determine the purification processes used in manufacturing and are important for quality control.
Challenging to characterize
saRNAs can be particularly challenging to characterize because they are even larger than the typical size of mRNA vaccines, which are, in turn, much larger than traditional monoclonal antibodies, he adds.
“If we think of the size of these molecules expressed as hydrodynamic radius (RH), which is the radius of the RNA molecule including its water shell,” Myatt says, then an mRNA “is likely to have a RH of 150-200 Ångstrom. In our research we determine an saRNA to have a hydrodynamic radius of 400 Ångstrom,” he notes, adding that a monoclonal antibody, by comparison, might only have an RH of 50–60 Ångstrom.
RNA is also negatively charged and greatly affected by charge which, again, impacts the purification process.
The research looked at several techniques, including dynamic light scattering (DLS), size-exclusion chromatography-small angle scattering (SEC-SAXS), and circular dichroism (CD).
DLS, he says, is helpful for quickly assessing the hydrodynamic radius. SEC-SAXS allows for deeper characterization, including an indication of molecular size, compactness, and shape, while CD provides information about the way the mRNA structure forms loops and bulges.
Myatt explains that the RNA structure is quite dynamic, and these techniques allow for analysis of RNA molecules in different buffer formulations, at different pHs and different temperatures. “The information determined from these techniques has already aided the production of mRNA vaccine molecules at CPI,” he points out.