Helen Gunter from The BASE Facility of The University of Queensland in Australia presented at the ONT Biopharma Day. The session’s title was “Using nanopore sequencing for mRNA vaccine quality control: a journey from R&D to GMP.” They noted that since the COVID vaccines, there has been interest in synthetic mRNAs. Gunter spoke about the potential of mRNA. The BASE Facility produces mRNAs at scale. Gunter explained that the mRNA has 5’UTR with a 5′ Cap, a coding region, 3’UTR, and a polyA tail. Sequence optimization is needed to enhance stability and translation. The mRNA manufacture workflow, Gunter explained, includes plasmid amplification, linearization (restriction digestion), in vitro transcription, purification by chromatography, lipid nanoparticle formulation, and purification. USP has produced guidance for mRNA quality control and testing. In 2023, Gunter and team published a Nature Communications article for mRNA identity quality control. Since then, Oxford Nanopore Technologies (ONT) and The BASE Facility have formed a collaboration. Gunter shared data from plasmid release testing. Their trials suggested that sequence identity can be reliably confirmed and conformation of the plasmid can be elucidated. Interestingly, shorter sequencing runs were sufficient and more reliable. They obtained 1% sensitivity of subclonal populations and no difference between 2 and 72 hr runs. Gunter explained that modified bases in mRNA vaccines increase translation and reduce inflammation. The BASE Facility team used SQK-RNA004 for direct RNA sequencing and developed a workflow for mRNA sequencing. The most common error was deletion. Sequence identity could be confirmed through dot blots. The long reads also help confirm full length mRNAs are being produced. One current limitation of direct RNA sequencing is that the first ~10 nt on the 5′ end cannot be reliably identified. The abundance of a cap can be investigated and correlates with the first nt. LNP encapsulation and mRNA integrity can be investigated through the purification of mRNAs and sequencing. Multivalent mRNA vaccines can be evaluated through sequencing, too. Gunter concluded that ONT nanopore sequencing can currently address most of USP’s mRNA quality control requirements and several additional tests are in development. The GridION-Q offers stable protocols that are suitable for this setting. Gunter also mentioned that they had developed mRNA standards at the BASE Facility.
